The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil.

PubMed

Sánchez-Aragó, María; Cuezva, José M

2011-02-08

Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU). The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells. We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable. Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

'Hints' in the killer protein gasdermin D: unveiling the secrets of gasdermins driving cell death.

PubMed

Qiu, Shiqiao; Liu, Jing; Xing, Feiyue

2017-04-01

Pyroptosis is a lytic form of cell death distinguished from apoptosis, ferroptosis, necrosis, necroptosis, NETosis, oncosis, pyronecrosis and autophagy. Proinflammatory caspases cleave a gasdermin D (GSDMD) protein to generate a 31 kDa N-terminal domain. The cleavage relieves the intramolecular inhibition on the gasdermin-N domain, which then moves to the plasma membrane to exhibit pore-forming activity. Thus, GSDMD acts as the final and direct executor of pyroptotic cell death. Owing to the selective targeting of the inner leaflet of the plasma membrane with the pore-forming that determines pyroptotic cell death, GSDMD could be a potential target to control cell death or extracellular bacterial infections. Intriguingly, other gasdermin family members also share similar N-terminal domains, but they present different cell death programs. Herein, we summarize features and functions of the novel player proteins in cell death, including GSDMD triggering pyroptosis, Gsdma3/GSDMA initiating autophagy/apoptosis and DFNA5 inducing apoptosis/secondary necrosis. The gasdermin N terminus appears to be a novel pore-forming protein. This provides novel insight into the underlying roles and mechanisms of lytic or nonlytic forms of programmed cell death, as well as their potential applications in inflammation-associated diseases.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

PubMed

Joglekar, Mugdha V; Trivedi, Prerak M; Kay, Thomas W; Hawthorne, Wayne J; O'Connell, Philip J; Jenkins, Alicia J; Hardikar, Anandwardhan A; Thomas, Helen E

2016-04-01

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

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.

Determination of cellular injury and death thresholds following exposure to high voltage 10ns electrical pulses

NASA Astrophysics Data System (ADS)

Ibey, Bennett L.; Roth, Caleb C.; Bernhard, Joshua A.; Pakhomov, Andrei G.; Wilmink, Gerald J.; Pakhomova, Olga

2011-03-01

Intense, nanosecond-duration electric pulses (nsEP) have been introduced as a novel modality to alter cellular function, with a mechanism of action qualitatively different from micro- and millisecond duration pulses used in electroporation. In this study, we determined the thresholds for plasma membrane injury (within 15 minutes) and cell death (at 24 hours) for 4 different cell types (CHO-K1, HeLa, Jurkat and U937). Plasma membrane injury was measured by flow cytometry using two fluorescent dyes, namely Annexin V-FITC, which binds to phosphatidylserine (PS) upon its externalization (subtle membrane injury), and propidium iodide (PI), which is typically impermeable to the cell, but enters when large pores are formed in the plasma membrane. In all cell types, 10-ns pulses caused phosphatidylserine (PS) externalization at low doses (<150kV/cm and 100 pulses for each cell type) and no PI uptake. Jurkat and U937 cell lines showed substantial cell death without uptake of PI (15 minutes post exposure) suggesting either delayed permeabilization due to swelling, or damage to intracellular components. In CHO-K1 and HeLa cell lines, PI uptake occurred at low doses relative to that necessary to cause cell death suggesting a necrotic death similar to longer pulse exposures. These findings suggest that nanosecond pulses may be beneficial in applications that require selective elimination of specific cell types.

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

The slow cell death response when screening chemotherapeutic agents.

PubMed

Blois, Joseph; Smith, Adam; Josephson, Lee

2011-09-01

To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay. With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry. Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72 h. Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72 h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.

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.

bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship.

PubMed

Strasser, A; Harris, A W; Cory, S

1991-11-29

Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.

METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

PubMed Central

Escamez, Sacha; André, Domenique; Zhang, Bo; Bollhöner, Benjamin; Pesquet, Edouard; Tuominen, Hannele

2016-01-01

ABSTRACT We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells. PMID:26740571

‘Hints' in the killer protein gasdermin D: unveiling the secrets of gasdermins driving cell death

PubMed Central

Qiu, Shiqiao; Liu, Jing; Xing, Feiyue

2017-01-01

Pyroptosis is a lytic form of cell death distinguished from apoptosis, ferroptosis, necrosis, necroptosis, NETosis, oncosis, pyronecrosis and autophagy. Proinflammatory caspases cleave a gasdermin D (GSDMD) protein to generate a 31 kDa N-terminal domain. The cleavage relieves the intramolecular inhibition on the gasdermin-N domain, which then moves to the plasma membrane to exhibit pore-forming activity. Thus, GSDMD acts as the final and direct executor of pyroptotic cell death. Owing to the selective targeting of the inner leaflet of the plasma membrane with the pore-forming that determines pyroptotic cell death, GSDMD could be a potential target to control cell death or extracellular bacterial infections. Intriguingly, other gasdermin family members also share similar N-terminal domains, but they present different cell death programs. Herein, we summarize features and functions of the novel player proteins in cell death, including GSDMD triggering pyroptosis, Gsdma3/GSDMA initiating autophagy/apoptosis and DFNA5 inducing apoptosis/secondary necrosis. The gasdermin N terminus appears to be a novel pore-forming protein. This provides novel insight into the underlying roles and mechanisms of lytic or nonlytic forms of programmed cell death, as well as their potential applications in inflammation-associated diseases. PMID:28362726

Bim regulates alloimmune-mediated vascular injury through effects on T-cell activation and death.

PubMed

von Rossum, Anna; Enns, Winnie; Shi, Yu P; MacEwan, Grace E; Malekesmaeli, Mehrnoush; Brinkman, Ryan; Choy, Jonathan C

2014-06-01

Bim is a proapoptotic Bcl-2 protein known to downregulate immune responses and to also be required for antigen-induced T-cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T-cell responses in a model of vascular rejection. Bim was required for proliferation of CD4 and CD8 T cells, and for interleukin-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T-cell activation, whereas a complete elimination of Bim was required to prevent CD4 T-cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim(+/-), but not Bim(-/-), graft recipients. T-cell proliferation in response to allograft arteries was significantly reduced in both Bim(+/-) and Bim(-/-) mice, but cell death was attenuated only in Bim(-/-) animals. Bim controls both T-cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries. © 2014 American Heart Association, Inc.

Bim Regulates Alloimmune-Mediated Vascular Injury Through Effects on T Cell Activation and Death

PubMed Central

von Rossum, Anna; Enns, Winnie; Shi, Yu P.; MacEwan, Grace E.; Malekesmaeli, Mehrnoush; Brinkman, Ryan; Choy, Jonathan C.

2014-01-01

Objective Bim is a pro-apoptotic Bcl-2 protein known to down-regulate immune responses and to also be required for antigen-induced T cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T cell responses in a model of vascular rejection. Approach and Results Bim was required for proliferation of CD4 and CD8 T cells, and for IL-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T cell activation whereas a complete elimination of Bim was required to prevent CD4 T cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim+/−, but not Bim−/−, graft recipients. T cell proliferation in response to allograft arteries was significantly reduced in both Bim+/− and Bim−/− mice, but cell death was attenuated only in Bim−/− animals. Conclusions Bim controls both T cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries. PMID:24700126

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PubMed

Shin, Yoo Seob; Cha, Hyun Young; Lee, Bok-Soon; Kang, Sung Un; Hwang, Hye Sook; Kwon, Hak Cheol; Kim, Chul-Ho; Choi, Eun Chang

2016-04-01

The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

PubMed

Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-07-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

PubMed Central

Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-01-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

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

The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil

PubMed Central

2011-01-01

Background Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU). Methods The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells. Results We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable. Conclusions Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression. PMID:21303518

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

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

PubMed Central

Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

2017-01-01

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

PubMed

Hernández-Tiedra, Sonia; Fabriàs, Gemma; Dávila, David; Salanueva, Íñigo J; Casas, Josefina; Montes, L Ruth; Antón, Zuriñe; García-Taboada, Elena; Salazar-Roa, María; Lorente, Mar; Nylandsted, Jesper; Armstrong, Jane; López-Valero, Israel; McKee, Christopher S; Serrano-Puebla, Ana; García-López, Roberto; González-Martínez, José; Abad, José L; Hanada, Kentaro; Boya, Patricia; Goñi, Félix; Guzmán, Manuel; Lovat, Penny; Jäättelä, Marja; Alonso, Alicia; Velasco, Guillermo

2016-11-01

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ 9 -tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

Induction of cell death by magnetic particles in response to a gradient magnetic field inside a uniform magnetic field

NASA Astrophysics Data System (ADS)

Amaya-Jaramillo, Carlos David; Pérez-Portilla, Adriana Patricia; Serrano-Olmedo, José Javier; Ramos-Gómez, Milagros

2017-10-01

A new instrument based on a magnetic force produced by an alternating magnetic field gradient, which is obtained through Maxwell coils, inside a constant field magnet has been designed and used to produce cell death. We have determined the interaction of microparticles and cells under different conditions such as incubation time with microparticles, particle size, magnetic field exposition time, and different current waveforms at different frequencies to produce a magnetic field gradient. We determined that the highest rate of cell death occurs at a frequency of 1 Hz with a square waveform and 1 h of irradiation. This method could be of great interest to remove cancer cells due mainly to the alterations in stiffness observed in the membranes of the tumor cells. Cancer cells can be eliminated in response to the forces caused by the movement of magnetic nanoparticles of the appropriate size under the application of a specific magnetic field. [Figure not available: see fulltext.

Cell death features induced in Leishmania major by 1,3,4-thiadiazole derivatives.

PubMed

Ardestani, Sussan K; Poorrajab, Fatemeh; Razmi, Sepideh; Foroumadi, Alireza; Ajdary, Soheila; Gharegozlou, Behnaz; Behrouzi-Fardmoghadam, Mina; Shafiee, Abbas

2012-10-01

Under a variety of stress conditions, Leishmania species display some morphological and biochemical features characteristic of mammalian programmed cell death or necrosis. Nitroheteroaryl-1,3,4-thiadiazoles induce cell death in Leishmania major (L. major). Putative mechanisms of action of these compounds were investigated in vitro at cellular and molecular levels. We used colorimetric assay to measure acid phosphatase activity which is an indicator of cell viability in the promastigotes. The mode of toxicity was determined by detection of phosphatidylserine translocation to the surface, evaluation of cell membrane integrity, and in situ dUTP nick end-labeling assay. We also determined poly-ADP-ribose polymerase-like protein (PARP) level in the parasites after treatment. A significant reduction of acid phosphatase level, one of the most crucial and virulent factors of the parasite was found in parasites treated with 1,3,4-thiadiazole derivatives. In addition, 1,3,4-thiadiazole derivatives induced loss of plasma membrane integrity, DNA breakage, proteolysis of PARP and necrotic-like death in the parasites. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro cytotoxic effects of benzalkonium chloride in corticosteroid injection suspension.

PubMed

Davis, Daniel; Cyriac, Mathew; Ge, Dongxia; You, Zongbing; Savoie, Felix H

2010-01-01

Some deleterious effects on cartilage and even severe arthropathy have been reported after intra-articular corticosteroid injections. The objective of the present in vitro study was to determine if an injectable corticosteroid suspension is toxic to articular chondrocytes and synovial cells. Human and bovine articular chondrocytes, bovine synovial cells, mouse C3H10T1/2 cells, and human osteosarcoma MG-63 cells were treated for thirty minutes in monolayer or suspension culture with an injectable corticosteroid suspension or its chemical components, including betamethasone sodium phosphate, betamethasone acetate, and benzalkonium chloride (as preservative). Cell viability was determined by means of microscopy or flow cytometry analysis. In monolayer culture, the betamethasone corticosteroids per se did not cause cell death, whereas benzalkonium chloride caused death of articular chondrocytes. In suspension culture, betamethasone sodium phosphate at dosages of as high as 6 mg/mL did not cause significant death of human or bovine articular chondrocytes (p > 0.05). In contrast, benzalkonium chloride caused a death rate of 10.6% in human articular chondrocytes at a dosage of 10 microg/mL (p < 0.01), 21.0% at a dosage of 13.3 microg/mL (p < 0.01), and 99.3% and 99.4% at dosages of 20 and 200 microg/mL, respectively (p < 0.001 for both). Similarly, benzalkonium chloride caused death of bovine articular chondrocytes, bovine synovial cells, C3H10T1/2 cells, and MG-63 cells in a dose-dependent manner. When treated with a combination of betamethasone sodium phosphate and 200 microg/mL benzalkonium chloride, >99% of human or bovine articular chondrocytes were dead (p < 0.001). The injectable corticosteroid suspension caused death in in vitro culture of human and bovine articular chondrocytes as well as bovine synovial cells because of its preservative benzalkonium chloride. The betamethasone corticosteroids per se did not cause significant chondrocyte death under the conditions tested.

Guidelines and recommendations on yeast cell death nomenclature

PubMed Central

Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J.; Breitenbach, Michael; Burhans, William C.; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F.; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B.; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W.; Grant, Chris M.; Greenwood, Michael T.; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M.; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P.; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A.; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D.; Outeiro, Tiago F.; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F.; Sharon, Amir; Sigrist, Stephan J.; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M.; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B.; Tuite, Mick; Vögtle, F.-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J.; Zhao, Richard Y.; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

2018-01-01

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research. PMID:29354647

Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

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

Kim, Sung Hun; Yoo, Chong Il; Medical Research Institute, College of Medicine, Pusan National University, Pusan, 602-739

2006-09-01

The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatmentmore » caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.« less

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells

PubMed Central

Rock, Nathan; Chintala, Shravan K.

2008-01-01

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a compete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 uM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma. PMID:18243176

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.

Histological and Finite Element Analysis of Cell Death due to Irreversible Electroporation

PubMed Central

Long, G.; Bakos, G.; Shires, P. K.; Gritter, L.; Crissman, J. W.; Harris, J. L.; Clymer, J. W.

2014-01-01

Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10μs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism. PMID:24000980

Inner ear supporting cells protect hair cells by secreting HSP70

PubMed Central

May, Lindsey A.; Kramarenko, Inga I.; Brandon, Carlene S.; Voelkel-Johnson, Christina; Roy, Soumen; Truong, Kristy; Francis, Shimon P.; Monzack, Elyssa L.; Lee, Fu-Shing; Cunningham, Lisa L.

2013-01-01

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70–/– mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress. PMID:23863716

Apoptosis in lung injury and remodeling.

PubMed

Li, Xiaopeng; Shu, Ruijie; Filippatos, Gerasimos; Uhal, Bruce D

2004-10-01

The mode of cell death termed apoptosis, sometimes referred to as programmed cell death, is as critical a determinant of cell population size as is cell proliferation. Although best characterized in cells of the immune system, apoptosis is now known to be a key factor in the maintenance of normal cell turnover within structural cells in the parenchyma of virtually every organ. Recent interest in apoptosis in the lung has sparked a surge of investigations designed to determine the roles of apoptosis in lung development, injury, and remodeling. Of particular recent interest are the roles of apoptosis in disease pathogenesis and resolution, in which the concept of apoptosis as a "programmed" cell death, i.e., genetically determined, is often more accurately viewed as "inappropriate cell suicide" with regard to its extent and/or timing. Data accumulating over the past decade have made clear the complexity of the control of lung cell apoptosis; concepts of the regulation of apoptosis originally determined in classical cell culture models are often, but not always, applicable to structural cells. For this reason, each of the many cell types of the lung must be studied as a potentially new subject with its own idiosyncrasies yet to be discovered. In light of the large volume of literature now available, this article focuses on the roles of apoptosis in three pathophysiological contexts: acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. Each section presents key data describing the evidence for apoptosis in the lung, its possible relevance to disease pathogenesis, and proposed mechanisms that might suggest potential avenues for therapeutic intervention.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

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

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death.

PubMed

Ramírez-Barrantes, Ricardo; Córdova, Claudio; Gatica, Sebastian; Rodriguez, Belén; Lozano, Carlo; Marchant, Ivanny; Echeverria, Cesar; Simon, Felipe; Olivero, Pablo

2018-01-01

The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose-response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca 2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca 2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca 2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca 2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

Diagnosis of Cell Death by Means of Infrared Spectroscopy

PubMed Central

Zelig, Udi; Kapelushnik, Joseph; Moreh, Raymond; Mordechai, Shaul; Nathan, Ilana

2009-01-01

Abstract Fourier transform infrared (FTIR) spectroscopy has been established as a fast spectroscopic method for biochemical analysis of cells and tissues. In this research we aimed to investigate FTIR's utility for identifying and characterizing different modes of cell death, using leukemic cell lines as a model system. CCRF-CEM and U937 leukemia cells were treated with arabinoside and doxorubicin apoptosis inducers, as well as with potassium cyanide, saponin, freezing-thawing, and H2O2 necrosis inducers. Cell death mode was determined by various gold standard biochemical methods in parallel with FTIR-microscope measurements. Both cell death modes exhibit large spectral changes in lipid absorbance during apoptosis and necrosis; however, these changes are similar and thus cannot be used to distinguish apoptosis from necrosis. In contrast to the above confounding factor, our results reveal that apoptosis and necrosis can still be distinguished by the degree of DNA opaqueness to infrared light. Moreover, these two cell death modes also can be differentiated by their infrared absorbance, which relates to the secondary structure of total cellular protein. In light of these findings, we conclude that, because of its capacity to monitor multiple biomolecular parameters, FTIR spectroscopy enables unambiguous and easy analysis of cell death modes and may be useful for biochemical and medical applications. PMID:19804743

The cell on the edge of life and death: Crosstalk between autophagy and apoptosis.

PubMed

Kasprowska-Liśkiewicz, Daniela

2017-09-21

Recently, the crosstalk between autophagy and apoptosis has attracted broader attention. Basal autophagy serves to maintain cell homeostasis, while the upregulation of this process is an element of stress response that enables the cell to survive under adverse conditions. Autophagy may also determine the fate of the cell through its interactions with cell death pathways. The protein networks that control the initiation and the execution phase of these two processes are highly interconnected. Several scenarios for the crosstalk between autophagy and apoptosis exist. In most cases, the activation of autophagy represents an attempt of the cell to cope with stress, and protects the cell from apoptosis or delays its initiation. Generally, the simultaneous activation of pro-survival and pro-death pathways is prevented by the mutual inhibitory crosstalk between autophagy and apoptosis. But in some circumstances, autophagy or the proteins of the core autophagic machinery may promote cellular demise through excessive self-digestion (so-called "autophagic cell death") or by stimulating the activation of other cell death pathways. It is controversial whether cells actually die via autophagy, which is why the term "autophagic cell death" has been under intense debate lately. This review summarizes the recent findings on the multilevel crosstalk between autophagy and apoptosis in aspects of common regulators, mutual inhibition of these processes, the stimulation of apoptosis by autophagy or autophagic proteins and finally the role of autophagy as a death-execution mechanism.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

PubMed

Aits, Sonja; Gustafsson, Lotta; Hallgren, Oskar; Brest, Patrick; Gustafsson, Mattias; Trulsson, Maria; Mossberg, Ann-Kristin; Simon, Hans-Uwe; Mograbi, Baharia; Svanborg, Catharina

2009-03-01

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

Vinblastine and diethylstilboestrol tested in the in vitro mammalian cell micronucleus test (MNvit) at Swansea University UK in support of OECD draft Test Guideline 487.

PubMed

Johnson, George E; Jenkins, Gareth J; Thomas, Adam D; Doak, Shareen H

2010-10-29

The known aneugens vinblastine and diethylstilboestrol (DES) were tested in the in vitro micronucleus assay, with and without cytokinesis block in Chinese hamster CHO cells, at the laboratories of Swansea University, Swansea, UK. These experiments were carried out to determine the suitability of the cell death and cytostasis measures used in the assay, as recommended in the draft OECD Test Guideline 487, 2007. Both compounds were positive in the assay without cytokinesis block at concentrations giving approximately 50% or less cell death and cytostasis, using relative population doublings and relative increase in cell counts. Moreover, both compounds were positive in the assay with cytokinesis block at concentrations giving approximately 50% cell death and cytostasis, using replicative index. Vinblastine was also positive for mitotic slippage, causing micronuclei in mononucleate cells with cytokinesis block. Relative population doublings and relative increase in cell counts were appropriate measures of cell death and cytostasis for the non-cytokinesis block in vitro micronucleus assay. In the cytokinesis blocked micronucleus assay, replicative index and cytokinesis block proliferation index were suitable cell death and cytostasis measures. Copyright © 2009 Elsevier B.V. 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.

Estrogen attenuates glutamate-induced cell death by inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels

PubMed Central

Sribnick, Eric A.; Del Re, Angelo M.; Ray, Swapan K.; Woodward, John J.; Banik, Naren L.

2009-01-01

Estrogen-mediated neuroprotection is observed in neurodegenerative disease and neurotrauama models; however, determining a mechanism for these effects has been difficult. We propose that estrogen may limit cell death in the nervous system tissue by inhibiting increases in intracellular free Ca2+. Here, we present data using VSC 4.1 cell line, a ventral spinal motoneuron and neuroblastoma hybrid cell line. Treatment with 1 mM glutamate for 24 h induced apoptosis. When cells were pre-treated with 100 nM 17β-estradiol (estrogen) for 1 h and then co-treated with glutamate, apoptotic death was significantly attenuated. Estrogen also prevented glutamate-mediated changes in resting membrane potential and membrane capacitance. Treatment with either 17α-estradiol or cell impermeable estrogen did not mimic the findings seen with estrogen. Glutamate treatment significantly increased both intracellular free Ca2+ and the activities of downstream proteases such as calpain and caspase-3. Estrogen attenuated both the increases in intracellular free Ca2+ and protease activities. In order to determine the pathway responsible for estrogen-mediated inhibition of these increases in intracellular free Ca2+, cells were treated with several Ca2+ entry inhibitors, but only the L-type Ca2+ channel blocker nifedipine demonstrated cytoprotective effects comparable to estrogen. To expand these findings, cells were treated with the L-type Ca2+ channel agonist FPL 64176, which increased both cell death and intracellular free Ca2+, and estrogen inhibited both effects. From these observations, we conclude that estrogen limits glutamate-induced cell death in VSC 4.1 cells through effects on L-type Ca2+ channels, inhibiting Ca2+ influx as well as activation of the pro-apoptotic proteases calpain and caspase-3. PMID:19389388

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

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.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line[S

PubMed Central

Podbielska, Maria; Szulc, Zdzisław M.; Kurowska, Ewa; Hogan, Edward L.; Bielawski, Jacek; Bielawska, Alicja; Bhat, Narayan R.

2016-01-01

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated “donor” cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer. PMID:27623848

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.

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

Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability.

PubMed

Beebe, Stephen J; Chen, Yeong-Jer; Sain, Nova M; Schoenbach, Karl H; Xiao, Shu

2012-01-01

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.

Single-cell imaging of the heat-shock response in colon cancer cells suggests that magnitude and length rather than time of onset determines resistance to apoptosis.

PubMed

Ramapathiran, Lavanya; Bernas, Tytus; Walter, Franziska; Williams, Linda; Düssmann, Heiko; Concannon, Caoimhín G; Prehn, Jochen H M

2014-02-01

Targeting the proteasome is a valuable approach for cancer therapy, potentially limited by pro-survival pathways that are induced in parallel to cell death. Whether these pro-survival pathways are activated in all cells, show different activation kinetics in sensitive versus resistant cells or interact functionally with cell death pathways is unknown. We monitored activation of the heat-shock response (HSR), a key survival pathway induced by proteasome inhibition, relative to apoptosis activation in HCT116 colon cancer cells expressing enhanced green fluorescent protein (EGFP) under the control of the HSP70 promoter. Single-cell and high-content time-lapse imaging of epoxomicin treatment revealed that neither basal activity nor the time of onset of the HSR differed between resistant and sensitive populations. However, resistant cells had significantly higher and prolonged reporter activity than those that succumbed to cell death. p53 deficiency protected against cell death but failed to modulate the HSR. By contrast, inhibition of the HSR significantly increased the cytotoxicity of epoxomicin. Our data provide novel insights into the kinetics and heterogeneity of the HSR during proteasome inhibition, suggesting that the HSR modulates cell death signalling unidirectionally.

NEMO Inhibits Programmed Necrosis in an NFκB-Independent Manner by Restraining RIP1

PubMed Central

Legarda, Diana; Ting, Adrian T.

2012-01-01

TNF can trigger two opposing responses: cell survival and cell death. TNFR1 activates caspases that orchestrate apoptosis but some cell types switch to a necrotic death when treated with caspase inhibitors. Several genes that are required to orchestrate cell death by programmed necrosis have been identified, such as the kinase RIP1, but very little is known about the inhibitory signals that keep this necrotic cell death pathway in check. We demonstrate that T cells lacking the regulatory subunit of IKK, NFκB essential modifier (NEMO), are hypersensitive to programmed necrosis when stimulated with TNF in the presence of caspase inhibitors. Surprisingly, this pro-survival activity of NEMO is independent of NFκB-mediated gene transcription. Instead, NEMO inhibits necrosis by binding to ubiquitinated RIP1 to restrain RIP1 from engaging the necrotic death pathway. In the absence of NEMO, or if ubiquitination of RIP1 is blocked, necrosis ensues when caspases are blocked. These results indicate that recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response. PMID:22848449

Guidelines and recommendations on yeast cell death nomenclature.

PubMed

Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J; Breitenbach, Michael; Burhans, William C; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W; Grant, Chris M; Greenwood, Michael T; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D; Outeiro, Tiago F; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F; Sharon, Amir; Sigrist, Stephan J; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B; Tuite, Mick; Vögtle, F-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J; Zhao, Richard Y; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

2018-01-01

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.

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

Galán-Malo, Patricia; Vela, Laura; Gonzalo, Oscar

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x{sub L} and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x{sub L} gene was silenced in A549 cells, vincristinemore » induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x{sub L} or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x{sub L} switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x{sub L}/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types. Highlights: ► Vincristine induces cell death by apoptosis or mitotic catastrophe. ► Apoptosis-proficient cells die by apoptosis during mitosis upon vincristine treatment. ► p53wt apoptosis-deficient cells undergo apoptosis from a G1-like tetraploid state. ► p53mt apoptosis-deficient cells can survive and divide giving rise to 8N cells.« less

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.

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

Cell birth, cell death, cell diversity and DNA breaks: how do they all fit together?

NASA Technical Reports Server (NTRS)

Gilmore, E. C.; Nowakowski, R. S.; Caviness, V. S. Jr; Herrup, K.

2000-01-01

Substantial death of migrating and differentiating neurons occurs within the developing CNS of mice that are deficient in genes required for repair of double-stranded DNA breaks. These findings suggest that large-scale, yet previously unrecognized, double-stranded DNA breaks occur normally in early postmitotic and differentiating neurons. Moreover, they imply that cell death occurs if the breaks are not repaired. The cause and natural function of such breaks remains a mystery; however, their occurrence has significant implications. They might be detected by histological methods that are sensitive to DNA fragmentation and mistakenly interpreted to indicate cell death when no relationship exists. In a broader context, there is now renewed speculation that DNA recombination might be occurring during neuronal development, similar to DNA recombination in developing lymphocytes. If this is true, the target gene(s) of recombination and their significance remain to be determined.

Cell proliferation is a key determinant of the outcome of FOXO3a activation

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

Poulsen, Raewyn C., E-mail: raewyn.poulsen@gmail.com; Carr, Andrew J.; Hulley, Philippa A.

2015-06-19

The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media,more » FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail. - Highlights: • FOXO3a activity can result in either promotion of cell survival or apoptosis. • The outcome of FOXO3a activation differs in proliferating compared to non-proliferating cells. • Proliferating cells are susceptible to FOXO3a-mediated apoptosis. • Inhibition of cell proliferation by FOXO3a promotes cell survival.« less

The effects of monobromobimane on neuronal cell death in the hippocampus after transient global cerebral ischemia in rats.

PubMed

Abe, Tsutomu; Takagi, Norio; Nakano, Midori; Takeo, Satoshi

2004-03-11

Calcium accumulation and free radical formation in the mitochondria are suggested to result in opening of the mitochondrial permeability transition pore that may be an initial step in neuronal cell death. The purpose of the present study was to determine whether monobromobimane (MBM) was a possible protective agent against neuronal cell death after transient global ischemia and the swelling of isolated hippocampal mitochondria. Infusion of MBM (1 or 3 microg) to cerebral ventricles 30 min before ischemia attenuated the expression of TUNEL-labeled cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion dose-dependently. Treatment with MBM inhibited an increase in caspase-3-like activity at 48 h of reperfusion in the hippocampus. MBM (30-300 microM) also inhibited an enhanced swelling rate induced by Ca2+ and phenylarsineoxide in the isolated hippocampal mitochondria. These results suggest that in vivo treatment with MBM may protect against neuronal cell death through inhibition of the mitochondrial swelling and caspase-3-dependent apoptotic pathway.

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.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection.

PubMed

Iampietro, Mathieu; Younan, Patrick; Nishida, Andrew; Dutta, Mukta; Lubaki, Ndongala Michel; Santos, Rodrigo I; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

2017-05-01

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases

PubMed Central

Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Pickett, Chillian; Sumin, Li; Jones, Jocelyn; Chen, Han; Webb, Brian; Choi, Jae; Zhou, You; Zimmerman, Matthew C.; Franco, Rodrigo

2013-01-01

The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone and 1-methyl-4-phenylpyridinium (MPP+) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2•−), oxidative stress and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation was determined in the cytosol, intermembrane (IMS) and mitochondrial matrix compartments, using dihydroethidine derivatives, the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and mitochondrial matrix prior to cell death. MPP+ and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on ROS steady state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm) and dopaminergic cell death induced by MPP+ or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP+ or rotenone, induced the transcriptional activation the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2•− in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP+ does not depend directly on mitochondrial O2•− formation. PMID:23602909

Quantifying cell turnover using CFSE data.

PubMed

Ganusov, Vitaly V; Pilyugin, Sergei S; de Boer, Rob J; Murali-Krishna, Kaja; Ahmed, Rafi; Antia, Rustom

2005-03-01

The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based "Smith-Martin" model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S+G2+M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett-Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle.

Can dead bacterial cells be defined and are genes expressed after cell death?

PubMed

Trevors, J T

2012-07-01

There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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.

ER stress responses in the absence of apoptosome: a comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts.

PubMed

Deegan, Shane; Saveljeva, Svetlana; Gupta, Sanjeev; MacDonald, David C; Samali, Afshin

2014-08-29

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9(+/+) and casp9(-/-) MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9(-/-) cells as compared with casp9(+/+) MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9(-/-) MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Zebrafish hair cell mechanics and physiology through the lens of noise-induced hair cell death

NASA Astrophysics Data System (ADS)

Coffin, Allison B.; Xu, Jie; Uribe, Phillip M.

2018-05-01

Hair cells are exquisitely sensitive to auditory stimuli, but also to damage from a variety of sources including noise trauma and ototoxic drugs. Mammals cannot regenerate cochlear hair cells, while non-mammalian vertebrates exhibit robust regenerative capacity. Our research group uses the lateral line system of larval zebrafish to explore the mechanisms underlying hair cell damage, identify protective therapies, and determine molecular drivers of innate regeneration. The lateral line system contains externally located sensory organs called neuromasts, each composed of ˜8-20 hair cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and share similar susceptibility to ototoxic damage. In the last decade, the lateral line has emerged as a powerful model system for understanding hair cell death mechanisms and for identifying novel protective compounds. Here we demonstrate that the lateral line is a tractable model for noise-induced hair cell death. We have developed a novel noise damage system capable of inducing over 50% loss of lateral line hair cells, with hair cell death occurring in a dose- and time-dependent manner. Cell death is greatest 72 hours post-exposure. However, early signs of hair cell damage, including changes in membrane integrity and reduced mechanotransduction, are apparent within hours of noise exposure. These features, early signs of damage followed by delayed hair cell death, are consistent with mammalian data, suggesting that noise acts similarly on zebrafish and mammalian hair cells. In our future work we will use our new model system to investigate noise damage events in real time, and to develop protective therapies for future translational research.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

PubMed

Seki, Keisuke; Cueno, Marni E; Kamio, Noriaki; Saito, Yuko; Kamimoto, Atsushi; Kurita-Ochiai, Tomoko; Ochiai, Kuniyasu

2016-06-01

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Mortality in children, adolescents and adults with sickle cell anemia in Rio de Janeiro, Brazil.

PubMed

Lobo, Clarisse Lopes de Castro; Nascimento, Emilia Matos do; Jesus, Leonardo José Carvalho de; Freitas, Thiago Gotelip de; Lugon, Jocemir Ronaldo; Ballas, Samir K

To determine the mortality rate of children, adolescents and adults with sickle cell anemia in Rio de Janeiro, Brazil. The number of deaths, the mortality rate and the causes of deaths in patients with sickle cell anemia who were treated and followed up at our institution for 15 years were determined and compared to data available for the Brazilian population. The overall number of deaths was 281 patients with a mortality rate of 16.77%. Survival probability was significantly higher in females. The number of deaths and the mortality rate were age-specific with a significant increase in the 19- to 29-year-old age group. The remaining life expectancy of the patients with sickle cell anemia was less than that of Brazilians at large. The gap between the two was about 20 years for ages between one and five years with this gap decreasing to ten years after the age of 65 years. The most common causes of death were infection, acute chest syndrome, overt stroke, organ damage and sudden death during painful crises. To the best of our knowledge, this is the first Brazilian study in a single institution in Rio de Janeiro; the mortality rate was 18.87% among adult patients with sickle cell anemia. The mortality rates in children and adults are higher than those reported in developed countries of the northern hemisphere. Copyright © 2017 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.

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

PubMed

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

2017-05-01

24(S)-Hydroxycholesterol (24S-OHC) has diverse physiological and pathological functions. In particular, cytotoxic effects of 24S-OHC in neuronal cells are important in development of neurodegenerative diseases. 24S-OHC induces necroptosis-like cell death in SH-SY5Y cells expressing little caspase-8. In the present study, 24S-OHC was found to induce apoptosis as determined by caspase-3 activation in all-trans-retinoic acid (atRA)-treated SH-SY5Y cells in which expression of caspase-8 was induced. 24S-OHC-induced cell death was inhibited by α-tocopherol (α-Toc) but not by α-tocotrienol (α-Toc3) in SH-SY5Y cells regardless of whether cells were treated with atRA. In contrast, cumene hydroperoxide (CumOOH)-induced cell death was significantly inhibited by α-Toc and α-Toc3. In atRA-treated SH-SY5Y cells, generation of reactive oxygen species (ROS) was induced by stimulation with CumOOH but was not induced by stimulation with 24S-OHC. These results suggest that inhibition of 24S-OHC-induced cell death by α-Toc cannot be explained by its radical scavenging antioxidant activity. Esterification of 24S-OHC followed by lipid droplet (LD) formation due to acyl-CoA:cholesterol acyltransferase 1 (ACAT1) are key events in 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells as demonstrated by inhibition of cell death by ACAT1 inhibitor. LD number was not changed by treatment with either α-Toc or α-Toc3. The different physical properties of α-Toc and α-Toc3 may account for their different inhibitory effects on 24S-OHC-induced cell death. Copyright © 2016 Elsevier Ltd. All rights reserved.

Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells

PubMed Central

Passante, E; Würstle, M L; Hellwig, C T; Leverkus, M; Rehm, M

2013-01-01

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein–protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future. PMID:23933815

Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

PubMed Central

Kaiser, Christina L.; Chapman, Brittany J.; Guidi, Jessica L.; Terry, Caitlin E.; Mangiardi, Dominic A.; Cotanche, Douglas A.

2008-01-01

Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue. To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point. PMID:18487027

Methuosis

PubMed Central

Maltese, William A.; Overmeyer, Jean H.

2015-01-01

Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death. PMID:24726643

Bubbling cell death: A hot air balloon released from the nucleus in the cold.

PubMed

Chang, Nan-Shan

2016-06-01

Cell death emanating from the nucleus is largely unknown. In our recent study, we determined that when temperature is lowered in the surrounding environment, apoptosis stops and bubbling cell death (BCD) occurs. The study concerns the severity of frostbite. When exposed to severe cold and strong ultraviolet (UV) irradiation, people may suffer serious damages to the skin and internal organs. This ultimately leads to limb amputations, organ failure, and death. BCD is defined as "formation of a single bubble from the nucleus per cell and release of this swelling bubble from the cell surface to extracellular space that causes cell death." When cells are subjected to UV irradiation and/or brief cold shock (4℃ for 5 min) and then incubated at room temperature or 4℃ for time-lapse microscopy, each cell releases an enlarging nuclear gas bubble containing nitric oxide. Certain cells may simultaneously eject hundreds or thousands of exosome-like particles. Unlike apoptosis, no phosphatidylserine flip-over, mitochondrial apoptosis, damage to Golgi complex, and chromosomal DNA fragmentation are shown in BCD. When the temperature is increased back at 37℃, bubble formation stops and apoptosis restarts. Mechanistically, proapoptotic WW domain-containing oxidoreductase and p53 block the protective TNF receptor adaptor factor 2 that allows nitric oxide synthase 2 to synthesize nitric oxide and bubble formation. In this mini-review, updated knowledge in cell death and the proposed molecular mechanism for BCD are provided. © 2016 by the Society for Experimental Biology and Medicine.

Bubbling cell death: A hot air balloon released from the nucleus in the cold

PubMed Central

2016-01-01

Cell death emanating from the nucleus is largely unknown. In our recent study, we determined that when temperature is lowered in the surrounding environment, apoptosis stops and bubbling cell death (BCD) occurs. The study concerns the severity of frostbite. When exposed to severe cold and strong ultraviolet (UV) irradiation, people may suffer serious damages to the skin and internal organs. This ultimately leads to limb amputations, organ failure, and death. BCD is defined as “formation of a single bubble from the nucleus per cell and release of this swelling bubble from the cell surface to extracellular space that causes cell death.” When cells are subjected to UV irradiation and/or brief cold shock (4℃ for 5 min) and then incubated at room temperature or 4℃ for time-lapse microscopy, each cell releases an enlarging nuclear gas bubble containing nitric oxide. Certain cells may simultaneously eject hundreds or thousands of exosome-like particles. Unlike apoptosis, no phosphatidylserine flip-over, mitochondrial apoptosis, damage to Golgi complex, and chromosomal DNA fragmentation are shown in BCD. When the temperature is increased back at 37℃, bubble formation stops and apoptosis restarts. Mechanistically, proapoptotic WW domain-containing oxidoreductase and p53 block the protective TNF receptor adaptor factor 2 that allows nitric oxide synthase 2 to synthesize nitric oxide and bubble formation. In this mini-review, updated knowledge in cell death and the proposed molecular mechanism for BCD are provided. PMID:27075929

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells.

PubMed

Choi, Doo Jin; Kim, Sun-Lim; Choi, Ji Won; Park, Yong Il

2014-07-25

Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H2O2)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated. Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting. Maysin pretreatment reduced the cytotoxic effect of H2O2 on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H2O2-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5-50 μg/ml) for 2h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H2O2 (200 μM)-insulted cells. These results suggest that CS maysin has neuroprotective effects against oxidative stress (H2O2)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

The study of hydrogen peroxide level under cisplatin action using genetically encoded sensor hyper

NASA Astrophysics Data System (ADS)

Belova, A. S.; Orlova, A. G.; Maslennikova, A. V.; Brilkina, A. A.; Balalaeva, I. V.; Antonova, N. O.; Mishina, N. M.; Shakhova, N. M.; Belousov, V. V.

2014-03-01

The aim of the work was to study the participation of hydrogen peroxide in reaction of cervical cancer cell line HeLa Kyoto on cisplatin action. Determination of hydrogen peroxide level was performed using genetically encoded fluorescent sensor HyPer2. The dependence of cell viability on cisplatin concentration was determined using MTT assay. Mechanisms of cell death as well as HyPer2 reaction was revealed by flow cytometry after 6-hours of incubation with cisplatin in different concentrations. Cisplatin used in low concentrations had no effect on hydrogen peroxide level in HeLa Kyoto cells. Increase of HyPer2 fluorescence was detected only after exposure with cisplatin in high concentration. The reaction was not the consequence of cell death.

Antiproliferative Activity and Induction of Apoptosis in Human Melanoma Cells by Houttuynia cordata Thunb Extract.

PubMed

Yanarojana, Mongkol; Nararatwanchai, Thamthiwat; Thairat, Sarut; Tancharoen, Salunya

2017-12-01

To analyze the apoptotic effect of Houttuynia cordata Thunb (HCT) extract on human melanoma A375 cells and its underlying mechanisms. The effects of HCT on cell death were determined using the MTT assay. Hoechst 33342 staining was conducted to confirm the detection of cell apoptosis. Caspase-3 and caspase-8 mRNA and cleaved protein levels were investigated by RT-PCR and western blotting, respectively. The release of high mobility group box 1 (HMGB1) and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by ELISA. Caspase-3 and caspase-8 specific inhibitors suppressed HCT-induced cell death. HCT increased caspase-3 and caspase-8 mRNA, protein levels, and caspase activities in a concentration- and time-dependent manner. HCT induced MAPK phosphorylation in a time-dependent fashion. Pretreatment of cells with a selective inhibitor of p38 MAPK reduced apoptosis and reversed the levels of HMGB1 release in response to HCT treatment. HCT induces A375 programmed cell death by activating the caspase-dependent pathway and by p38 phosphorylation associated with HMGB1 reduction. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

PubMed

Schelman, William R; Andres, Robert D; Sipe, Kimberly J; Kang, Evan; Weyhenmeyer, James A

2004-09-28

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.

Combined selenium and vitamin C deficiency causes cell death in guinea pig skeletal muscle.

PubMed

Hill, Kristina E; Motley, Amy K; May, James M; Burk, Raymond F

2009-03-01

Combined antioxidant deficiencies of selenium and vitamin E or vitamin E and vitamin C in guinea pigs result in clinical illness. We hypothesized that combined selenium and vitamin C deficiency would have clinical consequences because in vitro interactions of these antioxidant nutrients have been reported. Because guinea pigs are dependent on dietary vitamin C, weanling male guinea pigs were fed selenium-deficient or control diet for 15 weeks before imposing vitamin C deficiency. Four dietary groups were formed and studied 3 weeks later: controls, vitamin C deficient, selenium deficient, and doubly deficient. Deficiencies were confirmed by determinations of glutathione peroxidase activity and vitamin C concentration in liver and skeletal muscle. Plasma creatine phosphokinase activity and liver, kidney, heart, and quadriceps histopathology were determined. Doubly deficient animals had moderately severe skeletal muscle cell death as judged by histopathology and plasma creatine phosphokinase activity of 6630 +/- 4400 IU/L (control, 70 + or - 5; vitamin C deficient, 95 + or - 110; selenium deficient, 280 + or - 250). Liver, kidney, and heart histology was normal in all groups. Muscle alpha-tocopherol levels were not depressed in the doubly deficient group, but muscle F2 isoprostane concentrations were elevated in them and correlated with markers of cell death. We conclude that combining selenium and vitamin C deficiencies in the guinea pig causes cell death in skeletal muscle that is more severe than the injury caused by selenium deficiency. The elevation of muscle F2 isoprostanes is compatible with the cell death being caused by oxidative stress.

Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

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

Choi, Sunga; Lim, Mi-Hee; Kim, Ki Mo

2011-12-15

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specificmore » features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer. -- Highlights: Black-Right-Pointing-Pointer We studied the mechanism which cordycepin-induced cell death association with estrogen receptor (ER) in breast cancer cells, MDA-MB-231 and MCF-7. Black-Right-Pointing-Pointer The cordycepin-induced cell death in MDA-MB-231 cells was associated with the mitochondria-mediated apoptotic pathway. Black-Right-Pointing-Pointer Cordycepin treatment also resulted in autophagy in MCF-7 cells, associated with induction of autophagosome formation. Black-Right-Pointing-Pointer The different cordycepin-mediated cell death pathways are irrespective of the ER response. Black-Right-Pointing-Pointer Cordycepin proves a clinically useful, ER-independent chemotherapeutic agent for human breast cancer cells.« less

An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer

PubMed Central

Wang, Guangji; Liu, Huiying; Wu, Xiaolan; Wang, Qiong; Liu, Miao; Liao, Ke; Wu, Mengqiu; Cheng, Xuefang; Hao, Haiping

2012-01-01

NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1+ A549 cells and H596-NQO1 cells, but not in NQO1− H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC. PMID:22848731

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

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce.

PubMed

Helmersson, Andreas; von Arnold, Sara; Burg, Kornel; Bozhkov, Peter V

2004-10-01

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

Induction of cell death in a glioblastoma line by hyperthermic therapy based on gold nanorods

PubMed Central

Fernandez Cabada, Tamara; Sanchez Lopez de Pablo, Cristina; Martinez Serrano, Alberto; del Pozo Guerrero, Francisco; Serrano Olmedo, Jose Javier; Ramos Gomez, Milagros

2012-01-01

Background Metallic nanorods are promising agents for a wide range of biomedical applications. In this study, we developed an optical hyperthermia method capable of inducing in vitro death of glioblastoma cells. Methods The procedure used was based on irradiation of gold nanorods with a continuous wave laser. This kind of nanoparticle converts absorbed light into localized heat within a short period of time due to the surface plasmon resonance effect. The effectiveness of the method was determined by measuring changes in cell viability after laser irradiation of glioblastoma cells in the presence of gold nanorods. Results Laser irradiation in the presence of gold nanorods induced a significant decrease in cell viability, while no decrease in cell viability was observed with laser irradiation or incubation with gold nanorods alone. The mechanism of cell death mediated by gold nanorods during photothermal ablation was analyzed, indicating that treatment compromised the integrity of the cell membrane instead of initiating the process of programmed cell death. Conclusion The use of gold nanorods in hyperthermal therapies is very effective in eliminating glioblastoma cells, and therefore represents an important area of research for therapeutic development. PMID:22619509

Mitomycin C-induced apoptosis in cultured human Tenon's capsule fibroblasts.

PubMed

Kim, J W; Kim, S K; Song, I H; Kim, I T

1999-06-01

To investigate the mitomycin C-induced apoptotic cell death of fibroblasts, the primarily cultured human Tenon's capsule fibroblasts were exposed to a clinically used dosage of 0.4 mg/ml of mitomycin C for 5 minutes. TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay and electron microscopic studies were performed to determine the extent of mitomycin C-induced apoptosis. A flow cytometric study was performed to quantify the apoptotic cell population over time. The TUNEL stains were positive and electron microscopy showed features of apoptotic cell death in some fibroblasts 3 and 5 days after treatment. Flow cytometric analysis using Annexin V-propidium iodide double staining detected apoptotic cells 3 days after treatment. These apoptotic cell populations increased at 4 days and were sustained for one week. This study revealed that the clinical effects of mitomycin C on fibroblasts may be mediated not only by antiproliferative but also apoptotic cell death to some degree. Therefore, the apoptotic cell death of fibroblasts induced by mitomycin C should be considered to properly understand the mechanism of wound healing after trabeculectomy with adjunctive mitomycin C.

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.

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

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

PubMed

Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei-Wei; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-09-01

Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidopsis.

PubMed

Gao, Zhen; Daneva, Anna; Salanenka, Yuliya; Van Durme, Matthias; Huysmans, Marlies; Lin, Zongcheng; De Winter, Freya; Vanneste, Steffen; Karimi, Mansour; Van de Velde, Jan; Vandepoele, Klaas; Van de Walle, Davy; Dewettinck, Koen; Lambrecht, Bart N; Nowack, Moritz K

2018-05-28

Flowers have a species-specific functional life span that determines the time window in which pollination, fertilization and seed set can occur. The stigma tissue plays a key role in flower receptivity by intercepting pollen and initiating pollen tube growth toward the ovary. In this article, we show that a developmentally controlled cell death programme terminates the functional life span of stigma cells in Arabidopsis. We identified the leaf senescence regulator ORESARA1 (also known as ANAC092) and the previously uncharacterized KIRA1 (also known as ANAC074) as partially redundant transcription factors that modulate stigma longevity by controlling the expression of programmed cell death-associated genes. KIRA1 expression is sufficient to induce cell death and terminate floral receptivity, whereas lack of both KIRA1 and ORESARA1 substantially increases stigma life span. Surprisingly, the extension of stigma longevity is accompanied by only a moderate extension of flower receptivity, suggesting that additional processes participate in the control of the flower's receptive life span.

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

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

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

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.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

PubMed

Soares, Júlia Ribeiro; José Tenório de Melo, Edésio; da Cunha, Maura; Fernandes, Kátia Valevski Sales; Taveira, Gabriel Bonan; da Silva Pereira, Lidia; Pimenta, Samy; Trindade, Fernanda Gomes; Regente, Mariana; Pinedo, Marcela; de la Canal, Laura; Gomes, Valdirene Moreira; de Oliveira Carvalho, André

2017-01-01

Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef 1 -Saccharomyces cerevisiae interaction. ApDef 1 -S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ApDef 1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef 1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef 1 induced death. ApDef 1 -S. cerevisiae interaction resulted in membrane permeabilization, H 2 O 2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ApDef 1 -S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef 1 -S. cerevisiae interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes.

PubMed

Aswad, Fred; Dennert, Gunther

2006-09-01

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.

Cell death pathways of particulate matter toxicity.

PubMed

Peixoto, Milena Simões; de Oliveira Galvão, Marcos Felipe; Batistuzzo de Medeiros, Silvia Regina

2017-12-01

Humans are exposed to various complex mixtures of particulate matter (PM) from different sources. Long-term exposure to high levels of these particulates has been linked to a diverse range of respiratory and cardiovascular diseases that have resulted in hospital admission. The evaluation of the effects of PM exposure on the mechanisms related to cell death has been a challenge for many researchers. Therefore, in this review, we have discussed the effects of airborne PM exposure on mechanisms related to cell death. For this purpose, we have compiled literature data on PM sources, the effects of exposure, and the assays and models used for evaluation, in order to establish comparisons between various studies. The analysis of this collected data suggested divergent responses to PM exposure that resulted in different cell death types (apoptosis, autophagy, and necrosis). In addition, PM induced oxidative stress within cells, which appeared to be an important factor in the determination of cell fate. When the levels of reactive oxygen species were overpowering, the cellular fate was directed toward cell death. This may be the underlying mechanism of the development or exacerbation of respiratory diseases, such as emphysema and chronic obstructive pulmonary diseases. In addition, PM was shown to cause DNA damage and the resulting mutations increased the risk of cancer. Furthermore, several conditions should be considered in the assessment of cell death in PM-exposed models, including the cell culture line, PM composition, and the interaction of the different cells types in in vivo models. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of animal anti-apoptotic gene Ced-9 enhances tolerance during Glycine max L.-Bradyrhizobium japonicum interaction under saline stress but reduces nodule formation.

PubMed

Robert, Germán; Muñoz, Nacira; Melchiorre, Mariana; Sánchez, Federico; Lascano, Ramiro

2014-01-01

The mechanisms by which the expression of animal cell death suppressors in economically important plants conferred enhanced stress tolerance are not fully understood. In the present work, the effect of expression of animal antiapoptotic gene Ced-9 in soybean hairy roots was evaluated under root hairs and hairy roots death-inducing stress conditions given by i) Bradyrhizobium japonicum inoculation in presence of 50 mM NaCl, and ii) severe salt stress (150 mM NaCl), for 30 min and 3 h, respectively. We have determined that root hairs death induced by inoculation in presence of 50 mM NaCl showed characteristics of ordered process, with increased ROS generation, MDA and ATP levels, whereas the cell death induced by 150 mM NaCl treatment showed non-ordered or necrotic-like characteristics. The expression of Ced-9 inhibited or at least delayed root hairs death under these treatments. Hairy roots expressing Ced-9 had better homeostasis maintenance, preventing potassium release; increasing the ATP levels and controlling the oxidative damage avoiding the increase of reactive oxygen species production. Even when our results demonstrate a positive effect of animal cell death suppressors in plant cell ionic and redox homeostasis under cell death-inducing conditions, its expression, contrary to expectations, drastically inhibited nodule formation even under control conditions.

Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species.

PubMed

Brinkman, Cassandra L; Schmidt-Malan, Suzannah M; Karau, Melissa J; Greenwood-Quaintance, Kerryl; Hassett, Daniel J; Mandrekar, Jayawant N; Patel, Robin

2016-01-01

Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.

A simple and powerful approach for isolation of Arabidopsis mutants with increased tolerance to H2O2-induced cell death.

PubMed

Gechev, Tsanko; Mehterov, Nikolay; Denev, Iliya; Hille, Jacques

2013-01-01

A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins. Copyright © 2013 Elsevier Inc. All rights reserved.

Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

PubMed

Cesari, Stella; Moore, John; Chen, Chunhong; Webb, Daryl; Periyannan, Sambasivam; Mago, Rohit; Bernoux, Maud; Lagudah, Evans S; Dodds, Peter N

2016-09-06

Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

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

Influence of injury severity on the rate and magnitude of the T lymphocyte and neuronal response to facial nerve axotomy.

PubMed

Ha, Grace K; Parikh, Shivani; Huang, Zhi; Petitto, John M

2008-08-13

The temporal relationship between severity of peripheral axonal injury and T lymphocyte trafficking to the neuronal cell bodies of origin in the brain has been unclear. We sought to test the hypothesis that greater neuronal death induced by disparate forms of peripheral nerve injury would result in differential patterns of T cell infiltration and duration at the cell bodies of origin in the brain and that these measures would correlate with the magnitude of neuronal death over time and cumulative neuronal loss. To test this hypothesis, we compared the time course of CD3(+) T cell infiltration and neuronal death (assessed by CD11b(+) perineuronal microglial phagocytic clusters) following axonal crush versus axonal resection injuries, two extreme variations of facial nerve axotomy that result in mild versus severe neuronal loss, respectively, in the facial motor nucleus. We also quantified the number of facial motor neurons present at 49 days post-injury to determine whether differences in the levels of neuronal death between nerve crush and resection correlated with differences in cumulative neuronal loss. Between 1 and 7 days post-injury when levels of neuronal death were minimal, we found that the rate of accumulation and magnitude of the T cell response was similar following nerve crush and resection. Differences in the T cell response were apparent by 14 days post-injury when the level of neuronal death following resection was substantially greater than that seen in crush injury. For nerve resection, the peak of neuronal death at 14 days post-resection was followed by a maximal T cell response one week later at 21 days. Differences in the level of neuronal death between the two injuries across the time course tested reflected differences in cumulative neuronal loss at 49 days post-injury. Altogether, these data suggest that the trafficking of T cells to the injured FMN is dependent upon the severity of peripheral nerve injury and associated neuronal death.

Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

PubMed

Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

2014-02-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. © 2014 FEBS.

Curcumin Induces Pancreatic Adenocarcinoma Cell Death via Reduction of the Inhibitors of Apoptosis

PubMed Central

Osterman, Carlos J. Díaz; Gonda, Amber; Stiff, TessaRae; Sigaran, Ulysses; Valenzuela, Malyn May Asuncion; Bennit, Heather R. Ferguson; Moyron, Ron B.; Khan, Salma; Wall, Nathan R.

2015-01-01

Objectives The inhibitor of apoptosis (IAP) proteins are critical modulators of chemotherapeutic resistance in various cancers. To address the alarming emergence of chemotherapeutic resistance in pancreatic cancer, we investigated the efficacy of the turmeric derivative curcumin in reducing IAP protein and mRNA expression resulting in pancreatic cancer cell death. Methods The pancreatic adenocarcinoma cell line PANC-1 was used to assess curcumin’s effects in pancreatic cancer. Curcumin uptake was measured by spectral analysis and fluorescence microscopy. AlamarBlue and Trypan blue exclusion assays were used to determine PANC-1 cell viability following curcumin treatment. Visualization of PANC-1 cell death was performed using Hoffman Modulation Contrast microscopy. Western blot and PCR analyses were used to evaluate curcumin’s effects on IAP protein and mRNA expression. Results Curcumin enters PANC-1 cells and is ubiquitously present within the cell following treatment. Furthermore, curcumin reduces cell viability and induces morphological changes characteristic of cell death. Additionally, curcumin decreases IAP protein and mRNA expression in PANC-1 cells. Conclusions These data demonstrate that PANC-1 cells are sensitive to curcumin treatment. Furthermore, curcumin as a potential therapeutic tool for overcoming chemotherapeutic resistance mediated by IAPs, supports a role for curcumin as part of the therapeutic approach for pancreatic cancer. PMID:26348467

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

PubMed

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

2008-09-22

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

Necroptosis: a potential, promising target and switch in acute pancreatitis.

PubMed

Wang, Gang; Qu, Feng-Zhi; Li, Le; Lv, Jia-Chen; Sun, Bei

2016-02-01

Pancreatic acinar cell death is the major pathophysiological change in early acute pancreatitis (AP), and the death modalities are important factors determining its progression and prognosis. During AP, acinar cells undergo two major modes of death, including necrosis and apoptosis. Acinar necrosis can lead to intensely local and systemic inflammatory responses, which both induce and aggravate the lesion. Necrosis has long been considered an unregulated, and passive cell death process. Since the effective interventions of necrosis are difficult to perform, its relevant studies have not received adequate attention. Necroptosis is a newly discovered cell death modality characterized by both necrosis and apoptosis, i.e., it is actively regulated by special genes, while has the typical morphological features of necrosis. Currently, necroptosis is gradually becoming an important topic in the fields of inflammatory diseases. The preliminary results from necroptosis in AP have confirmed the existence of acinar cell necroptosis, which may be a potential target for effectively regulating inflammatory injuries and improving its outcomes; however, the functional changes and mechanisms of necroptosis still require further investigation. This article reviewed the progress of necroptosis in AP to provide a reference for deeply understanding the pathogenic mechanisms of AP and identifying new therapeutic targets.

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.

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.

Microscopic analysis of cell death by metabolic stress-induced autophagy in prostate cancer

NASA Astrophysics Data System (ADS)

Changou, Chun; Cheng, R. Holland; Bold, Richard; Kung, Hsing-Jien; Chuang, Frank Y. S.

2013-02-01

Autophagy is an intracellular recycling mechanism that helps cells to survive against environmental stress and nutritional starvation. We have recently shown that prostate cancers undergo metabolic stress and caspase-independent cell death following exposure to arginine deiminase (ADI, an enzyme that degrades arginine in tissue). The aims of our current investigation into the application of ADI as a novel cancer therapy are to identify the components mediating tumor cell death, and to determine the role of autophagy (stimulated by ADI and/or rapamycin) on cell death. Using advanced fluorescence microscopy techniques including 3D deconvolution and superresolution structured-illumination microscopy (SIM), we show that prostate tumor cells that are killed after exposure to ADI for extended periods, exhibit a morphology that is distinct from caspase-dependent apoptosis; and that autophagosomes forming as a result of ADI stimulation contain DAPI-stained nuclear material. Fluorescence imaging (as well as cryo-electron microscopy) show a breakdown of both the inner and outer nuclear membranes at the interface between the cell nucleus and aggregated autophagolysosomes. Finally, the addition of N-acetyl cysteine (or NAC, a scavenger for reactive oxygen species) effectively abolishes the appearance of autophagolysosomes containing nuclear material. We hope to continue this research to understand the processes that govern the survival or death of these tumor cells, in order to develop methods to improve the efficacy of cancer pharmacotherapy.

Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies.

PubMed

Lasorsa, E; Smonksey, M; Kirk, J S; Rosario, S; Hernandez-Ilizaliturri, F J; Ellis, L

2015-12-10

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Kim, Hyun-Bok; Kim, Seong Yun; Cho, Kyung-Ok

2011-12-01

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50µM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

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.

Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells.

PubMed

Slaidina, Maija; Lehmann, Ruth

2017-01-23

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Sulforaphane Prevents Angiotensin II-Induced Testicular Cell Death via Activation of NRF2.

PubMed

Wang, Yonggang; Wu, Hao; Xin, Ying; Bai, Yang; Kong, Lili; Tan, Yi; Liu, Feng; Cai, Lu

2017-01-01

Although angiotensin II (Ang II) was reported to facilitate sperm motility and intratesticular sperm transport, recent findings shed light on the efficacy of Ang II in stimulating inflammatory events in testicular peritubular cells, effect of which may play a role in male infertility. It is still unknown whether Ang II can induce testicular apoptotic cell death, which may be a more direct action of Ang II in male infertility. Therefore, the present study aims to determine whether Ang II can induce testicular apoptotic cell death and whether this action can be prevented by sulforaphane (SFN) via activating nuclear factor (erythroid-derived 2)-like 2 (NRF2), the governor of antioxidant-redox signalling. Eight-week-old male C57BL/6J wild type (WT) and Nrf2 gene knockout mice were treated with Ang II, in the presence or absence of SFN. In WT mice, SFN activated testicular NRF2 expression and function, along with a marked attenuation in Ang II-induced testicular oxidative stress, inflammation, endoplasmic reticulum stress, and apoptotic cell death. Deletion of the Nrf2 gene led to a complete abolishment of these efficacies of SFN. The present study indicated that Ang II may result in testicular apoptotic cell death, which can be prevented by SFN via the activation of NRF2.

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

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

PubMed

Christopher, Karen L; Pedler, Michelle G; Shieh, Biehuoy; Ammar, David A; Petrash, J Mark; Mueller, Niklaus H

2014-02-01

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically. Copyright © 2013 Elsevier B.V. All rights reserved.

Inhibition of autophagy enhances DENSpm-induced apoptosis in human colon cancer cells in a p53 independent manner.

PubMed

Gurkan, Ajda Coker; Arisan, Elif Damla; Yerlikaya, Pinar Obakan; Ilhan, Halime; Unsal, Narcin Palavan

2018-06-01

One of the recently developed polyamine (PA) analogues, N 1 ,N 11 -diethylnorspermine (DENSpm), has been found to act as an apoptotic inducer in melanoma, breast, prostate and colon cancer cells. Also, its potential to induce autophagy has been established. Unfolded protein responses and starvation of amino acids are known to trigger autophagy. As yet, however, the molecular mechanism underlying PA deficiency-induced autophagy is not fully clarified. Here, we aimed to determine the apoptotic effect of DENSpm after autophagy inhibition by 3-methyladenine (3-MA) or siRNA-mediated Beclin-1 silencing in colon cancer cells. The apoptotic effects of DENSpm after 3-MA treatment or Beclin-1 silencing were determined by PI and AnnexinV/PI staining in conjunction with flow cytometry. Intracellular PA levels were measured by HPLC, whereas autophagy and the expression profiles of PA key players were determined in HCT116, SW480 and HT29 colon cancer cells by Western blotting. We found that DENSpm-induced autophagy was inhibited by 3-MA treatment and Beclin-1 silencing, and that apoptotic cell death was increased by PA depletion and spermidine/spermine N 1 -acetyltransferase (SSAT) upregulation. We also found that autophagy inhibition led to DENSpm-induced apoptosis through Atg5 down-regulation, p62 degradation and LC3 lipidation in both HCT116 and SW480 cells. p53 deficiency did not alter the response of the colon cancer cells to DENSpm-induced apoptotic cell death under autophagy suppression conditions. From our results we conclude that DENSpm-induced apoptotic cell death is increased when autophagy is inhibited by 3-MA or Beclin-1 siRNA through PA depletion and PA catabolic activation in colon cancer cells, regardless p53 mutation status.

TESTIN Induces Rapid Death and Suppresses Proliferation in Childhood B Acute Lymphoblastic Leukaemia Cells

PubMed Central

Weeks, Robert J.; Ludgate, Jackie L.; LeMée, Gwenn; Morison, Ian M.

2016-01-01

Background Childhood acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Despite high cure rates, side effects and late consequences of the intensive treatments are common. Unquestionably, the identification of new therapeutic targets will lead to safer, more effective treatments. We identified TES promoter methylation and transcriptional silencing as a very common molecular abnormality in childhood ALL, irrespective of molecular subtype. The aims of the present study were to demonstrate that TES promoter methylation is aberrant, to determine the effects of TES re-expression in ALL, and to determine if those effects are mediated via TP53 activity. Methods Normal fetal and adult tissue DNA was isolated and TES promoter methylation determined by Sequenom MassARRAY. Quantitative RT-PCR and immunoblot were used to confirm re-expression of TES in ALL cell lines after 5’-aza-2’-deoxycytidine (decitabine) exposure or transfection with TES expression plasmids. The effects of TES re-expression on ALL cells were investigated using standard cell proliferation, cell death and cell cycle assays. Results In this study, we confirm that the TES promoter is unmethylated in normal adult and fetal tissues. We report that decitabine treatment of ALL cell lines results in demethylation of the TES promoter and attendant expression of TES mRNA. Re-expression of TESTIN protein in ALL cells using expression plasmid transfection results in rapid cell death or cell cycle arrest independent of TP53 activity. Conclusions These results suggest that TES is aberrantly methylated in ALL and that re-expression of TESTIN has anti-leukaemia effects which point to novel therapeutic opportunities for childhood ALL. PMID:26985820

Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements.

PubMed

Trumpaitė-Vanagienė, Rita; Čebatariūnienė, Alina; Tunaitis, Virginijus; Pūrienė, Alina; Pivoriūnas, Augustas

2018-02-01

To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann's Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed. The extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway. The apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by alternative methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species

PubMed Central

Brinkman, Cassandra L.; Schmidt-Malan, Suzannah M.; Karau, Melissa J.; Greenwood-Quaintance, Kerryl; Hassett, Daniel J.; Mandrekar, Jayawant N.

2016-01-01

Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the ‘electricidal effect’, in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS. PMID:27992529

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.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

PubMed

Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

2016-04-01

Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells.

PubMed

Fernandes, Kimberly A; Harder, Jeffrey M; Kim, Jessica; Libby, Richard T

2013-07-01

The AP1 family transcription factor JUN is an important molecule in the neuronal response to injury. In retinal ganglion cells (RGCs), JUN is upregulated soon after axonal injury and disrupting JUN activity delays RGC death. JUN is known to participate in the control of many different injury response pathways in neurons, including pathways controlling cell death and axonal regeneration. The role of JUN in regulating genes involved in cell death, ER stress, and regeneration was tested to determine the overall importance of JUN in regulating RGC response to axonal injury. Genes from each of these pathways were transcriptionally controlled following axonal injury and Jun deficiency altered the expression of many of these genes. The differentially expressed genes included, Atf3, Ddit3, Ecel1, Gadd45α, Gal, Hrk, Pten, Socs3, and Sprr1a. Two of these genes, Hrk and Atf3, were tested for importance in RGC death using null alleles of each gene. Disruption of the prodeath Bcl2 family member Hrk did not affect the rate or amount of RGC death after axonal trauma. Deficiency in the ATF/CREB family transcription factor Atf3 did lessen the amount of RGC death after injury, though it did not provide long term protection to RGCs. Since JUN's dimerization partner determines its transcriptional targets, the expression of several candidate AP1 family members were examined. Multiple AP1 family members were induced by axonal injury and had a different expression profile in Jun deficient retinas compared to wildtype retinas (Fosl1, Fosl2 and Jund). Overall, JUN appears to play a multifaceted role in regulating RGC response to axonal injury. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fas Versatile Signaling and Beyond: Pivotal Role of Tyrosine Phosphorylation in Context-Dependent Signaling and Diseases

PubMed Central

Chakrabandhu, Krittalak; Hueber, Anne-Odile

2016-01-01

The Fas/FasL system is known, first and foremost, as a potent apoptosis activator. While its proapoptotic features have been studied extensively, evidence that the Fas/FasL system can elicit non-death signals has also accumulated. These non-death signals can promote survival, proliferation, migration, and invasion of cells. The key molecular mechanism that determines the shift from cell death to non-death signals had remained unclear until the recent identification of the tyrosine phosphorylation in the death domain of Fas as the reversible signaling switch. In this review, we present the connection between the recent findings regarding the control of Fas multi-signals and the context-dependent signaling choices. This information can help explain variable roles of Fas signaling pathway in different pathologies. PMID:27799932

Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

PubMed

Bruno, P; Calastretti, A; Priulla, M; Asnaghi, L; Scarlatti, F; Nicolin, A; Canti, G

2007-10-01

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

Length of intact plasma membrane determines the diffusion properties of cellular water.

PubMed

Eida, Sato; Van Cauteren, Marc; Hotokezaka, Yuka; Katayama, Ikuo; Sasaki, Miho; Obara, Makoto; Okuaki, Tomoyuki; Sumi, Misa; Nakamura, Takashi

2016-01-11

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = -0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death.

Length of intact plasma membrane determines the diffusion properties of cellular water

PubMed Central

Eida, Sato; Van Cauteren, Marc; Hotokezaka, Yuka; Katayama, Ikuo; Sasaki, Miho; Obara, Makoto; Okuaki, Tomoyuki; Sumi, Misa; Nakamura, Takashi

2016-01-01

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = −0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death. PMID:26750342

Circulating endothelial progenitor cells and cardiovascular outcomes.

PubMed

Werner, Nikos; Kosiol, Sonja; Schiegl, Tobias; Ahlers, Patrick; Walenta, Katrin; Link, Andreas; Böhm, Michael; Nickenig, Georg

2005-09-08

Endothelial progenitor cells derived from bone marrow are believed to support the integrity of the vascular endothelium. The number and function of endothelial progenitor cells correlate inversely with cardiovascular risk factors, but the prognostic value associated with circulating endothelial progenitor cells has not been defined. The number of endothelial progenitor cells positive for CD34 and kinase insert domain receptor (KDR) was determined with the use of flow cytometry in 519 patients with coronary artery disease as confirmed on angiography. After 12 months, we evaluated the association between baseline levels of endothelial progenitor cells and death from cardiovascular causes, the occurrence of a first major cardiovascular event (myocardial infarction, hospitalization, revascularization, or death from cardiovascular causes), revascularization, hospitalization, and death from all causes. A total of 43 participants died, 23 from cardiovascular causes. A first major cardiovascular event occurred in 214 patients. The cumulative event-free survival rate increased stepwise across three increasing baseline levels of endothelial progenitor cells in an analysis of death from cardiovascular causes, a first major cardiovascular event, revascularization, and hospitalization. After adjustment for age, sex, vascular risk factors, and other relevant variables, increased levels of endothelial progenitor cells were associated with a reduced risk of death from cardiovascular causes (hazard ratio, 0.31; 95 percent confidence interval, 0.16 to 0.63; P=0.001), a first major cardiovascular event (hazard ratio, 0.74; 95 percent confidence interval, 0.62 to 0.89; P=0.002), revascularization (hazard ratio, 0.77; 95 percent confidence interval, 0.62 to 0.95; P=0.02), and hospitalization (hazard ratio, 0.76; 95 percent confidence interval, 0.63 to 0.94; P=0.01). Endothelial progenitor-cell levels were not predictive of myocardial infarction or of death from all causes. The level of circulating CD34+KDR+ endothelial progenitor cells predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk. Copyright 2005 Massachusetts Medical Society.

Molecular Determinants and Clinical Implications of Breast Cancer Dormancy

DTIC Science & Technology

2014-12-01

repair mediates resistance of hair follicle bulge stem cells to DNA-damage- induced cell death. Nat Cell Biol 2010; 12: 572–582. 7. Chiruvella KK1...on the role of cellular dormancy in promoting cancer aggressiveness and drug resistance in recurred breast cancer. We aimed to determine the impact...period, we have successfully established a reliable in vitro breast cancer dormancy cell model. Using this model, we tested and confirmed the

The anti-fibrotic agent pirfenidone synergizes with cisplatin in killing tumor cells and cancer-associated fibroblasts.

PubMed

Mediavilla-Varela, Melanie; Boateng, Kingsley; Noyes, David; Antonia, Scott J

2016-03-02

Anti-fibrotic drugs such as pirfenidone have been developed for the treatment of idiopathic pulmonary fibrosis. Because activated fibroblasts in inflammatory conditions have similar characteristics as cancer-associated fibroblasts (CAFs) and CAFs contribute actively to the malignant phenotype, we believe that anti-fibrotic drugs have the potential to be repurposed as anti-cancer drugs. The effects of pirfenidone alone and in combination with cisplatin on human patient-derived CAF cell lines and non-small cell lung cancer (NSCLC) cell lines were examined. The impact on cell death in vitro as well as tumor growth in a mouse model was determined. Annexin V/PI staining and Western blot analysis were used to characterize cell death. Synergy was assessed with the combination index method using Calcusyn software. Pirfenidone alone induced apoptotic cell death in lung CAFs at a high concentration (1.5 mg/mL). However, co-culture in vitro experiments and co-implantation in vivo experiments showed that the combination of low doses of cisplatin (10 μM) and low doses of pirfenidone (0.5 mg/mL), in both CAFs and tumors, lead to increased cell death and decreased tumor progression, respectively. Furthermore, the combination of cisplatin and pirfenidone in NSCLC cells (A549 and H157 cells) leads to increased apoptosis and synergistic cell death. Our studies reveal for the first time that the combination of cisplatin and pirfenidone is active in preclinical models of NSCLC and therefore may be a new therapeutic approach in this disease.

Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-04-01

Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture? A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency. hESCs are vulnerable to massive cell death upon cellular detachment and dissociation. hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control. 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs. H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs. These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death. This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare. Nil. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax.

PubMed

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

2017-12-19

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca 2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP 3 R). The cell-permeable Bcl-2/IP 3 R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca 2+ -release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC 50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP 3 R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP 3 R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca 2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca 2+ -dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca 2+ -signaling events via its BH4 domain.

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.

Vitamin D protects keratinocytes from deleterious effects of ionizing radiation.

PubMed

Langberg, M; Rotem, C; Fenig, E; Koren, R; Ravid, A

2009-01-01

Radiotherapy can induce severe skin responses that may limit the clinically acceptable radiation dose. The responses include erythema, dry and moist desquamation, erosions and dermal-epidermal blister formation. These effects reflect injury to, and reproductive failure of, epidermal cells and may also be due to dysregulation of the tissue remodelling process caused by excessive proteolytic activity. Calcitriol, the hormonally active vitamin D metabolite, protects keratinocytes from programmed cell death induced by various noxious stimuli. To examine whether calcitriol protects proliferating keratinocytes from the damage inflicted by ionizing radiation under conditions similar to those employed during radiotherapy. Autonomously proliferating HaCaT keratinocytes, used as a model for basal layer keratinocytes, were irradiated using a linear accelerator. Cell death was monitored by vital staining, executioner caspase activation, lactic dehydrogenase release and colony formation assay. Induction of matrix metalloproteinase-9 was assessed by gelatinase activity assay and mRNA determination. Levels of specific proteins were determined by immunoblotting. Treatment with calcitriol inhibited both caspase-dependent and -independent programmed cell death occurring within 48 h of irradiation and increased the colony formation capacity of irradiated cells. These effects may be attributable to inhibition of the c-Jun NH(2)-terminal kinase cascade and to upregulation of the truncated antiapoptotic isoform of p63. Treatment with the hormone also attenuated radiation-induced increase in matrix metalloproteinase-9 protein and mRNA levels. The results of this study suggest that active vitamin D derivatives may attenuate cell death and excessive proteolytic activity in the epidermis due to exposure to ionizing radiation in the course of radiotherapy.

The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death.

PubMed

Yu, Xiaoli; Tang, Junli; Wang, Qunqing; Ye, Wenwu; Tao, Kai; Duan, Shuyi; Lu, Chenchen; Yang, Xinyu; Dong, Suomeng; Zheng, Xiaobo; Wang, Yuanchao

2012-10-01

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Oxidative Pentose Phosphate Pathway Inhibition Is A Key Determinant of Antimalarial Induced Cancer Cell Death

PubMed Central

Salas, Eduardo; Roy, Srirupa; Marsh, Timothy; Rubin, Brian; Debnath, Jayanta

2015-01-01

Despite immense interest in employing antimalarials as autophagy inhibitors to treat cancer, it remains unclear if these agents act predominantly via autophagy inhibition or whether other pathways direct their anti-cancer properties. By comparing the treatment effects of the antimalarials chloroquine (CQ) and quinacrine (Q) on KRAS mutant lung cancer cells, we demonstrate that inhibition of the oxidative arm of the pentose phosphate pathway (oxPPP) is required for antimalarial induced apoptosis. Despite inhibiting autophagy, neither CQ treatment nor RNAi against autophagy regulators (ATGs) promote cell death. In contrast, Q triggers high levels of apoptosis, both in vitro and in vivo, and this phenotype requires both autophagy inhibition and p53-dependent inhibition of the oxPPP. Simultaneous genetic targeting of the oxPPP and autophagy is sufficient to trigger apoptosis in lung cancer cells, including cells lacking p53. Thus, in addition to reduced autophagy, oxPPP inhibition serves as an important determinant of antimalarial cytotoxicity in cancer cells. PMID:26434592

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

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

Mizobuchi, Masahide; Ogata, Hiroaki; Hatamura, Ikuji

2007-10-12

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10{sup -4} M), or 10% cyclodextrin, for 6 h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number ofmore » terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.« less

Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses.

PubMed

Garg, Abhishek D; Agostinis, Patrizia

2017-11-01

The immunogenicity of cancer cells is an emerging determinant of anti-cancer immunotherapy. Beyond developing immunostimulatory regimens like dendritic cell-based vaccines, immune-checkpoint blockers, and adoptive T-cell transfer, investigators are beginning to focus on the immunobiology of dying cancer cells and its relevance for the success of anticancer immunotherapies. It is currently accepted that cancer cells may die in response to anti-cancer therapies through regulated cell death programs, which may either repress or increase their immunogenic potential. In particular, the induction of immunogenic cancer cell death (ICD), which is hallmarked by the emission of damage-associated molecular patterns (DAMPs); molecules analogous to pathogen-associated molecular patterns (PAMPs) acting as danger signals/alarmins, is of great relevance in cancer therapy. These ICD-associated danger signals favor immunomodulatory responses that lead to tumor-associated antigens (TAAs)-directed T-cell immunity, which paves way for the removal of residual, treatment-resistant cancer cells. It is also emerging that cancer cells succumbing to ICD can orchestrate "altered-self mimicry" i.e. mimicry of pathogen defense responses, on the levels of nucleic acids and/or chemokines (resulting in type I interferon/IFN responses or pathogen response-like neutrophil activity). In this review, we exhaustively describe the main molecular, immunological, preclinical, and clinical aspects of immunosuppressive cell death or ICD (with respect to apoptosis, necrosis and necroptosis). We also provide an extensive historical background of these fields, with special attention to the self/non-self and danger models, which have shaped the field of cell death immunology. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Label-free monitoring of cell death induced by oxidative stress in living human cells using terahertz ATR spectroscopy

PubMed Central

Zou, Yi; Liu, Qiao; Yang, Xia; Huang, Hua-Chuan; Li, Jiang; Du, Liang-Hui; Li, Ze-Ren; Zhao, Jian-Heng; Zhu, Li-Guo

2017-01-01

We demonstrated that attenuated total reflectance terahertz time-domain spectroscopy (ATR THz-TDS) is able to monitor oxidative stress response of living human cells, which is proven in this work that it is an efficient non-invasive, label-free, real-time and in situ monitoring of cell death. Furthermore, the dielectric constant and dielectric loss of cultured living human breast epithelial cells, and along with their evolution under oxidative stress response induced by high concentration of H2O2, were quantitatively determined in the work. Our observation and results were finally confirmed using standard fluorescence-labeled flow cytometry measurements and visible fluorescence imaging. PMID:29359084

Cellular mechanisms of estradiol-mediated sexual differentiation of the brain.

PubMed

Wright, Christopher L; Schwarz, Jaclyn S; Dean, Shannon L; McCarthy, Margaret M

2010-09-01

Gonadal steroids organize the developing brain during a perinatal sensitive period and have enduring consequences for adult behavior. In male rodents testicular androgens are aromatized in neurons to estrogens and initiate multiple distinct cellular processes that ultimately determine the masculine phenotype. Within specific brain regions, overall cell number and dendritic morphology are the principal targets for hormonal organization. Recent advances have been made in elucidating the cellular mechanisms by which the neurological underpinnings of sexually dimorphic physiology and behavior are determined. These include estradiol-mediated prostaglandin synthesis, presynaptic release of glutamate, postsynaptic changes in glutamate receptors and changes in cell adhesion molecules. Sex differences in cell death are mediated by hormonal modulation of survival and death factors such as TNFalpha and Bcl-2/BAX. Copyright 2010 Elsevier Ltd. All rights reserved.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

PubMed

Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

2013-02-01

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

PubMed Central

Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

2013-01-01

Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to evaluate whether intra-articular injection of a mixture of bupivacaine and hyaluronan after arthroscopic surgery may protect against bupivacaine’s chondrotoxicity. PMID:22427617

Cell Death and Serum Markers of Collagen Metabolism during Cardiac Remodeling in Cavia porcellus Experimentally Infected with Trypanosoma cruzi

PubMed Central

Castro-Sesquen, Yagahira E.; Gilman, Robert H.; Paico, Henry; Yauri, Verónica; Angulo, Noelia; Ccopa, Fredy; Bern, Caryn

2013-01-01

We studied cell death by apoptosis and necrosis in cardiac remodeling produced by Trypanosoma cruzi infection. In addition, we evaluated collagen I, III, IV (CI, CIII and CIV) deposition in cardiac tissue, and their relationship with serum levels of procollagen type I carboxy-terminal propeptide (PICP) and procollagen type III amino-terminal propeptide (PIIINP). Eight infected and two uninfected guinea pigs were necropsied at seven time points up to one year post-infection. Cell death by necrosis and apoptosis was determined by histopathological observation and terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Deposition of cardiac collagen types was determined by immunohistochemistry and serum levels of PICP, PIIINP, and anti-T. cruzi IgG1 and IgG2 by ELISA. IgG2 (Th1 response) predominated throughout the course of infection; IgG1 (Th2 response) was detected during the chronic phase. Cardiac cell death by necrosis predominated over apoptosis during the acute phase; during the chronic phase, both apoptosis and necrosis were observed in cardiac cells. Apoptosis was also observed in lymphocytes, endothelial cells and epicardial adipose tissue, especially in the chronic phase. Cardiac levels of CI, CIII, CIV increased progressively, but the highest levels were seen in the chronic phase and were primarily due to increase in CIII and CIV. High serum levels of PICP and PIIINP were observed throughout the infection, and increased levels of both biomarkers were associated with cardiac fibrosis (p = 0.002 and p = 0.038, respectively). These results confirm the role of apoptosis in cell loss mainly during the chronic phase and the utility of PICP and PIIINP as biomarkers of fibrosis in cardiac remodeling during T. cruzi infection. PMID:23409197

Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans▿

PubMed Central

Shin, Kwang-Soo; Kwon, Nak-Jung; Kim, Young Hwan; Park, Hee-Soo; Kwon, Gi-Seok; Yu, Jae-Hyuk

2009-01-01

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (ΔflbA) mutant autolyzed completely after a 3-day incubation, the ΔflbA ΔchiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ΔflbA-induced autolysis. However, importantly, both ΔflbA and ΔflbA ΔchiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the ΔflbA and ΔflbA ΔchiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, β-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed. PMID:19286987

Minocycline causes widespread cell death and increases microglial labeling in the neonatal mouse brain.

PubMed

Strahan, J Alex; Walker, William H; Montgomery, Taylor R; Forger, Nancy G

2017-06-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a > tenfold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex, septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3 to P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose 1 week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial "inhibitor" increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 753-766, 2017. © 2016 Wiley Periodicals, Inc.

Minocycline Causes Widespread Cell Death and Increases Microglial Labeling in the Neonatal Mouse Brain

PubMed Central

Strahan, J. Alex; Walker, William H.; Montgomery, Taylor R.; Forger, Nancy G.

2016-01-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms, and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a >10-fold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex (S1), septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3-P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose one week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial “inhibitor” increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. PMID:27706925

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

Sex and Violence in Neuroscience.

ERIC Educational Resources Information Center

Barnes, Deborah M.

1988-01-01

Describes advances made in the understanding of how sex hormones may modify various cognitive skills, how normal brain signaling mechanisms may cause nerve cell death, and how many cells appear to hold genetic agents which determine their own destruction. (RT)

IKKβ and NFκB transcription govern lymphoma cell survival through AKT-induced plasma membrane trafficking of GLUT1

PubMed Central

Sommermann, Thomas; O’Neill, Kathleen; Plas, David R.; Cahir-McFarland, Ellen

2011-01-01

All cancer cells require increased nutrient uptake to support proliferation. Here we investigated the signals that govern glucose uptake in B-cell lymphomas and determined that the protein kinase IKKβ induced GLUT1 membrane trafficking in both viral and spontaneous B-cell lymphomas. IKKβ induced AKT activity, while IKKβ-driven NFκB transcription was required for GLUT1 surface localization downstream of AKT. Activated NFκB promoted AKT-mediated phosphorylation of the GLUT1 regulator, AKT Substrate 160kD (AS160), but was not required for AKT phosphorylation of the mammalian target of rapamycin (mTOR) regulator Tuberous Sclerosis 2 (TSC2). In Epstein Barr virus (EBV) transformed B-cells, NFκB inhibition repressed glucose uptake and induced caspase-independent cell death associated with autophagy. After NFκB inhibition, an alternate carbon source ameliorated both autophagy and cell death, whereas autophagy inhibitors specifically accelerated cell death. Taken together, the results suggest that NFκB signaling establishes a metabolic program supporting proliferation and apoptosis resistance by driving glucose import. PMID:21987722

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway.

PubMed

Lee, Jisun; Lee, Seul; Kim, Sun-Lim; Choi, Ji Won; Seo, Jeong Yeon; Choi, Doo Jin; Park, Yong Il

2014-12-05

Despite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). Maysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. Maysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200 μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. These results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

TNF Signaling through RIP1 Kinase Enhances SN38-Induced Death in Colon Adenocarcinoma.

PubMed

Cabal-Hierro, Lucia; O'Dwyer, Peter J

2017-04-01

Elucidation of TNF-directed mechanisms for cell death induction and maintenance of tumor growth has revealed a role for receptor-interacting protein kinases 1 and 3 (RIPK1/RIP1 and RIPK3/RIP3), components of the necrosome complex, as determinants of cell fate. Here, the participation of TNF signaling was analyzed with regard to the cytotoxic action of different DNA-damaging agents in a panel of colon cancer cells. While most of these cell lines were insensitive to TNF, combination with these drugs increased sensitivity by inducing cell death and DNA damage, especially in the case of the topoisomerase inhibitor SN38. Changes in levels of RIP1 and RIP3 occurred following monotherapy with SN38 or in combination with TNF. Downregulation of RIP1 resulted in increased resistance to SN38, implying a requirement for RIP1 in mediating cytotoxicity through the TNF/TNFR signaling pathway. Downregulation of RIP1 in a xenograft model impaired tumor growth inhibition from SN38 treatment, suggesting the potential of RIP1 to determine the clinical outcome of irinotecan treatment. These results indicate that TNF plays a key role in determining the cytotoxic effectiveness of SN38 in colorectal cancer and suggests a re-evaluation of TNF-based interventions to enhance therapeutic efficacy. Implications: The capacity of RIP1 to influence drug sensitivity suggests RIP1 may have biomarker potential. Mol Cancer Res; 15(4); 395-404. ©2017 AACR . ©2017 American Association for Cancer Research.

A Novel Method for Assessing Sex-Specific and Genotype-Specific Response to Injury in Astrocyte Culture

PubMed Central

Liu, Mingyue; Oyarzabal, Esteban; Yang, Rui; Murphy, Stephanie J; Hurn, Patricia D.

2008-01-01

Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1−3 day-old male and female, WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences. PMID:18436308

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.

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

PubMed

Riaz Ahmed, Kausar Begam; Kanduluru, Ananda Kumar; Feng, Li; Fuchs, Philip L; Huang, Peng

2017-05-01

Metastatic melanoma is the most aggressive of all skin cancers and is associated with poor prognosis owing to lack of effective treatments. 25-epi Ritterostatin GN1N is a novel antitumor agent with yet undefined mechanisms of action. We sought to delineate the antitumor mechanisms of 25-epi Ritterostatin GN1N in melanoma cells to determine the potential of this compound as a treatment for melanoma. Activation of the endoplasmic reticulum (ER) stress protein glucose-regulated protein 78 (GRP78) has been associated with increased melanoma progression, oncogenic signaling, drug resistance, and suppression of cell death. We found that 25-epi Ritterostatin GN1N induced cell death in melanoma cells at nanomolar concentrations, and this cell death was characterized by inhibition of GRP78 expression, increased expression of the ER stress marker CHOP, loss of mitochondrial membrane potential, and lipidation of the autophagy marker protein LC3B. Importantly, normal melanocytes exhibited limited sensitivity to 25-epi Ritterostatin GN1N. Subsequent in vivo results demonstrated that 25-epi Ritterostatin GN1N reduced melanoma growth in mouse tumor xenografts and did not affect body weight, suggesting minimal toxicity. In summary, our findings indicate that 25-epi Ritterostatin GN1N causes ER stress and massive autophagy, leading to collapse of mitochondrial membrane potential and cell death in melanoma cells, with minimal effects in normal melanocytes. Thus, 25-epi Ritterostatin GN1N is a promising anticancer agent that warrants further investigation.

Detection of leachables and cytotoxicity after exposure to methacrylate- and epoxy-based root canal sealers in vitro.

PubMed

Lodienė, Greta; Kopperud, Hilde M; Ørstavik, Dag; Bruzell, Ellen M

2013-10-01

Root canal sealing materials may have toxic potential in vitro depending on the cell line, cytotoxicity assay, material chemistry, and degree of polymer curing. The aims of the present study were to detect leaching components from epoxy- or methacrylate-based root canal sealers and to investigate the degree of cytotoxicity after exposure to extracts from these materials. Qualitative determination of substances released from the materials was performed by gas- and liquid chromatography/mass spectrometry. Submandibular salivary gland acinar cell death (apoptosis/necrosis) was determined using a fluorescence staining/microscopy technique. The major leachable monomer from the epoxy-based material was bisphenol-A diglycidyl ether (BADGE), whereas leachables from the methacrylate-based materials were mainly triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), hydroxyethyl methacrylate (HEMA), and polyethyleneglycol dimethacrylate (PEGDMA). Exposure to diluted extracts of cured methacrylate-based materials caused a postexposure time-dependent increase in cell death. This effect was not demonstrated as a result of exposure to undiluted extract of cured epoxy-based material. Extracts of all fresh materials induced apoptosis significantly, but at lower dilutions of the epoxy- than the methacrylate-based materials. The degree of leaching, determined from the relative chromatogram peak heights of eluates from the methacrylate-based sealer materials, corresponded with the degree of cell death induced by extracts of these materials. © 2013 Eur J Oral Sci.

Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

PubMed

Garcia-Garcia, Aracely; Zavala-Flores, Laura; Rodriguez-Rocha, Humberto; Franco, Rodrigo

2012-12-15

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

PubMed

Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

2009-07-01

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

Role of microglia in ethanol’s apoptotic action on hypothalamic neuronal cells in primary cultures

PubMed Central

Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol’s apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol’s neurotoxic action. Methods Ethanol’s neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia conditioned media. Ethanol’s apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol’s apoptotic action. Results We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol’s ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol’s neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented microglia-derived media’s ability to induce neuronal death. Conclusions These results suggest that ethanol’s apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol’s neurotoxic action. PMID:20662807

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

PubMed

Galluzzi, Lorenzo; Vitale, Ilio; Aaronson, Stuart A; Abrams, John M; Adam, Dieter; Agostinis, Patrizia; Alnemri, Emad S; Altucci, Lucia; Amelio, Ivano; Andrews, David W; Annicchiarico-Petruzzelli, Margherita; Antonov, Alexey V; Arama, Eli; Baehrecke, Eric H; Barlev, Nickolai A; Bazan, Nicolas G; Bernassola, Francesca; Bertrand, Mathieu J M; Bianchi, Katiuscia; Blagosklonny, Mikhail V; Blomgren, Klas; Borner, Christoph; Boya, Patricia; Brenner, Catherine; Campanella, Michelangelo; Candi, Eleonora; Carmona-Gutierrez, Didac; Cecconi, Francesco; Chan, Francis K-M; Chandel, Navdeep S; Cheng, Emily H; Chipuk, Jerry E; Cidlowski, John A; Ciechanover, Aaron; Cohen, Gerald M; Conrad, Marcus; Cubillos-Ruiz, Juan R; Czabotar, Peter E; D'Angiolella, Vincenzo; Dawson, Ted M; Dawson, Valina L; De Laurenzi, Vincenzo; De Maria, Ruggero; Debatin, Klaus-Michael; DeBerardinis, Ralph J; Deshmukh, Mohanish; Di Daniele, Nicola; Di Virgilio, Francesco; Dixit, Vishva M; Dixon, Scott J; Duckett, Colin S; Dynlacht, Brian D; El-Deiry, Wafik S; Elrod, John W; Fimia, Gian Maria; Fulda, Simone; García-Sáez, Ana J; Garg, Abhishek D; Garrido, Carmen; Gavathiotis, Evripidis; Golstein, Pierre; Gottlieb, Eyal; Green, Douglas R; Greene, Lloyd A; Gronemeyer, Hinrich; Gross, Atan; Hajnoczky, Gyorgy; Hardwick, J Marie; Harris, Isaac S; Hengartner, Michael O; Hetz, Claudio; Ichijo, Hidenori; Jäättelä, Marja; Joseph, Bertrand; Jost, Philipp J; Juin, Philippe P; Kaiser, William J; Karin, Michael; Kaufmann, Thomas; Kepp, Oliver; Kimchi, Adi; Kitsis, Richard N; Klionsky, Daniel J; Knight, Richard A; Kumar, Sharad; Lee, Sam W; Lemasters, John J; Levine, Beth; Linkermann, Andreas; Lipton, Stuart A; Lockshin, Richard A; López-Otín, Carlos; Lowe, Scott W; Luedde, Tom; Lugli, Enrico; MacFarlane, Marion; Madeo, Frank; Malewicz, Michal; Malorni, Walter; Manic, Gwenola; Marine, Jean-Christophe; Martin, Seamus J; Martinou, Jean-Claude; Medema, Jan Paul; Mehlen, Patrick; Meier, Pascal; Melino, Sonia; Miao, Edward A; Molkentin, Jeffery D; Moll, Ute M; Muñoz-Pinedo, Cristina; Nagata, Shigekazu; Nuñez, Gabriel; Oberst, Andrew; Oren, Moshe; Overholtzer, Michael; Pagano, Michele; Panaretakis, Theocharis; Pasparakis, Manolis; Penninger, Josef M; Pereira, David M; Pervaiz, Shazib; Peter, Marcus E; Piacentini, Mauro; Pinton, Paolo; Prehn, Jochen H M; Puthalakath, Hamsa; Rabinovich, Gabriel A; Rehm, Markus; Rizzuto, Rosario; Rodrigues, Cecilia M P; Rubinsztein, David C; Rudel, Thomas; Ryan, Kevin M; Sayan, Emre; Scorrano, Luca; Shao, Feng; Shi, Yufang; Silke, John; Simon, Hans-Uwe; Sistigu, Antonella; Stockwell, Brent R; Strasser, Andreas; Szabadkai, Gyorgy; Tait, Stephen W G; Tang, Daolin; Tavernarakis, Nektarios; Thorburn, Andrew; Tsujimoto, Yoshihide; Turk, Boris; Vanden Berghe, Tom; Vandenabeele, Peter; Vander Heiden, Matthew G; Villunger, Andreas; Virgin, Herbert W; Vousden, Karen H; Vucic, Domagoj; Wagner, Erwin F; Walczak, Henning; Wallach, David; Wang, Ying; Wells, James A; Wood, Will; Yuan, Junying; Zakeri, Zahra; Zhivotovsky, Boris; Zitvogel, Laurence; Melino, Gerry; Kroemer, Guido

2018-03-01

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Monitoring of thermal dose during ablation therapy using quantum dot-mediated fluorescence thermometry.

PubMed

Bensalah, Karim; Tuncel, Altug; Hanson, Willard; Stern, Joshua; Han, Bumsoo; Cadeddu, Jeffrey

2010-12-01

The objective of this study was to demonstrate the feasibility of quantum dot (QD)-mediated fluorescence thermometry to monitor thermal dose in an in-vitro thermal ablation zone generated by laser-heated gold nanoshells (LGNS). Hyperthermic cell death of human prostate cancer cell line (PC-3) was determined after various heating settings and correlated to the thermal conditions using an Arrhenius model prior to LGNS ablation. PC-3 cells with gold nanoshells (GNS) and QDs were exposed to a near-infrared laser and QD excitation light. When the cells were heated by GNS, local temperature was measured using the temperature-dependent fluorescence intensity of QDs. Using the predetermined Arrhenius model, the thermal dose (i.e., cell death of PC-3 cells) by LGNS was estimated with local temperatures measured with QD-mediated thermometry. The estimated thermal dose was confirmed with calcein-acetoxy-methylester viability assay. For PC-3 cell line, the activation energy and frequency factor of the Arrhenius model were 86.78 kcal/mol and 6.35 × 10(55) Hz, respectively. During LGNS ablation of PC-3 cells, QD-mediated temperature measurement showed that the temperature of the laser spot increased rapidly to ∼58 °C ± 4 °C. The estimated thermal dose showed that cell death reached to ∼90% in 120 seconds. The death cell zone observed after staining corresponded to a peak area of the temperature profile generated after analysis of the QD fluorescence intensity. This study shows that the QD fluorescence thermometry can accurately monitor the PC-3 cell death by LGNS ablation. This approach holds promises for a better monitoring of thermal ablation procedures in clinical practice.

Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives.

PubMed

Wang, Qiong; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Buzzacott, Peter; Belhomme, Marac; Theron, Michaël

2015-07-01

Excessive reactive oxygen species (ROS) is considered a consequence of hyperoxia and a major contributor to diving-derived vascular endothelial damage and decompression sickness. The aims of this work were: 1) to directly observe endothelial ROS production during simulated air dives as well as its relation with both mitochondrial activity and cell survival; and 2) to determine which ambient factor during air diving (hydrostatic pressure or oxygen and/or nitrogen partial pressure) is responsible for the observed modifications. In vitro diving simulation was performed with bovine arterial endothelial cells under real-time observation. The effects of air diving, hydrostatic, oxygen and nitrogen pressures, and N-acetylcysteine (NAC) treatment on mitochondrial ROS generation, mitochondrial membrane potential and cellular survival during simulation were investigated. Vascular endothelial cells performing air diving simulation suffered excessive mitochondrial ROS, mitochondrial depolarization, and cell death. These effects were prevented by NAC: after NAC treatment, the cells presented no difference in damage from nondiving cells. Oxygen diving showed a higher effect on ROS generation but lower impacts on mitochondrial depolarization and cell death than hydrostatic or nitrogen diving. Nitrogen diving had no effect on the inductions of ROS, mito-depolarization, or cell death. This study is the first direct observation of mitochondrial ROS production, mitochondrial membrane potential and cell survival during diving. Simulated air SCUBA diving induces excessive ROS production, which leads to mitochondrial depolarization and endothelial cell death. Oxygen partial pressure plays a crucial role in the production of ROS. Deleterious effects of hyperoxia-induced ROS are potentiated by hydrostatic pressure. These findings hold new implications for the pathogenesis of diving-derived endothelial dysfunction.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

PubMed Central

Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2016-01-01

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

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

Relation between parvovirus B19 infection and fetal mortality and spontaneous abortion.

PubMed

Shabani, Zahra; Esghaei, Maryam; Keyvani, Hossein; Shabani, Fateme; Sarmadi, Fateme; Mollaie, Hamidreza; Monavari, Seyed Hamidreza

2015-01-01

Infection with parvovirus B19 may cause fetal losses including spontaneous abortion, intrauterine fetal death and non-immune hydrops fetalis. The aim of this study is to determine the frequency of parvovirus B19 in formalin fixed placental tissues in lost fetuses using real-time PCR method. In this cross-sectional study, 100 formalin fixed placental tissues with unknown cause of fetal death were determined using real-time PCR method after DNA extraction. Six out of 100 cases (6%) were positive for parvovirus B19 using real-time PCR. Gestational age of all positive cases was less than 20 weeks with a mean of 12.3 weeks. Three cases have a history of abortion and all of positive cases were collected in spring. Mean age of positive cases were 28 years. Parvovirus B19 during pregnancy can infect red precursor cells and induces apoptosis or lyses these cells that resulting in anemia and congestive heart failure leading to fetal death. Management of parvovirus B19 infection in pregnant women is important because immediate diagnosis and transfusion in hydropsic fetuses can decrease the risk of fetal death.

Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous cell carcinoma.

PubMed

Aghbali, Amirala; Hosseini, Sepideh Vosough; Delazar, Abbas; Gharavi, Nader Kalbasi; Shahneh, Fatemeh Zare; Orangi, Mona; Bandehagh, Ali; Baradaran, Behzad

2013-08-01

Development of novel therapeutic modalities is crucial for the treatment of oral squamous cell carcinoma (OSCC). Recent scientific studies have been focused on herbal medicines as potent anti-cancer drug candidates. This study is the first to investigate the cytotoxic effects and the mechanism of cell death induced by grape seed extract (GSE) in oral squamous cell carcinoma (KB cells). MTT (3-(4,5-dimetylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) and trypan blue assays were performed in KB cells as well as human umbilical vein endothelial cells (HUVEC) were used to analyze the cytotoxic activity of GSE. Furthermore, the apoptosis-inducing action of the extract was determined by TUNEL, DNA fragmentation and cell death analysis. Statistical significance was determined by analysis of variance (ANOVA), followed by Duncan's test at a significance level of P≤0.05. The results showed apoptotic potential of GSE, confirmed by significant inhibition of cell growth and viability in a dose- and time- dependent manner without inducing damage to non-cancerous cell line HUVEC. The results of this study suggest that this plant contains potential bioactive compound(s) for the treatment of oral squamous cell carcinoma.

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.

Morphine protects SH-SY5Y human neuroblastoma cells against 6-hydroxydopamine-induced cell damage: involvement of anti-oxidant, calcium blocking, and anti-apoptotic properties.

PubMed

Elyasi, Leila; Eftekhar-Vaghefi, Seyed Hassan; Esmaeili-Mahani, Saeed

2014-06-01

Parkinson's disease is a neurodegenerative disorder characterized by progressive and selective death of dopaminergic neurons. Understanding the neuroprotective effects of chemical reagents has attracted increasing attention. The μ opioid agonist morphine exerts both toxic and protective effects. However, until recently, the neuroprotective role of morphine against 6-hydroxydopamine (6-OHDA)-induced cell death has not been studied. Here, we investigated the effects of morphine on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson's disease. Cell damage was induced by 150 μM 6-OHDA, and the cells' viability was examined by MTT assay. Intracellular calcium, reactive oxygen species (ROS), and mitochondrial membrane potential were determined by the fluorescence spectrophotometry method. Fragmented DNA and biochemical markers of apoptosis were also determined by gel electrophoresis and immunoblotting, respectively. The data showed that 6-OHDA caused a loss of cell viability and mitochondrial membrane potential. In addition, intracellular ROS and calcium levels, activated caspase-3, Bax:Bcl-2 ratio, cytochrome c release, as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Incubation of SH-SY5Y cells with morphine (100 μM) elicited a protective effect and reduced biochemical markers of cell damage and death. These results suggest that morphine has neuroprotective effects against 6-OHDA-induced neurotoxicity, and such effects are accompanied by its anti-oxidant, calcium blocking, and anti-apoptotic properties.

Coordinated induction of cell survival signaling in the inflamed microenvironment of the prostate.

PubMed

McIlwain, David W; Zoetemelk, Marloes; Myers, Jason D; Edwards, Marshé T; Snider, Brandy M; Jerde, Travis J

2016-06-01

Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation. We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3). Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens. The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation. © 2016 Wiley Periodicals, Inc.

Genistein abrogates G2 arrest induced by curcumin in p53 deficient T47D cells

PubMed Central

2012-01-01

Background The high cost and low level of cancer survival urge the finding of new drugs having better mechanisms. There is a high trend of patients to be “back to nature” and use natural products as an alternative way to cure cancer. The fact is that some of available anticancer drugs are originated from plants, such as taxane, vincristine, vinblastine, pacitaxel. Curcumin (diferuloylmethane), a dietary pigment present in Curcuma longa rizhome is reported to induce cell cycle arrest in some cell lines. Other study reported that genistein isolated from Glycine max seed inhibited phosphorylation of cdk1, gene involved during G2/M transition and thus could function as G2 checkpoint abrogator. The inhibition of cdk1 phosphorylation is one of alternative strategy which could selectively kill cancer cells and potentially be combined with DNA damaging agent such as curcumin. Methods T47D cell line was treated with different concentrations of curcumin and genistein, alone or in combination; added together or with interval time. Flow Cytometry and MTT assay were used to evaluate cell cycle distribution and viability, respectively. The presence of apoptotic cells was determined using acridine orange-ethidium bromide staining. Results In this study curcumin induced G2 arrest on p53 deficient T47D cells at the concentration of 10 μM. Increasing concentration up to 30 μM increased the number of cell death. Whilst genistein alone at low concentration (≤10 μM) induced cell proliferation, addition of genistein (20 μM) 16 h after curcumin resulted in more cell death (89%), 34% higher than that administered at the same time (56%). The combination treatment resulted in apoptotic cell death. Combining curcumin with high dose of genistein (50 μM) induced necrotic cells. Conclusions Genistein increased the death of curcumin treated T47D cells. Appropriate timing of administration and concentration of genistein determine the outcome of treatment and this method could potentially be developed as an alternative strategy for treatment of p53 defective cancer cells. PMID:23351311

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.

PubMed

Brannelly, Laura A; Roberts, Alexandra A; Skerratt, Lee F; Berger, Lee

2018-05-16

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Zinc deficiency mediates alcohol-induced apoptotic cell death in the liver of rats through activating ER and mitochondrial cell death pathways

PubMed Central

Sun, Qian; Zhong, Wei; Zhang, Wenliang; Li, Qiong; Sun, Xiuhua; Tan, Xiaobing; Sun, Xinguo; Dong, Daoyin

2015-01-01

Hepatic zinc deficiency has been well documented in alcoholic patients, but the mechanisms by which zinc deficiency mediates cell death have not been well defined. The objectives of this study were to determine whether alcohol perturbs subcellular zinc homeostasis and how organelle zinc depletion may link with cell death pathways. Wistar rats were pair-fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure significantly reduced zinc level in isolated hepatic endoplasmic reticulum (ER) and mitochondria. Among the detected zinc transporters, ER Zrt/Irt-like protein (ZIP)13 and mitochondrial ZIP8, which transport zinc from ER and mitochondria to cytosol, were significantly increased. Mitochondrial zinc transporter (ZnT) 4, which transports zinc from cytosol to mitochondria, was also increased. ER phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4, and C/EBP homologous protein were significantly upregulated, and mitochondrial cytochrome c release and Bax insertion were detected in association with caspase-3 activation and apoptotic cell death. To define the role of zinc deficiency in ER and mitochondrial stress, H4IIEC3 cells were treated with 3 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine for 6 h with or without supplementation with zinc or N-acetylcysteine (NAC). The results demonstrated that zinc deprivation induced caspase-3 activation and apoptosis in association with ER and mitochondria dysfunction, which were inhibited by zinc as low as 10 μM but not by 2 mM NAC. These results suggest that chronic ethanol exposure induced in ER and mitochondrial zinc deficiency might activate intrinsic cell death signaling pathway, which could not be effectively rescued by antioxidant treatment. PMID:25767260

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.

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

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

Lombardo, Tomás; Cavaliere, Victoria; Costantino, Susana N.

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{submore » 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect associated with superoxide levels as assessed by flow cytometry. ► Synergism between arsenite and MG132 in U937 leukemia cell line. ► Synergism turned into antagonism by low levels of hydrogen peroxide. ► Resistance to arsenic cytotoxicity linked to early superoxide anion increased levels.« less

Sensitization of vascular smooth muscle cell to TNF-{alpha}-mediated death in the presence of palmitate

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

Rho, Mun-Chual; Ah Lee, Kyeong; Mi Kim, Sun

2007-05-01

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-{alpha}-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokinemore » did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-{alpha}-mediated nuclear factor kappa B (NF-{kappa}B) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-{kappa}B subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-{kappa}B predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-{alpha}-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.« less

Role of intracellular freezing in the death of cells cooled at supraoptimal rates. [Preservation of erythrocytes, bone marrow cells, and yeasts by freezing

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

Mazur, P.

1976-01-01

Cooling velocity is one of the major factors that determines whether viable cells can be frozen to temperatures that permit indefinite storage. Cooling either too slowly or too rapidly tends to be damaging. Optimum cooling rates are reported for mouse marrow stem cells, yeast, and human red cells.

Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway.

PubMed

Li, Bao-Xia; Wang, Heng-Bang; Qiu, Miao-Zhen; Luo, Qiu-Yun; Yi, Han-Jie; Yan, Xiang-Lei; Pan, Wen-Tao; Yuan, Lu-Ping; Zhang, Yu-Xin; Xu, Jian-Hua; Zhang, Lin; Yang, Da-Jun

2018-03-12

Ovarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer. CCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation. APG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts. Our results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.

Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures.

PubMed

Heinrich, A; Balszuweit, F; Thiermann, H; Kehe, K

2009-12-15

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180-200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISA(plus) (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant. To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISA(plus) and the ToxiLight Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300 microM for 30 min, Lewisite (L) 60 microM for 5 min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent. This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.

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

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

Cell death and morphogenesis during early mouse development: Are they interconnected?

PubMed Central

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-01-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. PMID:25640415

Photochemical Targeting Of Phagocytic Trabecular Meshwork Cells Using Chlorin E6 Coupled Microspheres

NASA Astrophysics Data System (ADS)

Latina, M. A.; Kobsa, P. H.; Rakestraw, S. L.; Crean, E. A.; Hasan, T.; Yarmush, M. L.

1989-03-01

We have investigated a novel and efficient delivery system utilizing photosensitizer-coupled-latex microspheres to photochemically target and kill phagocytic trabecular meshwork (TM) cells. TM cells are the most actively phagocytic cells within the anterior chamber of the eye and are located within an optically accessible discrete band. This delivery system, along with the property of cell photocytosis, will achieve double selectivity by combining preferential localization of the photosensitizer to the target cells with spatial localization of illumination on the target cells. All experiments were performed with preconfluent bovine TM cells, 3rd to 4th passage, plated in 15 mm wells. Chlorin e6 monoethylene diamine monoamide was conjugated to the surface of 1.0 Am MX Duke Scientific fluorescent latex microspheres. Spectroscopic analysis revealed an average of 1.3 x 10 -17 moles of chlorin e6 per microsphere. TM cells were incubated for 18 hours with 5 x 10 7 microspheres/ml in MEM with 10% FCS, washed with MEM, and irradiated through fresh media using an argon-pumped dye laser emitting .2 W at 660 nm. A dose-survival study indicated that energy doses of 10 J/cm2 or greater resulted in greater than 95% cell death as determined by ethidium bromide exclusion. Cell death could be demonstrated as early as 4 hours post-irradiation. TM cells incubated with a solution of chlorin e6 at a concentration equal to that conjugated to the microspheres showed no cell death. Unirradiated controls also showed no cell death.

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

8-C-(E-phenylethenyl)quercetin from onion/beef soup induces autophagic cell death in colon cancer cells through ERK activation.

PubMed

Zhao, Yueliang; Fan, Daming; Zheng, Zong-Ping; Li, Edmund T S; Chen, Feng; Cheng, Ka-Wing; Wang, Mingfu

2017-02-01

Quercetin, a flavonoid, widely distributed in edible fruits and vegetables, was reported to effectively inhibit 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) formation in a food model (roast beef patties) with itself being converted into a novel compound 8-C-(E-phenylethenyl)quercetin (8-CEPQ). Here we investigated whether 8-CEPQ could be formed in a real food system, and tested its anticancer activity in human colon cancer cell lines. LC-MS was applied for the determination of 8-CEPQ formation in onion/beef soup. Anticancer activity of 8-CEPQ was evaluated by using cell viability assay and flow cytometry. Results showed that 8-CEPQ suppressed proliferation and caused G 2 phase arrest in colon cancer cells. Based on immunofluorescent staining assay, western blot assay, and RNA knockdown data, we found that 8-CEPQ did not cause apoptotic cell death. Instead, it induced autophagic cell death. Moreover, treatment with 8-CEPQ induced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK phosphorylation by the mitogen-activated protein kinase kinase (MEK)/ERK inhibitor U0126 attenuated 8-CEPQ-induced autophagy and reversed 8-CEPQ-mediated cell growth inhibition. Our results demonstrate that 8-CEPQ, a novel quercetin derivative, could be formed in onion/beef soup. 8-CEPQ inhibited colon cancer cell growth by inducing autophagic cell death through ERK activation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma membrane changes during programmed cell deaths

PubMed Central

Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai

2018-01-01

Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity. PMID:29076500

Preventing Exertional Death in Military Trainees: Recommendations and Treatment Algorithms From a Multidisciplinary Working Group.

PubMed

Webber, Bryant J; Casa, Douglas J; Beutler, Anthony I; Nye, Nathaniel S; Trueblood, Wesley E; O'Connor, Francis G

2016-04-01

Despite aggressive prevention programs and strategies, nontraumatic exertional sudden death events in military training continue to prove a difficult challenge for the Department of Defense. In November 2014, the 559th Medical Group at Joint Base San Antonio-Lackland, Texas, hosted a working group on sudden exertional death in military training. Their objectives were three-fold: (1) determine best practices to prevent sudden exertional death of military trainees, (2) determine best practices to establish safe and ethical training environments for military trainees with sickle cell trait, and (3) develop field-ready algorithms for managing military trainees who collapse during exertion. This article summarizes the major findings and recommendations of the working group. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

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

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.

PubMed

Brincks, Erik L; Kucaba, Tamara A; James, Britnie R; Murphy, Katherine A; Schwertfeger, Kathryn L; Sangwan, Veena; Banerjee, Sulagna; Saluja, Ashok K; Griffith, Thomas S

2015-12-01

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression. © 2015 FEBS.

Triptolide Enhances the Tumoricidal Activity of TRAIL Against Renal Cell Carcinoma

PubMed Central

James, Britnie R.; Murphy, Katherine A.; Schwertfeger, Kathryn L.; Sangwan, Veena; Banerjee, Sulagna; Saluja, Ashok K.; Griffith, Thomas S.

2015-01-01

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. TNF-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) has been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased HSP70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression. PMID:26426449

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

PubMed

Gordeev, Serguei A; Bykova, Tatiana V; Zubova, Svetlana G; Bystrova, Olga A; Martynova, Marina G; Pospelov, Valery A; Pospelova, Tatiana V

2015-12-29

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Pummelo Protects Doxorubicin-Induced Cardiac Cell Death by Reducing Oxidative Stress, Modifying Glutathione Transferase Expression, and Preventing Cellular Senescence

PubMed Central

Chularojmontri, L.; Gerdprasert, O.; Wattanapitayakul, S. K.

2013-01-01

Citrus flavonoids have been shown to reduce cardiovascular disease (CVD) risks prominently due to their antioxidant effects. Here we investigated the protective effect of pummelo (Citrus maxima, CM) fruit juice in rat cardiac H9c2 cells against doxorubicin (DOX-) induced cytotoxicity. Four antioxidant compositions (ascorbic acid, hesperidin, naringin, and gallic acid) were determined by HPLC. CM significantly increased cardiac cell survival from DOX toxicity as evaluated by MTT assay. Reduction of cellular oxidative stress was monitored by the formation of DCF fluorescent product and total glutathione (GSH) levels. The changes in glutathione-S-transferase (GST) activity and expression were determined by enzyme activity assay and Western blot analysis, respectively. Influence of CM on senescence-associated β-galactosidase activity (SA-β-gal) was also determined. The mechanisms of cytoprotection involved reduction of intracellular oxidative stress, maintaining GSH availability, and enhanced GST enzyme activity and expression. DOX-induced cellular senescence was also attenuated by long-term CM treatment. Thus, CM fruit juice can be promoted as functional fruit to protect cells from oxidative cell death, enhance the phase II GSTP enzyme activity, and decrease senescence phenotype population induced by cardiotoxic agent such as DOX. PMID:23401708

Autophagic activity in BC3H1 cells exposed to yessotoxin.

PubMed

Korsnes, Mónica Suárez; Kolstad, Hilde; Kleiveland, Charlotte Ramstad; Korsnes, Reinert; Ørmen, Elin

2016-04-01

The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Deletion Of XIAP reduces the severity of acute pancreatitis via regulation of cell death and nuclear factor-κB activity

PubMed Central

Liu, Yong; Chen, Xiao-Dong; Yu, Jiang; Chi, Jun-Lin; Long, Fei-Wu; Yang, Hong-Wei; Chen, Ke-Ling; Lv, Zhao-Ying; Zhou, Bin; Peng, Zhi-Hai; Sun, Xiao-Feng; Li, Yuan; Zhou, Zong-Guang

2017-01-01

Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that inhibition of XIAP represents a potential therapeutic strategy for the treatment of acute pancreatitis. PMID:28300832

JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.

PubMed

Maślikowski, Bart M; Wang, Lizhen; Wu, Ying; Fielding, Ben; Bédard, Pierre-André

2017-01-01

The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant negative mutant TAM67 induced senescence, adipogenesis, or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEFs) whereas inhibition of JunD by short hairpin RNA (shRNA) specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEFs expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe sets upregulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPβ-regulated mediator of apoptosis in gamma interferon (IFN-γ)-induced cell death. Here, we show that inhibition of DAPK1 abrogates cell death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPβ was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPβ abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by chromatin immunoprecipitation (ChIP) assays, JunD was not detected on the DAPK1 promoter under any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells. Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-κB and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src-transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumor suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumor suppressor function of DAPK1. Copyright © 2016 American Society for Microbiology.

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

PubMed Central

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B.; Bultynck, Geert

2017-01-01

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain. PMID:29340082

A crucial role for the phosphorylation of STRAP at Ser188 by MPK38 in STRAP-dependent cell death through ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways

PubMed Central

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2014-01-01

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-β receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser188 via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys152 and Cys270 of STRAP and Cys339 and Cys377 of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser188 phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser188 phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser188 phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-β, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death. PMID:25485581

A crucial role for the phosphorylation of STRAP at Ser(188) by MPK38 in STRAP-dependent cell death through ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways.

PubMed

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2014-01-01

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-β receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser(188) via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys(152) and Cys(270) of STRAP and Cys(339) and Cys(377) of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser(188) phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser(188) phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser(188) phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-β, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death.

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

Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells.

PubMed

Tsai, Chung-Lin; Chiu, Ying-Ming; Ho, Tin-Yun; Hsieh, Chin-Tung; Shieh, Dong-Chen; Lee, Yi-Ju; Tsay, Gregory J; Wu, Yi-Ying

2018-04-01

Gastric cancer is one of the most common malignant cancers with a poor prognosis and high mortality rate worldwide. Current treatment of gastric cancer includes surgery and chemotherapy as the main modalities, but the potentially severe side-effects of chemotherapy present a considerable challenge. Gallic acid is a trihydroxybenzoic acid found to exert an anticancer effect against a variety of cancer cells. The purpose of this study was to determine the anti-cancer activity of Galla chinensis and its main component gallic acid on human gastric adenocarcinoma cells. MTT assay and cell death ELISA were used to determine the apoptotic effect of Gallic Chinensis and gallic acid on human gastric adenocarcinoma cells. To determine the pathway and relevant components by which gallic acid-induced apoptosis is mediated through, cells were transfected with siRNA (Fas, FasL, DR5, p53) using Lipofectamine 2000. Reults: Gallic Chinensis and gallic acid induced apoptosis of human gastric adenocarcinoma cells. Gallic acid induced up-regulation of Fas, FasL, and DR5 expression in AGS cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced gallic acid-induced cell death. In addition, p53 was shown to be involved in gallic acid-mediated Fas, FasL, and DR5 expression as well as cell apoptosis in AGS cells. These results suggest that gallic acid has a potential role in the treatment of gastric cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Increasing role of the cancer chemotherapeutic doxorubicin in cellular metabolism.

PubMed

Meredith, Ann-Marie; Dass, Crispin R

2016-06-01

The use of doxorubicin, a drug utilised for many years to treat a wide variety of cancers, has long been limited due to the significant toxicity that can occur not only during, but also years after treatment. It has multiple mechanisms of action including the intercalation of DNA, inhibition of topoisomerase II and the production of free radicals. We review the literature, with the aim of highlighting the role of drug concentration being an important determinant on the unfolding cell biological events that lead to cell stasis or death. The PubMed database was consulted to compile this review. It has been found that the various mechanisms of action at the disposal of doxorubicin culminate in either cell death or cell growth arrest through various cell biological events, such as apoptosis, autophagy, senescence and necrosis. Which of these events is the eventual cause of cell death or growth arrest appears to vary depending on factors such as the patient, cell and cancer type, doxorubicin concentration and the duration of treatment. Further understanding of doxorubicin's influence on cell biological events could lead to an improvement in the drug's efficacy and reduce toxicity. © 2016 Royal Pharmaceutical Society.

Activation of apoptotic pathways in the absence of cell death in an inner-ear immortomouse cell line

PubMed Central

Chen, Fu-Quan; Hill, Kayla; Guan, Ya-Jun; Schacht, Jochen; Sha, Su-Hua

2012-01-01

Aminoglycoside antibiotics and cisplatin (CDDP) are the major ototoxins of clinical medicine due to their capacity to cause significant, as well as permanent hearing loss by targeting the mammalian sensory cells. Understanding the pathogenesis of damage is the first step in designing effective prevention of drug-induced hearing loss. In-vitro systems greatly enhance the efficiency of biochemical and molecular investigations through ease of access and manipulation. HEI-OC1, an inner ear cell line derived from the immortomouse, expresses markers for auditory sensory cells and, therefore, is a potential tool to study the ototoxic mechanisms of drugs like aminoglycoside antibiotics and CDDP. HEI-OC1 cells (and also HeLa cells) efficiently take up fluorescently tagged gentamicin and respond to drug treatment with changes in cell death and survival signaling pathways. Within hours, the C-jun N-terminal kinase pathway and the transcription factor AP-1 were activated and at later times, the “executioner caspase”, caspase-3. These responses were robust and elicited by both gentamicin and kanamycin. However, despite the initiation of apoptotic pathways and transient changes in nuclear morphology, cell death was not observed following aminoglycoside treatment, while administration of CDDP lead to significant cell death as determined by flow cytometric measurements; β-galactosidase analysis ruled out senescence in gentamicin-treated cells. The ability to withstand treatment with aminoglycosides but not with CDDP suggests that this cell line might be helpful in providing some insight into the differential actions of the two ototoxic drugs. PMID:22240458

Metallodrug induced apoptotic cell death and survival attempts are characterizable by Raman spectroscopy

NASA Astrophysics Data System (ADS)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2014-09-01

Chrysotherapeutics are under investigation as new or additional treatments for different types of cancers. In this study, gold complexes were investigated for their anticancer potential using Raman spectroscopy. The aim of the study was to determine whether Raman spectroscopy could be used for the characterization of metallodrug-induced cell death. Symptoms of cell death such as decreased peak intensities of proteins bonds and phosphodiester bonds found in deoxyribose nucleic acids were evident in the principal component analysis of the spectra. Vibrational bands around 761 cm-1 and 1300 cm-1 (tryptophan, ethanolamine group, and phosphatidylethanolamine) and 1720 cm-1 (ester bonds associated with phospholipids) appeared in the Raman spectra of cervical adenocarcinoma (HeLa) cells after metallodrug treatment. The significantly (p < 0.05, one way analysis of variance) increased intensity of phosphatidylethanolamine after metallodrug treatment could be a molecular signature of induced apoptosis since both the co-regulated phosphatidylserine and phosphatidylethanolamine are externalized during cell death. Treated cells had significantly higher levels of glucose and glycogen vibrational peaks, indicative of a survival mechanism of cancer cells under chemical stress. Cancer cells excrete chemotherapeutics to improve their chances of survival and utilize glucose to achieve this. Raman spectroscopy was able to monitor a survival strategy of cancer cells in the form of glucose uptake to alleviate chemical stress. Raman spectroscopy was invaluable in obtaining molecular information generated by biomolecules affected by anticancer metallodrug treatments and presents an alternative to less reproducible, conventional biochemical assays for cytotoxicity analyses.

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

2018-05-01

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

Thymoquinone chemosensitizes colon cancer cells through inhibition of NF-κB.

PubMed

Zhang, Lida; Bai, Yangqiu; Yang, Yuxiu

2016-10-01

In the present study, the effects and molecular mechanisms of thymoquinone (TQ) on colon cancer cells were investigated. Cell viability was determined using a Cell Counting Kit-8 assay, and the results revealed that treatment with TQ significantly decreased cell viability in COLO205 and HCT116 cells in a dose-dependent manner. TQ treatment additionally sensitized COLO205 and HCT116 cells to cisplatin therapy in a concentration-dependent manner. To investigate the molecular mechanisms of TQ action, western blot analysis was used to determine the levels of phosphorylated p65 and nuclear factor-κB (NF-κB)-regulated gene products vascular endothelial growth factor (VEGF), c-Myc and B-cell lymphoma 2 (Bcl-2). The results indicated that TQ treatment significantly decreased the level of phosphorylated p65 in the nucleus, which indicated the inhibition of NF-κB activation by TQ treatment. Treatment with TQ also decreased the expression levels of VEGF, c-Myc and Bcl-2. In addition, the inhibition of NF-κB activation with a specific inhibitor, pyrrolidine dithiocarbamate, potentiated the induction of cell death and caused a chemosensitization effect of TQ in colon cancer cells. Overall, the results of the present study suggested that TQ induced cell death and chemosensitized colon cancer cells by inhibiting NF-κB signaling.

Thymoquinone chemosensitizes colon cancer cells through inhibition of NF-κB

PubMed Central

Zhang, Lida; Bai, Yangqiu; Yang, Yuxiu

2016-01-01

In the present study, the effects and molecular mechanisms of thymoquinone (TQ) on colon cancer cells were investigated. Cell viability was determined using a Cell Counting Kit-8 assay, and the results revealed that treatment with TQ significantly decreased cell viability in COLO205 and HCT116 cells in a dose-dependent manner. TQ treatment additionally sensitized COLO205 and HCT116 cells to cisplatin therapy in a concentration-dependent manner. To investigate the molecular mechanisms of TQ action, western blot analysis was used to determine the levels of phosphorylated p65 and nuclear factor-κB (NF-κB)-regulated gene products vascular endothelial growth factor (VEGF), c-Myc and B-cell lymphoma 2 (Bcl-2). The results indicated that TQ treatment significantly decreased the level of phosphorylated p65 in the nucleus, which indicated the inhibition of NF-κB activation by TQ treatment. Treatment with TQ also decreased the expression levels of VEGF, c-Myc and Bcl-2. In addition, the inhibition of NF-κB activation with a specific inhibitor, pyrrolidine dithiocarbamate, potentiated the induction of cell death and caused a chemosensitization effect of TQ in colon cancer cells. Overall, the results of the present study suggested that TQ induced cell death and chemosensitized colon cancer cells by inhibiting NF-κB signaling. PMID:27698868

Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells.

PubMed

Hahm, Eun-Ryeong; Lee, Joomin; Singh, Shivendra V

2014-11-01

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen-activated protein kinases (MAPK), including c-jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK, and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) in regulation of WA-induced apoptosis using human breast cancer cells. Exposure of MCF-7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese-superoxide dismutase conferred partial protection against WA-mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF-7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA-induced apoptosis in MCF-7 cells was partially but significantly blocked in the presence of a JNK-specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA-induced apoptosis in SUM159 cells. The WA-treated cells exhibited induction of long and short forms of Mcl-1. RNA interference of Mcl-1 alone triggered apoptosis. Furthermore, the WA-induced cell death in MCF-7 cells was modestly but significantly augmented by knockdown of the Mcl-1 protein. These observations indicate that: MAPK have cell line-specific role in cell death by WA, and Mcl-1 induction confers modest protection against WA-induced apoptosis. © 2013 Wiley Periodicals, Inc.

The Interplay of IL-21 and BAFF in the Formation and Maintenance of Human B Cell Memory

PubMed Central

Karnell, Jodi L.; Ettinger, Rachel

2011-01-01

To date, IL-21 stands out as the most influential cytokine for human B cell activation and differentiation. Indeed, when compared to other important B cell tropic cytokines such as IL-2, IL-4, IL-6 and IL-10, IL-21 is clearly the most potent in terms of its ability to influence humoral immune responses in humans. IL-21 has wide reaching actions in determining how B cells will respond to co-stimulation ranging from induction of cell death upon BCR crosslinking to potent induction of class switch recombination and plasma cell differentiation when CD40 molecules are co-engaged. Another crucial B cell factor, exemplified in recent clinical trials, is BAFF/BLys. BAFF plays a critical role in the survival of human B cells and plasma blasts and influences B cell expansion and migration. Recent evidence has shown that IL-21 and BAFF can work in concert to promote and perhaps maintain humoral immunity in humans. Notably, BAFF has the unique ability to substitute for CD40L activities in regard to IL-21-co-stimulation and differentiation of a specific B cell subpopulation located in the human splenic marginal zone. However, and perhaps surprisingly, BAFF signals do not have the capability to override IL-21-driven cell death events when BCR is engaged. In stark contrast, anti-CD40 ligation of B cells co-activated with IL-21 and anti-IgM not only reverses this aforementioned activation-induced cell death, but transforms this death signal into one that drives plasma cell differentiation. Here we will discuss these two critical B cell factors, IL-21 and BAFF, and their distinct and complimentary effects on human B cell responses. PMID:22566888

Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

PubMed Central

Zheng, Weiwen; Rasmussen, Ulla; Zheng, Siping; Bao, Xiaodong; Chen, Bin; Gao, Yuan; Guan, Xiong; Larsson, John; Bergman, Birgitta

2013-01-01

Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom. PMID:23822984

Die another way – non-apoptotic mechanisms of cell death

PubMed Central

Tait, Stephen W. G.; Ichim, Gabriel; Green, Douglas R.

2014-01-01

ABSTRACT Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death. PMID:24833670

Cell Death and Cell Death Responses in Liver Disease: Mechanisms and Clinical Relevance

PubMed Central

Luedde, Tom; Kaplowitz, Neil; Schwabe, Robert F.

2015-01-01

Summary Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets. PMID:25046161

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

PubMed

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

2013-01-25

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

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo.

PubMed

Cary, Zachary D; Willingham, Mark C; Lyles, Douglas S

2011-06-01

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

PubMed Central

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice. PMID:26352139

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway.

PubMed

You, Dalsan; Kim, Yunlim; Jang, Myoung Jin; Lee, Chunwoo; Jeong, In Gab; Cho, Yong Mee; Hwang, Jung Jin; Hong, Jun Hyuk; Ahn, Hanjong; Kim, Choung-Soo

2015-01-01

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.

Conserved features of cancer cells define their sensitivity of HAMLET-induced death; c-Myc and glycolysis

PubMed Central

Storm, Petter; Puthia, Manoj Kumar; Aits, Sonja; Urbano, Alexander; Northen, Trent; Powers, Scott; Bowen, Ben; Chao, Yinxia; Reindl, Wolfgang; Lee, Do Yup; Sullivan, Nancy Liu; Zhang, Jianping; Trulsson, Maria; Yang, Henry; Watson, James; Svanborg, Catharina

2014-01-01

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small hairpin RNA inhibition, proteomic and metabolomic technology we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted the sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, the HAMLET sensitivity was modified by the glycolytic state of the tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen Hexokinase 1, PFKFB1 and HIF1α modified HAMLET sensitivity. Hexokinase 1 was shown to bind HAMLET in a protein array containing approximately 8000 targets and Hexokinase activity decreased within 15 minutes of HAMLET treatment, prior to morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. The glycolytic machinery was modified and glycolysis was shifted towards the pentose phosphate pathway. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 minutes. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene-addiction or the Warburg effect. PMID:21643007

Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis.

PubMed

Storm, P; Aits, S; Puthia, M K; Urbano, A; Northen, T; Powers, S; Bowen, B; Chao, Y; Reindl, W; Lee, D Y; Sullivan, N L; Zhang, J; Trulsson, M; Yang, H; Watson, J D; Svanborg, C

2011-12-01

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here, we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small-hairpin RNA (shRNA) inhibition, proteomic and metabolomic technology, we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, HAMLET sensitivity was modified by the glycolytic state of tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen, hexokinase 1 (HK1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 and hypoxia-inducible factor 1α modified HAMLET sensitivity. HK1 was shown to bind HAMLET in a protein array containing ∼8000 targets, and HK activity decreased within 15 min of HAMLET treatment, before morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 min. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene addiction or the Warburg effect.

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.

Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development

PubMed Central

Koto, Akiko; Kuranaga, Erina

2009-01-01

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases’ role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death. PMID:19822670

Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds.

PubMed

Fourquin, Chloé; Beauzamy, Léna; Chamot, Sophy; Creff, Audrey; Goodrich, Justin; Boudaoud, Arezki; Ingram, Gwyneth

2016-09-15

Seed development in angiosperms demands the tightly coordinated development of three genetically distinct structures. The embryo is surrounded by the endosperm, which is in turn enclosed within the maternally derived seed coat. In Arabidopsis, final seed size is determined by early expansion of the coenocytic endosperm, which then cellularises and subsequently undergoes developmental programmed cell death, breaking down as the embryo grows. Endosperm breakdown requires the endosperm-specific basic helix-loop-helix transcription factor ZHOUPI. However, to date, the mechanism underlying the Arabidopsis endosperm breakdown process has not been elucidated. Here, we provide evidence that ZHOUPI does not induce the developmental programmed cell death of the endosperm directly. Instead ZHOUPI indirectly triggers cell death by regulating the expression of cell wall-modifying enzymes, thus altering the physical properties of the endosperm to condition a mechanical environment permitting the compression of the cellularised endosperm by the developing embryo. © 2016. Published by The Company of Biologists Ltd.

Bromelain-induced apoptosis in GI-101A breast cancer cells.

PubMed

Dhandayuthapani, Sivanesan; Perez, Honey Diaz; Paroulek, Alexandra; Chinnakkannu, Panneerselvam; Kandalam, Umadevi; Jaffe, Mark; Rathinavelu, Appu

2012-04-01

Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.

The importance of being dead: cell death mechanisms assessment in anti-sarcoma therapy.

PubMed

Rello-Varona, Santiago; Herrero-Martín, David; Lagares-Tena, Laura; López-Alemany, Roser; Mulet-Margalef, Núria; Huertas-Martínez, Juan; Garcia-Monclús, Silvia; García Del Muro, Xavier; Muñoz-Pinedo, Cristina; Tirado, Oscar Martínez

2015-01-01

Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called "autophagic cell death" or "mitotic catastrophe") have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review, our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

Cell Death in C. elegans Development.

PubMed

Malin, Jennifer Zuckerman; Shaham, Shai

2015-01-01

Cell death is a common and important feature of animal development, and cell death defects underlie many human disease states. The nematode Caenorhabditis elegans has proven fertile ground for uncovering molecular and cellular processes controlling programmed cell death. A core pathway consisting of the conserved proteins EGL-1/BH3-only, CED-9/BCL2, CED-4/APAF1, and CED-3/caspase promotes most cell death in the nematode, and a conserved set of proteins ensures the engulfment and degradation of dying cells. Multiple regulatory pathways control cell death onset in C. elegans, and many reveal similarities with tumor formation pathways in mammals, supporting the idea that cell death plays key roles in malignant progression. Nonetheless, a number of observations suggest that our understanding of developmental cell death in C. elegans is incomplete. The interaction between dying and engulfing cells seems to be more complex than originally appreciated, and it appears that key aspects of cell death initiation are not fully understood. It has also become apparent that the conserved apoptotic pathway is dispensable for the demise of the C. elegans linker cell, leading to the discovery of a previously unexplored gene program promoting cell death. Here, we review studies that formed the foundation of cell death research in C. elegans and describe new observations that expand, and in some cases remodel, this edifice. We raise the possibility that, in some cells, more than one death program may be needed to ensure cell death fidelity. © 2015 Elsevier Inc. All rights reserved.

Glutamate-mediated excitotoxicity in neonatal hippocampal neurons is mediated by mGluR-induced release of Ca++ from intracellular stores and is prevented by estradiol

PubMed Central

Hilton, Genell D.; Nunez, Joseph L.; Bambrick, Linda; Thompson, Scott M.; McCarthy, Margaret M.

2008-01-01

Hypoxic/ischemic (HI) brain injury in newborn full-term and premature infants is a common and pervasive source of life time disabilities in cognitive and locomotor function. In the adult, HI induces glutamate release and excitotoxic cell death dependent on NMDA receptor activation. In animal models of the premature human infant, glutamate is also released following HI, but neurons are largely insensitive to NMDA or AMPA/kainic acid (KA) receptor-mediated damage. Using primary cultured hippocampal neurons we have determined that glutamate increases intracellular calcium much more than kainic acid. Moreover, glutamate induces cell death by activating Type I metabotropic glutamate receptors (mGluRs). Pretreatment of neurons with the gonadal steroid estradiol reduces the level of the Type I metabotropic glutamate receptors and completely prevents cell death, suggesting a novel therapeutic approach to excitotoxic brain damage in the neonate. PMID:17156362

Neuroprotective Compound from an Endophytic Fungus, Colletotrichum sp. JS-0367.

PubMed

Song, Ji Hoon; Lee, Changyeol; Lee, Dahae; Kim, Soonok; Bang, Sunghee; Shin, Myoung-Sook; Lee, Jun; Kang, Ki Sung; Shim, Sang Hee

2018-05-23

Colletotrichum sp. JS-0367 was isolated from Morus alba (mulberry), identified, and cultured on a large scale for chemical investigation. One new anthraquinone (1) and three known anthraquinones (2-4) were isolated and identified using spectroscopic methods including 1D/2D-NMR and HRESIMS. Although the neuroprotective effects of some anthraquinones have been reported, the biological activities of the four anthraquinones isolated in this study have not been reported. Therefore, the neuroprotective effects of these compounds were determined against murine hippocampal HT22 cell death induced by glutamate. Compound 4, evariquinone, showed strong protective effects against HT22 cell death induced by glutamate by the inhibition of intracellular ROS accumulation and Ca 2+ influx triggered by glutamate. Immunoblot analysis revealed that compound 4 reduced the phosphorylation of MAPKs (JNK, ERK1/2, and p38) induced by glutamate. Furthermore, compound 4 strongly attenuated glutamate-mediated apoptotic cell death.

Sepia Ink Oligopeptide Induces Apoptosis of Lung Cancer Cells via Mitochondrial Pathway.

PubMed

Wang, Xiaohua; Chen, Cheng; Zhou, Guoren; Ye, Jinjun; Yin, Rong; Feng, Dongjie; Zhang, Shuai; Wang, Xiaojun; Zhao, Xin; Zhang, Zhi

2018-01-01

Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism. Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting. We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO. Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO. © 2018 The Author(s). Published by S. Karger AG, Basel.

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.

Cell death and morphogenesis during early mouse development: are they interconnected?

PubMed

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-04-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

PubMed

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

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

Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin

Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by whichmore » ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.« less

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.

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.

Initial targets and cellular responses to PDT

NASA Astrophysics Data System (ADS)

Rodriguez, Myriam E.; Azizuddin, Kashif; Chiu, Song-mao; Delos Santos, Grace; Joseph, Sheeba; Xue, Liang-yan; Oleinick, Nancy L.

2007-02-01

Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals of Cleveland, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components are photodamaged. In most cancer cells, apoptosis is triggered by the initial photodamage; however, in cells deficient in one of the critical intermediates of apoptosis, this process does not occur, although the cells remain as sensitive to the lethal effects of Pc 4-PDT as the apoptosis-competent cells, when cell death is determined by colony formation. Here we report that an alternative death process, autophagy, is induced in all cells tested and becomes the dominant pathway for elimination of lethally damaged cells when apoptosis is compromised. The anti-apoptotic protein Bcl-2, when overexpressed, protects only apoptosis-competent cells against loss of clonogenicity, while the autophagy inhibitor 3-methyladenine provides a markedly greater protection to apoptosis-deficient cells. The results suggest that the primary determinant of cell death is not the final pathway for elimination of the cells but the initial photodamage to critical membrane targets. In attempts to identify those targets, we have studied the role of different membrane phospholipids in the localization of Pc 4. Cardiolipin (CL) is a phospholipid found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. Previous fluorescence resonance energy transfer studies revealed colocalization of Pc 4 and CL, which points to CL as a possible binding site and target for Pc 4. Unilamellar liposomes with different lipid compositions were used as membrane models to test the affinity of Pc 4. As revealed by the binding constants, Pc 4 does not display preferential binding to CL in these systems. Moreover, binding affinities appear to be independent of lipid composition. Localization of Pc 4 in mitochondrial membranes is likely determined by proteins or other factors not replicated in the liposomes. Studies in cells with modified CL content could report modified binding affinities.

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis,

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.

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.

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.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis.

PubMed

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-05-11

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis

PubMed Central

Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-01-01

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc–Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad–Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development. PMID:28492553

MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted-cell viability

NASA Astrophysics Data System (ADS)

Chan, Kannie W. Y.; Liu, Guanshu; Song, Xiaolei; Kim, Heechul; Yu, Tao; Arifin, Dian R.; Gilad, Assaf A.; Hanes, Justin; Walczak, Piotr; van Zijl, Peter C. M.; Bulte, Jeff W. M.; McMahon, Michael T.

2013-03-01

Biocompatible nanomaterials and hydrogels have become an important tool for improving cell-based therapies by promoting cell survival and protecting cell transplants from immune rejection. Although their potential benefit has been widely evaluated, at present it is not possible to determine, in vivo, if and how long cells remain viable following their administration without the use of a reporter gene. Here, we report a pH-nanosensor-based magnetic resonance imaging (MRI) technique that can monitor cell death in vivo non-invasively. We demonstrate that specific MRI parameters that change on cell death of microencapsulated hepatocytes are associated with the measured bioluminescence imaging radiance. Moreover, the readout from this pH-sensitive nanosensor can be directly co-registered with high-resolution anatomical images. All of the components of these nanosensors are clinical grade and hence this approach should be a translatable and universal modification of hydrogels.

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining.

PubMed

Byczkowska, Anna; Kunikowska, Anita; Kaźmierczak, Andrzej

2013-02-01

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green-yellow, yellow-orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.

What cell death does in development.

PubMed

Zakeri, Zahra; Penaloza, Carlos G; Smith, Kyle; Ye, Yixia; Lockshin, Richard A

2015-01-01

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

PubMed

Tezcan, Gulcin; Tunca, Berrin; Bekar, Ahmet; Yalcin, Murat; Sahin, Saliha; Budak, Ferah; Cecener, Gulsah; Egeli, Unal; Demir, Cevdet; Guvenc, Gokcen; Yilmaz, Gozde; Erkan, Leman Gizem; Malyer, Hulusi; Taskapilioglu, Mevlut Ozgur; Evrensel, Turkkan; Bilir, Ayhan

2015-03-01

Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

Impact of desiccation and heat exposure stress on Salmonella tolerance to acidic conditions.

PubMed

Richardson, Kurt E; Cox, Nelson A; Cosby, Douglas E; Berrang, Mark E

2018-02-01

In a recent study, the pH of commonly used Salmonella pre-enrichment media became acidic (pH 4.0 to 5.0) when feed or feed ingredients were incubated for 24 h. Acidic conditions have been reported to injure or kill Salmonella. In this study, cultures of four known feed isolates (S. montevideo, S. senftenberg, S. tennessee, and S. schwarzengrund) and four important processing plant isolates (S. typhimurium, S. enteritidis, S. infantis, and S. heidelberg) were grown on meat and bone meal and later subjected to desiccation and heat exposure to stress the microorganism. The impact of stress on the isolates ability to survive in acidic conditions ranging from pH 4.0 to 7.0 was compared to the non-stressed isolate. Cell injury was determined on xylose lysine tergitol 4 (XLT4) and cell death determined on nutrient agar (NA). When measured by cell death in non-stressed Salmonella, S. typhimurium was the most acid tolerant and S. heidelberg was the most acid sensitive whereas in stressed Salmonella, S. senftenberg was the most acid tolerant and S. tennessee was the most acid sensitive. The pH required to cause cell injury varied among isolates. With some isolates, the pH required for 50% cell death and 50% cell injury was similar. In other isolates, cell injury occurred at a more neutral pH. These findings suggest that the pH of pre-enrichment media may influence the recovery and bias the serotype of Salmonella recovered from feed during pre-enrichment.

Inhibition of caspases prevents ototoxic and ongoing hair cell death

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

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

PubMed

Wilkinson, Derek; Ramsdale, Mark

2011-10-01

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

Degenerative changes and cell death in long-living homo- and heterotopic transplants from embryonic germ layers of rat neocortex.

PubMed

Petrova, E S; Otellin, V A

2003-09-01

Morphological study of allotransplants of rat embryonic neocortex 14-18 months after transplantation into the neocortex, lateral cerebral ventricle, and sciatic nerve of adult animals revealed death of nerve and glial cells in the delayed postoperation period independently on the site of transplantation. After heterotopic transplantation the count of degenerated neurons was 2 times higher that after homotopic transplantation. In heterotopic transplants a considerable number of grafted neurons underwent reversible and irreversible degenerative changes accompanied by their premature aging. Neuronal death is probably determined by insufficiency of trophic influence from afferent structures and target tissues. We hypothesized that antiapoptotic preparations can be used for prevention of transplanted cell death. It was also found that degeneration of neurons was associated with impaired vascularization of transplants and pronounced immune reaction of the recipient in late posttransplantation period. Transplantation of embryonic brain structures can serve as a model system in studies concerning involutive and pathological processes in the central nervous system and in the search for factors improving survival of neurons.

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis, suggesting a link between POX and cancer cell survival.

Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism.

PubMed

Song, Ju-Xian; Choi, Mandy Yuen-Man; Wong, Kavin Chun-Kit; Chung, Winkie Wing-Yan; Sze, Stephen Cho-Wing; Ng, Tzi-Bun; Zhang, Kalin Yan-Bo

2012-01-21

Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

Apoptotic induction of skin cancer cell death by plant extracts.

PubMed

Thuncharoen, Walairat; Chulasiri, Malin; Nilwarangkoon, Sirinun; Nakamura, Yukio; Watanapokasin, Ramida

2013-01-01

The aim of the present study was to investigate the effects of plant extracts on cancer apoptotic induction. Human epidermoid carcinoma A431 cell line, obtained from the American Type Culture Collection (ATCC, Manassas, VA), was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 degrees C, 5% carbon dioxide (CO2). Plant extract solutions were obtained from S & J international enterprises public company limited. These plant extracts include 50% hydroglycol extracts from Etlingera elatior (Jack) R.M.Smith (torch ginger; EE), Rosa damascene (damask rose; DR) and Rafflesia kerrii Meijer (bua phut; RM). The cell viability, time and dose dependency were determined by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. A431 cells were treated with the plant extracts and stained with Hoechst 33342 fluorescent staining dye. Cell viability was demonstrated by the inhibitory concentration 50% (IC50). The anti-proliferative effects were shown to be dependent on time and dose. Typical characteristics of apoptosis which are cell morphological changes and chromatin condensation were clearly observed. The plant extracts was shown to be effective for anti-proliferation and induction of apoptosis cell death in skin cancer cells. Therefore, mechanisms underlying the cell death and its potential use for treatment of skin cancer will be further studied.

Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

USGS Publications Warehouse

Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

2012-01-01

Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

PubMed

Alexander, Bobby; Fishman, Andrew I; Eshghi, Majid; Choudhury, Muhammad; Konno, Sensuke

2013-09-01

Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

PubMed Central

Nicot, Christophe; Harrod, Robert

2000-01-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-dl-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-κB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia. PMID:11046153

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

PubMed Central

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-01-01

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed. PMID:27845895

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

PubMed

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-03-21

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Distinct p300-responsive mechanisms promote caspase-dependent apoptosis by human T-cell lymphotropic virus type 1 Tax protein.

PubMed

Nicot, C; Harrod, R

2000-11-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.

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.

VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

PubMed

Tenn, Catherine C; Wang, Yushan

2007-05-01

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

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.

Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

PubMed Central

Murali, V. K.; Ornelles, D. A.; Gooding, L. R.; Wilms, H. T.; Huang, W.; Tollefson, A. E.; Wold, W. S. M.

2014-01-01

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection. PMID:24198418

Cytotoxicity and radiosensitization effect of TRA-8 on radioresistant human larynx squamous carcinoma cells.

PubMed

Wu, F; Hu, Y; Long, J; Zhou, Y J; Zhong, Y H; Liao, Z K; Liu, S Q; Zhou, F X; Zhou, Y F; Xie, C H

2009-02-01

TRAIL induces apoptosis in a variety of tumorigenic and transformed cell lines, but not in many normal cells. Recent studies have demonstrated that death receptor 5 (DR5), one of the two death receptors bound by TRAIL, showed expression in most malignantly transformed cells. This study evaluated effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with ionizing radiation, on radioresistant human larynx squamous carcinoma cell line (Hep-2R). Cells were treated with TRA-8 alone or in combination with radiation, cell viability inhibition was measured by MTT assay, and the induction of apoptosis was determined by Annexin V staining. Radionsensitivity of Hep-2R cells treated with TRA-8 were investigated with long-term clonogenic assays. Regulation of DR5 expression in cells after radiation was analyzed by indirect immunofluorescence using murine TRA-8 in combination with flow cytometry. The results suggested that TRA-8 enhanced radionsensitivity of Hep-2R cells, and that TRA-8 regulated Hep-2R cell cycle arrest at G2/M phase. Irradiation up-regulated the expression of DR5, and when combined with TRA-8 yielded optimal survival benefit. Therefore, TRA-8 can be used in combination with irradiation in radioresistant human larynx squamous carcinoma cells. Monoclonal antibodies such as TRA-8 may play an important role in the development of an effective treatment strategy for patients with radioresistant cancers.

IGF-1 protects against dexamethasone-induced cell death in insulin secreting INS-1 cells independent of AKT/PKB phosphorylation.

PubMed

Avram, Diana; Ranta, Felicia; Hennige, Anita M; Berchtold, Susanne; Hopp, Sabine; Häring, Hans-Ulrich; Lang, Florian; Ullrich, Susanne

2008-01-01

Appropriate insulin secretion depends on beta-cell mass that is determined by the balance between cell proliferation and death. IGF-1 stimulates proliferation and protects against apoptosis. In contrast, glucocorticoids promote cell death. In this study we examined molecular interactions of the glucocorticoid dexamethasone (dexa) with IGF-1 signalling pathways in insulin secreting INS-1 cells. IGF-1 (50 ng/ml) increased the growth rate and stimulated BrdU incorporation, while dexa (100 nmol/l) inhibited cell growth, BrdU incorporation and induced apoptosis. Dexa-induced cell death was partially antagonized by IGF-1. This protection was further increased by LY294002 (10 micromol/l), an inhibitor of PI3 kinase. In contrast, MAP kinase inhibitor PD98059 (10 micromol/l) significantly reduced the protective effect of IGF-1. The analysis of signalling pathways by Western blotting revealed that dexa increased IRS-2 protein abundance while the expression of PI3K, PKB and ERK remained unchanged. Despite increased IRS-2 protein,IRS-2 tyrosine phosphorylation stimulated by IGF-1 was inhibited by dexa. Dexa treatment reduced basal PKB phosphorylation. However, IGF-1-mediated stimulation of PKB phosphorylation was not affected by dexa, but ERK phosphorylation was reduced. LY294002 restored IGF-1-induced ERK phosphorylation. These data suggest that dexa induces apoptosis in INS-1 cells by inhibiting phosphorylation of IRS-2, PKB and ERK. IGF-1 counteracts dexa-mediated apoptosis in the presence of reduced PKB but increased ERK phosphorylation. (c) 2008 S. Karger AG, Basel.

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death

PubMed Central

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-01-01

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL. PMID:27588473

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

PubMed

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-09-27

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL.

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

PubMed

Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

2014-03-01

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

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.

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.

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.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

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

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

Programmed cell death acts at different stages of Drosophila neurodevelopment to shape the central nervous system

PubMed Central

Desplan, Claude

2016-01-01

Nervous system development is a process that integrates cell proliferation, differentiation and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerge while integrating this information. PMID:27404003

Matrix regulation of skeletal cell apoptosis II: role of Arg-Gly-Asp-containing peptides.

PubMed

Perlot, Robert L; Shapiro, Irving M; Mansfield, Kyle; Adams, Christopher S

2002-01-01

This investigation was based on the assumption that arg-gly-asp (RGD)-containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC-3T3-E1 cells, and chick chondrocytes were incubated with purified RGD-containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC-3T3-E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage-sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS-mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled-RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells.

Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death.

PubMed

Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath

2010-02-01

We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.

Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells

PubMed Central

Liu, Chunming; Sherpa, Tshering

2013-01-01

Purpose To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Methods Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3′,5′-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Results Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. Conclusions These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels. PMID:23805033

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.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

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

Lin, Ming-Chung; Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan; Chen, Chia-Ling

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-likemore » cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase in peritoneal vascular permeability.« less

Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

NASA Astrophysics Data System (ADS)

Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

2009-02-01

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

Quercetin inhibits prostate cancer by attenuating cell survival and inhibiting anti-apoptotic pathways.

PubMed

Ward, Ashley B; Mir, Hina; Kapur, Neeraj; Gales, Dominique N; Carriere, Patrick P; Singh, Shailesh

2018-06-14

Despite recent advances in diagnosis and treatment, prostate cancer (PCa) remains the leading cause of cancer-related deaths in men. Current treatments offered in the clinics are often toxic and have severe side effects. Hence, to treat and manage PCa, new agents with fewer side effects or having potential to reduce side effects of conventional therapy are needed. In this study, we show anti-cancer effects of quercetin, an abundant bioflavonoid commonly used to treat prostatitis, and defined quercetin-induced cellular and molecular changes leading to PCa cell death. Cell viability was assessed using MTT. Cell death mode, mitochondrial outer membrane potential, and oxidative stress levels were determined by flow cytometry using Annexin V-7 AAD dual staining kit, JC-1 dye, and ROS detection kit, respectively. Antibody microarray and western blot were used to delineate the molecular changes induced by quercetin. PCa cells treated with various concentrations of quercetin showed time- and dose-dependent decrease in cell viability compared to controls, without affecting normal prostate epithelial cells. Quercetin led to apoptotic and necrotic cell death in PCa cells by affecting the mitochondrial integrity and disturbing the ROS homeostasis depending upon the genetic makeup and oxidative status of the cells. LNCaP and PC-3 cells that have an oxidative cellular environment showed ROS quenching after quercetin treatment while DU-145 showed rise in ROS levels despite having a highly reductive environment. Opposing effects of quercetin were also observed on the pro-survival pathways of PCa cells. PCa cells with mutated p53 (DU-145) and increased ROS showed significant reduction in the activation of pro-survival Akt pathway while Raf/MEK were activated in response to quercetin. PC-3 cells lacking p53 and PTEN with reduced ROS levels showed significant activation of Akt and NF-κB pathway. Although some of these changes are commonly associated with oncogenic response, the cumulative effect of these alterations is PCa cell death. Our results demonstrated quercetin exerts its anti-cancer effects by modulating ROS, Akt, and NF-κB pathways. Quercetin could be used as a chemopreventive option as well as in combination with chemotherapeutic drugs to improve clinical outcomes of PCa patients.

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

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

Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung

Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis inmore » T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.« less

Targeting Programmed Cell Death Using Small-Molecule Compounds to Improve Potential Cancer Therapy.

PubMed

Ke, Bowen; Tian, Mao; Li, Jingjing; Liu, Bo; He, Gu

2016-11-01

Evasion of cell death is one of the hallmarks of cancer cells, beginning with long-established apoptosis and extending to other new forms of cell death. An elaboration of cell death pathways thus will contribute to a better understanding of cancer pathogenesis and therapeutics. With the recent substantial biochemical and genetic explorations of cell death subroutines, their classification has switched from primarily morphological to more molecular definitions. According to their measurable biochemical features and intricate mechanisms, cell death subroutines can be divided into apoptosis, autophagic cell death, mitotic catastrophe, necroptosis, parthanatos, ferroptosis, pyroptosis, pyronecrosis, anoikis, cornification, entosis, and NETosis. Supportive evidence has gradually revealed the prime molecular mechanisms of each subroutine and thus providing series of possible targets in cancer therapy, while the intricate relationships between different cell death subroutines still remain to be clarified. Over the past decades, cancer drug discovery has significantly benefited from the use of small-molecule compounds to target classical modalities of cell death such as apoptosis, while newly identified cell death subroutines has also emerging their potential for cancer drug discovery in recent years. In this review, we comprehensively focus on summarizing 12 cell death subroutines and discussing their corresponding small-molecule compounds in potential cancer therapy. Together, these inspiring findings may provide more evidence to fill in the gaps between cell death subroutines and small-molecule compounds to better develop novel cancer therapeutic strategies. © 2016 Wiley Periodicals, Inc.

Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

PubMed

Wiman, K G; Zhivotovsky, B

2017-05-01

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

Intra- and Interdimeric Caspase-8 Self-Cleavage Controls Strength and Timing of CD95-Induced Apoptosis

PubMed Central

Kallenberger, Stefan M.; Beaudouin, Joël; Claus, Juliane; Fischer, Carmen; Sorger, Peter K.; Legewie, Stefan; Eils, Roland

2014-01-01

Apoptosis in response to the ligand CD95L (also known as Fas ligand) is initiated by caspase-8, which is activated by dimerization and self-cleavage at death-inducing signaling complexes (DISCs). Previous work indicated that the degree of substrate cleavage by caspase-8 determines whether a cell dies or survives in response to a death stimulus. To determine how a death ligand stimulus is effectively translated into caspase-8 activity, we assessed this activity over time in single cells with compartmentalized probes that are cleaved by caspase-8, and used multiscale modeling to simultaneously describe single-cell and population data with an ensemble of single-cell models. We derived and experimentally validated a minimal model in which cleavage of caspase-8 in the enzymatic domain occurs in an interdimeric manner through interaction between DISCs, whereas prodomain cleavage sites are cleaved in an intradimeric manner within DISCs. Modeling indicated that sustained membrane-bound caspase-8 activity is followed by transient cytosolic activity, which can be interpreted as a molecular timer mechanism reflected by a limited lifetime of active caspase-8. The activation of caspase-8 by combined intra- and interdimeric cleavage ensures weak signaling at low concentrations of CD95L and strongly accelerated activation at higher ligand concentrations, thereby contributing to precise control of apoptosis. PMID:24619646

Morphodynamics of a growing microbial colony driven by cell death

NASA Astrophysics Data System (ADS)

Ghosh, Pushpita; Levine, Herbert

2017-11-01

Bacterial cells can often self-organize into multicellular structures with complex spatiotemporal morphology. In this work, we study the spatiotemporal dynamics of a growing microbial colony in the presence of cell death. We present an individual-based model of nonmotile bacterial cells which grow and proliferate by consuming diffusing nutrients on a semisolid two-dimensional surface. The colony spreads by growth forces and sliding motility of cells and undergoes cell death followed by subsequent disintegration of the dead cells in the medium. We model cell death by considering two possible situations: In one of the cases, cell death occurs in response to the limitation of local nutrients, while the other case corresponds to an active death process, known as apoptotic or programmed cell death. We demonstrate how the colony morphology is influenced by the presence of cell death. Our results show that cell death facilitates transitions from roughly circular to highly branched structures at the periphery of an expanding colony. Interestingly, our results also reveal that for the colonies which are growing in higher initial nutrient concentrations, cell death occurs much earlier compared to the colonies which are growing in lower initial nutrient concentrations. This work provides new insights into the branched patterning of growing bacterial colonies as a consequence of complex interplay among the biochemical and mechanical effects.

Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons.

PubMed

Stankowska, Dorota L; Krishnamoorthy, Vignesh R; Ellis, Dorette Z; Krishnamoorthy, Raghu R

2017-06-01

Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of hippocampal neurons leading to memory deficits and cognitive decline. Studies suggest that levels of the vasoactive peptide endothelin-1 (ET-1) are increased in the brain tissue of Alzheimer's patients. Curcumin, the main ingredient of the spice turmeric, has been shown to have anti-inflammatory, anti-cancer, and neuroprotective effects. However, the mechanisms underlying some of these beneficial effects are not completely understood. The objective of this study was to determine if curcumin could protect hippocampal neurons from ET-1 mediated cell death and examine the involvement of c-Jun in this pathway. Primary hippocampal neurons from rat pups were isolated using a previously published protocol. Viability of the cells was measured by the live/dead assay. Immunoblot and immunohistochemical analyses were performed to analyze c-Jun levels in hippocampal neurons treated with either ET-1 or a combination of ET-1 and curcumin. Apoptotic changes were evaluated by immunoblot detection of cleaved caspase-3, cleaved fodrin, and a caspase 3/7 activation assay. ET-1 treatment produced a 2-fold increase in the levels of c-Jun as determined by an immunoblot analysis in hippocampal neurons. Co-treatment with curcumin significantly attenuated the ET-1 mediated increase in c-Jun levels. ET-1 caused increased neuronal cell death of hippocampal neurons indicated by elevation of cleaved caspase-3, cleaved fodrin and an increased activity of caspases 3 and 7 which was attenuated by co-treatment with curcumin. Blockade of JNK, an upstream effector of c-Jun by specific inhibitor SP600125 did not fully protect from ET-1 mediated activation of pro-apoptotic enzymes in primary hippocampal cells. Our data suggests that one mechanism by which curcumin protects against ET-1-mediated cell death is through blocking an increase in c-Jun levels. Other possible mechanisms include decreasing pro-apoptotic signaling activated by ET-1 in primary hippocampal neurons.

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.

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.

Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

PubMed

Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

2017-10-01

Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

PubMed Central

Simon, Priscilla S.; Bardhan, Kankana; Chen, May R.; Paschall, Amy V.; Lu, Chunwan; Bollag, Roni J.; Kong, Feng-Chong; Jin, JianYue; Kong, Feng-Ming; Waller, Jennifer L.; Pollock, Raphael E.; Liu, Kebin

2016-01-01

Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert its anti-tumor activity; however, the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity in the tumor microenvironment is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα induces tumor cell death in an autocrine manner. A sublethal dose of Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. Furthermore, we determined in a syngeneic sarcoma mouse model that radiation up-regulates IRF3, IFNβ, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which are associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Moreover, tumor-infiltrating T cells are in their active form since both the perforin and FasL pathways are activated in irradiated tumor tissues. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Therefore, radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation-mediated tumor suppression. PMID:27014915

Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

PubMed

Pizato, Nathalia; Luzete, Beatriz Christina; Kiffer, Larissa Fernanda Melo Vasconcelos; Corrêa, Luís Henrique; de Oliveira Santos, Igor; Assumpção, José Antônio Fagundes; Ito, Marina Kiyomi; Magalhães, Kelly Grace

2018-01-31

The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1β secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.

Bis-demethoxy curcumin analog nanoparticles: synthesis, characterization, and anticancer activity in vitro.

PubMed

Francis, Arul Prakash; Murthy, Prakhya Balakishna; Devas, Thiyagarajan

2014-07-01

We have optimized a protocol for the preparation of bisdemethoxy curcumin analog nanoparticles (BDMCA-NP) by the solvent assisted process. The structural similarities between bulk and nano BDMCA were determined by Co-TLC, NMR and F-TIR. This shows that our synthesis protocol enhanced the dispersibility and reduce the size of BDMCA without altering the integrity of functional moieties and structure, which is crucial for anticancer and antioxidant activities. The morphology and size of BDMCA-NP as determined by SEM, HRTEM and DLS was found to be around 80 nm. BDMCA-NP treated breast cancer cell lines (MCF 7) showed cell death as characterized by MTT assay. Flow cytometric analysis of BDMCA-NP treated MCF 7 cell lines showed an increase of cell count in G2/M phase indicates the cell cycle arrest. Western blot analysis revealed the presence of caspase 3, caspase 9, cleaved fragments of PARP and Bax proteins in the BDMCA-NP treated MCF 7 cell lines, but not in untreated cell lines. To recap, we have prepared BDMCA-NP by solvent assisted process, which exerted anticancer activity against breast cancer cells, which may be due to (i) enhanced dispersibility and surface: volume ratio, (ii) apoptosis (iii) mitochondrial pathway induced cell death, (iv) G2/M phase cell cycle arrest and (v) disassembly of mitotic spindle of the cancer cells. Thus, nano BDMCA can be used as a potent anticancer agent.

The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

PubMed Central

Mostowy, Serge; Shenoy, Avinash R.

2016-01-01

Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

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.

Xylogenesis in zinnia (Zinnia elegans) cell cultures: unravelling the regulatory steps in a complex developmental programmed cell death event.

PubMed

Iakimova, Elena T; Woltering, Ernst J

2017-04-01

Physiological and molecular studies support the view that xylogenesis can largely be determined as a specific form of vacuolar programmed cell death (PCD). The studies in xylogenic zinnia cell culture have led to many breakthroughs in xylogenesis research and provided a background for investigations in other experimental models in vitro and in planta . This review discusses the most essential earlier and recent findings on the regulation of xylem elements differentiation and PCD in zinnia and other xylogenic systems. Xylogenesis (the formation of water conducting vascular tissue) is a paradigm of plant developmental PCD. The xylem vessels are composed of fused tracheary elements (TEs)-dead, hollow cells with patterned lignified secondary cell walls. They result from the differentiation of the procambium and cambium cells and undergo cell death to become functional post-mortem. The TE differentiation proceeds through a well-coordinated sequence of events in which differentiation and the programmed cellular demise are intimately connected. For years a classical experimental model for studies on xylogenesis was the xylogenic zinnia (Zinnia elegans) cell culture derived from leaf mesophyll cells that, upon induction by cytokinin and auxin, transdifferentiate into TEs. This cell system has been proven very efficient for investigations on the regulatory components of xylem differentiation which has led to many discoveries on the mechanisms of xylogenesis. The knowledge gained from this system has potentiated studies in other xylogenic cultures in vitro and in planta. The present review summarises the previous and latest findings on the hormonal and biochemical signalling, metabolic pathways and molecular and gene determinants underlying the regulation of xylem vessels differentiation in zinnia cell culture. Highlighted are breakthroughs achieved through the use of xylogenic systems from other species and newly introduced tools and analytical approaches to study the processes. The mutual dependence between PCD signalling and the differentiation cascade in the program of TE development is discussed.

Optical cell cleaning with NIR femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

2015-03-01

Femtosecond laser microscopes have been used as both micro and nanosurgery tools. The optical knock-out of undesired cells in multiplex cell clusters shall be further reported on in this study. Femtosecond laser-induced cell death is beneficial due to the reduced collateral side effects and therefore can be used to selectively destroy target cells within monolayers, as well as within 3D tissues, all the while preserving cells of interest. This is an important characteristic for the application in stem cell research and cancer treatment. Non-precise damage compromises the viability of neighboring cells by inducing side effects such as stress to the cells surrounding the target due to the changes in the microenvironment, resulting from both the laser and laser-exposed cells. In this study, optimum laser parameters for optical cleaning by isolating single cells and cell colonies are exploited through the use of automated software control. Physiological equilibrium and cellular responses to the laser induced damages are also investigated. Cell death dependence on laser focus, determination and selectivity of intensity/dosage, controllable damage and cell recovery mechanisms are discussed.

Hepatic apoptotic activity following transient normothermic inflow occlusion and reperfusion in the swine model.

PubMed

Helling, T S; Edwards, C A; Helling, T S; Chang, C C; Hodges, M C; Dhar, A; VanWay, C

1999-09-01

Accelerated hepatic apoptosis was first described in portal vein-ligated livers but has since been reported in a variety of liver injuries. Because porto-prival states can induce apoptosis we sought to determine whether transient ischemic periods followed by reperfusion would trigger such cell death. The cytokines TNF-alpha and TGF-beta are known to facilitate apoptosis and are released in response to a number of stimuli including ischemia. We also investigated alterations in plasma and tissue levels of these cytokines which might lend support to their role in increased apoptotic activity following ischemia/reperfusion. Female pigs were used as the experimental model. Inflow occlusion of portal and hepatic arterial blood was performed to a portion of the swine liver directing the entire splanchnic flow to the remaining hepatic lobes for a period of 2 h. The livers were then reperfused and plasma and tissue samples taken for determination of apoptotic activity utilizing cell death immunoperoxidase staining of 3'-OH DNA ends generated by fragmentation and ELISA assay of histone-associated DNA fragments. Plasma and tissue levels of TNF-alpha and plasma levels of TGF-beta were determined by ELISA assay. An increase in apoptotic activity following reperfusion was seen at Day 2 and Day 4 compared to preischemic values by the cell death stain. The ELISA cell death assay showed an increase in apoptotic activity at 60 min, Day 2, and Day 4. Moreover, the ELISA cell death assay showed enhanced apoptotic activity in "hyperperfused" hepatic lobes compared to preischemic, or resting, liver. This was also observed when compared to sham-operated animals. Surprisingly, there was no detectable increase in plasma TNF-alpha or TGF-beta levels following ischemia/reperfusion, although homogenized liver TNF-alpha levels were increased at 60 min and Day 2 following reperfusion. We conclude that transient hepatic inflow occlusion followed by reperfusion can induce increased apoptotic activity in the swine model. Furthermore, increased apoptotic activity also occurs in the hyperperfused liver raising the possibility of a locally active factor or global hepatic expression of receptor activity in response to ischemia/reperfusion. This period of ischemia/reperfusion did not produce a detectable increase in circulating cytokine levels, and accelerated apoptosis could not be linked to heightened TNF-alpha or TGF-beta plasma activity. Higher tissue levels of TNF-alpha could reflect enhanced binding to TNF cell surface receptors or amplified receptor expression. Copyright 1999 Academic Press.

Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells.

PubMed

Cia, David; Vergnaud-Gauduchon, Juliette; Jacquemot, Nathalie; Doly, Michel

2014-09-01

To determine whether the green tea polyphenol epigallocatechin gallate (EGCG) could prevent H(2)O(2)-induced oxidative stress in primary rat retinal pigment epithelial cells. Primary cultures of retinal pigment epithelium (RPE) cells were established from Long-Evans newborn rats. RPE cells were pretreated with various concentrations of EGCG for 24 h before being exposed to hydrogen peroxide (H(2)O(2)) for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was quantified by flow cytometry using propidium iodide (PI). Treatment of RPE cells with EGCG alone does not affect the cell viability up to 50 µM. Exposure of RPE cells to 600 µM H(2)O(2) caused a significant decrease in cell viability; whereas pretreatment with 10, 25, and 50 µM EGCG significantly reduced this decrease in a dose-dependent manner. The proportion of PI-positive cells increased significantly in cultures treated with H(2)O(2) alone; whereas pretreatment of RPE cells with 50 µM EGCG significantly reduced H(2)O(2)-induced RPE cell death. Our study shows that EGCG pretreatment can protect primary rat RPE cells from H(2)O(2)-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H(2)O(2)-induced oxidative stress.

Apigenin, a dietary flavonoid, induces apoptosis, DNA damage, and oxidative stress in human breast cancer MCF-7 and MDA MB-231 cells.

PubMed

Vrhovac Madunić, Ivana; Madunić, Josip; Antunović, Maja; Paradžik, Mladen; Garaj-Vrhovac, Vera; Breljak, Davorka; Marijanović, Inga; Gajski, Goran

2018-05-01

Apigenin is found in several dietary plant foods such as vegetables and fruits. To investigate potential anticancer properties of apigenin on human breast cancer, ER-positive MCF-7 and triple-negative MDA MB-231 cells were used. Moreover, toxicological safety of apigenin towards normal cells was evaluated in human lymphocytes. Cytotoxicity of apigenin towards cancer cells was evaluated by MTT assay whereas further genotoxic and oxidative stress parameters were measured by comet and lipid peroxidation assays, respectively. In order to examine the type of cell death induced by apigenin, several biomarkers were used. Toxicological safety towards normal cells was evaluated by cell viability and comet assays. After the treatment with apigenin, we observed changes in cell morphology in a dose- (10 to 100 μM) and time-dependent manner. Moreover, apigenin caused cell death in both cell lines leading to significant toxicity and dominantly to apoptosis. Furthermore, apigenin proved to be genotoxic towards the selected cancer cells with a potential to induce oxidative damage to lipids. Of great importance is that no significant cytogenotoxic effects were detected in normal cells. The observed cytogenotoxic and pro-cell death activities of apigenin coupled with its low toxicity towards normal cells indicate that this natural product could be used as a future anticancer modality. Therefore, further analysis to determine the exact mechanism of action and in vivo studies on animal models are warranted.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

PubMed

Aouacheria, Abdel; Baghdiguian, Stephen; Lamb, Heather M; Huska, Jason D; Pineda, Fernando J; Hardwick, J Marie

2017-10-01

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Myocardial Autophagy after Severe Burn in Rats

PubMed Central

Zhang, Qiong; Shi, Xiao-hua; Huang, Yue-sheng

2012-01-01

Background Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn. Methods Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function. Results Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function. Conclusion Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction. PMID:22768082

Photoreceptor cell death and rescue in retinal detachment and degenerations

PubMed Central

Murakami, Yusuke; Notomi, Shoji; Hisatomi, Toshio; Nakazawa, Toru; Ishibashi, Tatsuro; Miller, Joan W.; Vavvas, Demetrios G.

2013-01-01

Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss. PMID:23994436

Patterns of cell death in the perinatal mouse forebrain.

PubMed

Mosley, Morgan; Shah, Charisma; Morse, Kiriana A; Miloro, Stephen A; Holmes, Melissa M; Ahern, Todd H; Forger, Nancy G

2017-01-01

The importance of cell death in brain development has long been appreciated, but many basic questions remain, such as what initiates or terminates the cell death period. One obstacle has been the lack of quantitative data defining exactly when cell death occurs. We recently created a "cell death atlas," using the detection of activated caspase-3 (AC3) to quantify apoptosis in the postnatal mouse ventral forebrain and hypothalamus, and found that the highest rates of cell death were seen at the earliest postnatal ages in most regions. Here we have extended these analyses to prenatal ages and additional brain regions. We quantified cell death in 16 forebrain regions across nine perinatal ages from embryonic day (E) 17 to postnatal day (P) 11 and found that cell death peaks just after birth in most regions. We found greater cell death in several regions in offspring delivered vaginally on the day of parturition compared with those of the same postconception age but still in utero at the time of collection. We also found massive cell death in the oriens layer of the hippocampus on P1 and in regions surrounding the anterior crossing of the corpus callosum on E18 as well as the persistence of large numbers of cells in those regions in adult mice lacking the pro-death Bax gene. Together these findings suggest that birth may be an important trigger of neuronal cell death and identify transient cell groups that may undergo wholesale elimination perinatally. J. Comp. Neurol. 525:47-64, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Dead Cert: Measuring Cell Death.

PubMed

Crowley, Lisa C; Marfell, Brooke J; Scott, Adrian P; Boughaba, Jeanne A; Chojnowski, Grace; Christensen, Melinda E; Waterhouse, Nigel J

2016-12-01

Many cells in the body die at specific times to facilitate healthy development or because they have become old, damaged, or infected. Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer. The ability to objectively measure cell death in a laboratory setting is therefore essential to understanding and investigating the causes and treatments of many human diseases and disorders. Often, it is sufficient to know the extent of cell death in a sample; however, the mechanism of death may also have implications for disease progression, treatment, and the outcomes of experimental investigations. There are a myriad of assays available for measuring the known forms of cell death, including apoptosis, necrosis, autophagy, necroptosis, anoikis, and pyroptosis. Here, we introduce a range of assays for measuring cell death in cultured cells, and we outline basic techniques for distinguishing healthy cells from apoptotic or necrotic cells-the two most common forms of cell death. We also provide personal insight into where these assays may be useful and how they may or may not be used to distinguish apoptotic cell death from other death modalities. © 2016 Cold Spring Harbor Laboratory Press.

Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe

PubMed Central

Nakaoka, Hidenori; Wakamoto, Yuichi

2017-01-01

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50–80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions. PMID:28632741

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions.

PubMed

Usami, N; Furusawa, Y; Kobayashi, K; Lacombe, S; Reynaud-Angelin, A; Sage, E; Wu, Ting-Di; Croisy, A; Guerquin-Kern, J-L; Le Sech, C

2008-07-01

This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions--hadrontherapy--with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 microM PtTC, and then irradiated by fast ions C(6+) and He(2+), with Linear Energy Transfer (LET) within range 2-70 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

Pathway of 3-MCPD-induced apoptosis in human embryonic kidney cells.

PubMed

Ji, Jian; Zhu, Pei; Sun, Chao; Sun, Jiadi; An, Lu; Zhang, Yinzhi; Sun, Xiulan

2017-01-01

3-Chloropropane-1,2-diol (3-MCPD) is a heat-produced contaminant formed during the preparation of soy sauce worldwide. The present investigation was conducted to determine the molecular aspects of 3-MCPD toxicity on human embryonic kidney cells (HEK293). Cell viability and apoptosis were assessed in response to exposure to 3-MCPD using the MTT assay and high-content screening (HCS). DNA damage, intracellular reactive oxygen species (ROS) and apoptosis-related proteins were evaluated. Genes related with apoptosis were detected by qPCR-array for further understanding the 3-MCPD induced cell apoptosis signaling pathway. Our results clearly showed that 3-MCPD treatment inhibits cell proliferation and reactive oxygen species generation. qPCR-array indicated that nine apoptotic genes were up-regulated more than 2-fold and six down-regulated more than 2-fold. Genes associated with the mitochondrial apoptotic pathway, especially BCL2 family genes, changed significantly, indicating that the mitochondrial apoptotic pathway is activated. Death receptor pathway-related genes, TNFRSF11B and TNFRSF1A, changed significantly, indicating that the death receptor pathway is also activated, resulting in the inhibition of cell growth and proliferation as well as induction of apoptosis. To sum up, the experiment results indicated that 3-MCPD induced HEK293 cell toxicity through the death receptor pathway and mitochondrial pathway.

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease.

PubMed

Wang, Tao; Lao, Uyen; Edgar, Bruce A

2009-09-07

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.

Nanosurface design of dental implants for improved cell growth and function

NASA Astrophysics Data System (ADS)

Pan, Hsu-An; Hung, Yao-Ching; Chiou, Jin-Chern; Tai, Shih-Ming; Chen, Hsin-Hung; Huang, G. Steven

2012-08-01

A strategy was proposed for the topological design of dental implants based on an in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 to 200 nm. MG63 osteoblasts were seeded on nanodot arrays and cultured for 3 days. Cell number, percentage undergoing apoptotic-like cell death, cell adhesion and cytoskeletal organization were evaluated. Nanodots with a diameter of approximately 50 nm enhanced cell number by 44%, minimized apoptotic-like cell death to 2.7%, promoted a 30% increase in microfilament bundles and maximized cell adhesion with a 73% increase in focal adhesions. An enhancement of about 50% in mineralization was observed, determined by von Kossa staining and by Alizarin Red S staining. Therefore, we provide a complete range of nanosurfaces for growing osteoblasts to discriminate their nanoscale environment. Nanodot arrays present an opportunity to positively and negatively modulate cell behavior and maturation. Our results suggest a topological approach which is beneficial for the design of dental implants.

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena.

PubMed

Pianigiani, E; Tognetti, L; Ierardi, F; Mariotti, G; Rubegni, P; Cevenini, G; Perotti, R; Fimiani, M

2016-06-01

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2 h of aortic clamping and banked within 12 h of harvesting (13-14 h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.

cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation

PubMed Central

2010-01-01

Background Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death. Results We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1. Conclusions We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast. PMID:21108829

Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration.

PubMed

Dunn, S R; Thomason, J C; Le Tissier, M D A; Bythell, J C

2004-11-01

Bleaching of reef building corals and other symbiotic cnidarians due to the loss of their dinoflagellate algal symbionts (=zooxanthellae), and/or their photosynthetic pigments, is a common sign of environmental stress. Mass bleaching events are becoming an increasingly important cause of mortality and reef degradation on a global scale, linked by many to global climate change. However, the cellular mechanisms of stress-induced bleaching remain largely unresolved. In this study, the frequency of apoptosis-like and necrosis-like cell death was determined in the symbiotic sea anemone Aiptasia sp. using criteria that had previously been validated for this symbiosis as indicators of programmed cell death (PCD) and necrosis. Results indicate that PCD and necrosis occur simultaneously in both host tissues and zooxanthellae subject to environmentally relevant doses of heat stress. Frequency of PCD in the anemone endoderm increased within minutes of treatment. Peak rates of apoptosis-like cell death in the host were coincident with the timing of loss of zooxanthellae during bleaching. The proportion of apoptosis-like host cells subsequently declined while cell necrosis increased. In the zooxanthellae, both apoptosis-like and necrosis-like activity increased throughout the duration of the experiment (6 days), dependent on temperature dose. A stress-mediated PCD pathway is an important part of the thermal stress response in the sea anemone symbiosis and this study suggests that PCD may play different roles in different components of the symbiosis during bleaching.

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

PubMed Central

Kovac, S; Domijan, A-M; Walker, M C; Abramov, A Y

2014-01-01

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na+] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets. PMID:25275601

A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death

PubMed Central

Minogue, Peter J.; Tong, Jun-Jie; Arora, Anita; Russell-Eggitt, Isabelle; Hunt, David M.; Moore, Anthony T.; Ebihara, Lisa; Beyer, Eric C.; Berthoud, Viviana M.

2009-01-01

PURPOSE To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. METHODS The GJA8 gene was directly sequenced. Formation of functional channels was assessed by two-microelectrode voltage-clamp. Connexin protein levels and distribution were assessed by immunoblotting and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. RESULTS Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in cell number and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. CONCLUSIONS Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function. PMID:19684000

Neuroprotective effects of phytosterols and flavonoids from Cirsium setidens and Aster scaber in human brain neuroblastoma SK-N-SH cells.

PubMed

Chung, Mi Ja; Lee, Sanghyun; Park, Yong Il; Lee, Jisun; Kwon, Ki Han

2016-03-01

We investigated the neuroprotective effects and action mechanism of three major compounds [daucosterol (Dau), pectolinarin (Pec), and astragalin (Ast)] isolated from edible plants against H2O2-induced cell death of human brain neuroblastoma SK-N-SH cells. Cytotoxicity was determined by MTT and lactate dehydrogenase (LDH) assays. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by TUNEL assay. The formation of reactive oxygen species (ROS), expression of antioxidant enzymes and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by 2,7-dichlorofluorescein diacetate (DCF-DA) assay, RT-PCR, and western blotting, respectively. The ethyl acetate fractions from Cirsium setidens (CSEA) and Aster scaber (ASEA) showed neuroprotective effects in SK-N-SH cells. The phytochemicals were isolated from CSEA and ASEA and identified by spectral analyses, as β-sitosterol, Dau, Pec, Ast, or isoquercitrin. Pretreatment with Dau, Pec, or Ast showed protective effects against H2O2-induced cell death and inhibited ROS generation by oxidative stress. HO-1 mRNA and protein levels were increased by the presence of H2O2 and were further elevated by pretreatment with Dau and Ast. Dau pretreatment resulted in further increases of H2O2-induced enhancement in levels of CAT and SOD2. Pretreatment with Dau, Pec, and Ast inhibited phosphorylation of MAPK, such as extracellular protein regulated protein kinase, p38, and c-Jun N-terminal kinase by H2O2. Dau exerts its neuroprotective effects by down regulation of MAPK pathways and upregulation of the HO-1, CAT and SOD2 antioxidant genes and is associated with reduced oxidative stress in SK-N-SH cells. Copyright © 2016 Elsevier Inc. All rights reserved.

tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize

USDA-ARS?s Scientific Manuscript database

Sex determination in maize is controlled by a developmental cascade leading to the formation of unisexual florets derived from an initially bisexual floral meristem. Abortion of pistil primordia in staminate florets is controlled by a tasselseed-mediated cell death process. Here, we describe the pos...

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

PubMed

Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

2011-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

Inhibitory Effect of Lycopene on Amyloid-β-Induced Apoptosis in Neuronal Cells.

PubMed

Hwang, Sinwoo; Lim, Joo Weon; Kim, Hyeyoung

2017-08-16

Alzheimer's disease (AD) is a fatal neurodegenerative disease. Brain amyloid-β deposition is a crucial feature of AD, causing neuronal cell death by inducing oxidative damage. Reactive oxygen species (ROS) activate NF-κB, which induces expression of Nucling. Nucling is a pro-apoptotic factor recruiting the apoptosome complex. Lycopene is an antioxidant protecting from oxidative stress-induced cell damage. We investigated whether lycopene inhibits amyloid-β-stimulated apoptosis through reducing ROS and inhibiting mitochondrial dysfunction and NF-κB-mediated Nucling expression in neuronal SH-SY5Y cells. We prepared cells transfected with siRNA for Nucling or nontargeting control siRNA to determine the role of Nucling in amyloid-β-induced apoptosis. The amyloid-β increased intracellular and mitochondrial ROS levels, apoptotic indices (p53, Bax/Bcl-2 ratio, caspase-3 cleavage), NF-kB activation and Nucling expression, while cell viability, mitochondrial membrane potential, and oxygen consumption rate decreased in SH-SY5Y cells. Lycopene inhibited these amyloid-β-induced alterations. However, amyloid-β did not induce apoptosis, determined by cell viability and apoptotic indices (p53, Bax/Bcl-2 ratio, caspase-3 cleavage), in the cells transfected with siRNA for Nucling. Lycopene inhibited apoptosis by reducing ROS, and by inhibiting mitochondrial dysfunction and NF-κB-target gene Nucling expression in neuronal cells. Lycopene may be beneficial for preventing oxidative stress-mediated neuronal death in patients with neurodegeneration.

Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

PubMed

Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

2018-04-01

Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

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

The Molecular Ecophysiology of Programmed Cell Death in Marine Phytoplankton

NASA Astrophysics Data System (ADS)

Bidle, Kay D.

2015-01-01

Planktonic, prokaryotic, and eukaryotic photoautotrophs (phytoplankton) share a diverse and ancient evolutionary history, during which time they have played key roles in regulating marine food webs, biogeochemical cycles, and Earth's climate. Because phytoplankton represent the basis of marine ecosystems, the manner in which they die critically determines the flow and fate of photosynthetically fixed organic matter (and associated elements), ultimately constraining upper-ocean biogeochemistry. Programmed cell death (PCD) and associated pathway genes, which are triggered by a variety of nutrient stressors and are employed by parasitic viruses, play an integral role in determining the cell fate of diverse photoautotrophs in the modern ocean. Indeed, these multifaceted death pathways continue to shape the success and evolutionary trajectory of diverse phytoplankton lineages at sea. Research over the past two decades has employed physiological, biochemical, and genetic techniques to provide a novel, comprehensive, mechanistic understanding of the factors controlling this key process. Here, I discuss the current understanding of the genetics, activation, and regulation of PCD pathways in marine model systems; how PCD evolved in unicellular photoautotrophs; how it mechanistically interfaces with viral infection pathways; how stress signals are sensed and transduced into cellular responses; and how novel molecular and biochemical tools are revealing the impact of PCD genes on the fate of natural phytoplankton assemblages.

Curcumin in Cell Death Processes: A Challenge for CAM of Age-Related Pathologies

PubMed Central

Salvioli, S.; Sikora, E.; Cooper, E. L.

2007-01-01

Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied phytochemical which has a variety of biological activities: anti-inflammatory and anti-oxidative. In this review we discuss the biological mechanisms and possible clinical effects of curcumin treatment on cancer therapy, and neurodegenerative diseases such as Alzheimer's Disease, with particular attention to the cell death processes induced by curcumin. Since oxidative stress and inflammation are major determinants of the aging process, we also argue that curcumin can have a more general effect that slows down the rate of aging. Finally, the effects of curcumin can be described as xenohormetic, since it activates a sort of stress response in mammalian cells. PMID:17549234

Oncolytic Reovirus in Canine Mast Cell Tumor

PubMed Central

Hwang, Chung Chew; Umeki, Saori; Kubo, Masahito; Hayashi, Toshiharu; Shimoda, Hiroshi; Mochizuki, Masami; Maeda, Ken; Baba, Kenji; Hiraoka, Hiroko; Coffey, Matt; Okuda, Masaru; Mizuno, Takuya

2013-01-01

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility. PMID:24073198

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells.

PubMed

Buravkova, L B; Rylova, Y V; Andreeva, E R; Kulikov, A V; Pogodina, M V; Zhivotovsky, B; Gogvadze, V

2013-10-01

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs. Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide. We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs. Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state. These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Measurement of cell death by oxidative stress in three-dimensional spheroids from trophoblast and in fragments of decidua tissue.

PubMed

Theuerkauf, Regine-Susanne; Ahammer, Helmut; Siwetz, Monika; Helige, Christine; Dohr, Gottfried; Walcher, Wolfgang; Palacio, José Ramón; Martinez, Paz; Sedlmayr, Peter

2010-05-01

We report a new morphometric method for measurement of the amount of cell death in three-dimensional multicellular spheroids of the trophoblast-like cell line AC1-M59 and of cultured pieces of decidua tissue (decidua spheroids) in response to a cytotoxic agent. The viability of the spheroids was assessed by adding propidium iodide to the culture medium at the end of the toxic treatment. On fluorescence and brightfield images of serial cryosections the areas of propidium iodide fluorescence and the entire corresponding spheroids were measured by applying digital image processing and ratiometrical quantification. As an example, we evaluated the cytotoxic effect of hydrogen peroxide on both types of spheroids. The relative potency of hydrogen peroxide to induce tissue damage was assessed quantitatively for determination of the minimal concentration that leads to an increase in cytotoxicity. The method presented suggests general applicability for in vitro determination of toxicity against tissues. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism

PubMed Central

2012-01-01

Background Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Methods Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Results Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. Conclusion The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells. PMID:22264378

Influence of P53 on the radiotherapy response of hepatocellular carcinoma

PubMed Central

Gomes, Ana R.; Abrantes, Ana M.; Brito, Ana F.; Laranjo, Mafalda; Casalta-Lopes, João E.; Gonçalves, Ana C.; Sarmento-Ribeiro, Ana B.; Tralhão, José G.

2015-01-01

Background/Aims Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. Methods Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. Results The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. Conclusions These results suggest that P53 plays a key role in the radiotherapy response of HCC. PMID:26527121

1,3,4-Thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones cause death in Trypanosoma cruzi through oxidative stress.

PubMed

Martins, Solange C; Lazarin-Bidóia, Danielle; Desoti, Vânia C; Falzirolli, Hugo; da Silva, Cleuza C; Ueda-Nakamura, Tania; Silva, Sueli de O; Nakamura, Celso V

2016-12-01

This work evaluated the in vitro and in vivo activity of TDZ 2 on Trypanosoma cruzi amastigotes and determined the possible mechanism of action of this compound on T. cruzi death. TDZ 2 inhibited T. cruzi proliferation in vitro and had low haemolytic potential. It also induced morphological and ultrastructural alterations. We observed a reduction of cell volume, the depolarization of the mitochondrial membrane, an increase in ROS production, lipoperoxidation of the cell membrane, lipid bodies formation and production of nitric oxide, a decrease in reduced thiols levels and, presence of autophagic vacuoles. The in vivo study found a reduction of parasitemia in animals treated with TDZ 2 alone or combined with benznidazole. Altogether, the alterations induced by TDZ 2 point to an oxidative stress condition that lead to T. cruzi cell death. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The machinery of Nod-like receptors: refining the paths to immunity and cell death.

PubMed

Saleh, Maya

2011-09-01

One of the fundamental aspects of the innate immune system is its capacity to discriminate between self and non-self or altered self, and to quickly respond by eliciting effector mechanisms that act in concert to restore normalcy. This capacity is determined by a set of evolutionarily conserved pattern recognition receptors (PRRs) that sense the presence of microbial motifs or endogenous danger signals, including tissue damage, cellular transformation or metabolic perturbation, and orchestrate the nature, duration and intensity of the innate immune response. Nod-like receptors (NLRs), a group of intracellular PRRs, are particularly essential as evident by the high incidence of genetic variations in their genes in various diseases of homeostasis. Here, I overview the signaling mechanisms of NLRs and discuss the mounting evidence of evolutionary conservation between their pathways and the cell death machinery. I also describe their effector functions that link the sensing of danger to the induction of inflammation, autophagy or cell death. © 2011 John Wiley & Sons A/S.

The quick and the dead: microbial demography at the yeast thermal limit.

PubMed

Maxwell, Colin S; Magwene, Paul M

2017-03-01

The niche of microorganisms is determined by where their populations can expand. Populations can fail to grow because of high death or low birth rates, but these are challenging to measure in microorganisms. We developed a novel technique that enables single-cell measurement of age-structured birth and death rates in the budding yeast, Saccharomyces cerevisiae, and used this method to study responses to heat stress in a genetically diverse panel of strains. We find that individual cells show significant heterogeneity in their rates of birth and death during heat stress. Genotype-by-environment effects on processes that regulate asymmetric cell division contribute to this heterogeneity. These lead to either premature senescence or early life mortality during heat stress, and we find that a mitochondrial inheritance defect explains the early life mortality phenotype of one of the strains we studied. This study demonstrates how the interplay of physiology, genetic variation and environmental variables influence where microbial populations survive and flourish. © 2016 John Wiley & Sons Ltd.

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.

Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

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

Li, Lingyun, E-mail: lingyunlee@126.com; Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004; Gao, Luyan

Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase inmore » the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.« less

Differential PKA activation and AKAP association determines cell fate in cancer cells

PubMed Central

2013-01-01

Background The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms. Methods Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects. Results We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation. Conclusions We had identified 2 cytoplasmic pathways dependent upon the same enzymatic activity with opposite effects on cell fate in terms of life and death. Understanding the specific mechanistic functions of IGF1R with respect to determining the PKA survival functions would have potential for impact upon the development of new therapeutic strategies by exploiting the IGF1R/cAMP-PKA survival signaling in cancer. PMID:24083380

RIP1 Inhibition Rescues from LPS-Induced RIP3-Mediated Programmed Cell Death, Distributed Energy Metabolism and Spatial Memory Impairment.

PubMed

Nikseresht, Sara; Khodagholi, Fariba; Nategh, Mohsen; Dargahi, Leila

2015-10-01

Receptor interacting protein 1 (RIP1) has a critical role in initiation of programmed necrosis or necroptosis. RIP1 in a close collaboration with RIP3 not only mediates necroptosis but also is involved in apoptosis and inflammatory signaling. However, the interpretation of the distinct function of RIP1 and RIP3 is complicated. Herein, we demonstrated that RIP1 inhibition in the context of LPS-induced neuroinflammation decreases RIP3 expression. Concomitant administration of Nec-1, specific inhibitor of RIP1, with LPS also attenuated the activating effect of RIP3 on metabolic enzymes, glutamate-ammonia ligase and glutamate dehydrogenase as bioenergetic determinants, in hippocampal and cortical cells. RIP1 inhibition possessed an anti-inflammatory effect and improved the antioxidant capacity against LPS. Interestingly, and opposed to some reports that necroptosis inhibition sensitizes cells to apoptosis, our results showed that RIP1 inhibition attenuates apoptotic cell death in response to LPS. The survival of neuronal function was also confirmed by measuring spontaneous alternations of rats in Y-maze. In conclusion, effects of RIP1 inhibition on RIP3 and cell death provide new approaches to ameliorate neuroinflammation and relative disorders.

The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells.

PubMed

Vera, Carolina Andrea; Oróstica, Lorena; Gabler, Fernando; Ferreira, Arturo; Selman, Alberto; Vega, Margarita; Romero, Carmen Aurora

2017-04-01

Ovarian cancer is the seventh most common cancer among women worldwide, causing approximately 120,000 deaths every year. Immunotherapy, designed to boost the body's natural defenses against cancer, appears to be a promising option against ovarian cancer. Calreticulin (CRT) is an endoplasmic reticulum (ER) resident chaperone that, translocated to the cell membrane after ER stress, allows cancer cells to be recognized by the immune system. The nerve growth factor (NGF) is a pro-angiogenic molecule overexpressed in this cancer. In the present study, we aimed to determine weather NGF has an effect in CRT translocation induced by cytotoxic and ER stress. We treated A2780 ovarian cancer cells with NGF, thapsigargin (Tg), an ER stress inducer and mitoxantrone (Mtx), a chemotherapeutic drug; CRT subcellular localization was analyzed by immunofluorescence followed by confocal microscopy. In order to determine NGF effect on Mtx and Tg-induced CRT translocation from the ER to the cell membrane, cells were preincubated with NGF prior to Mtx or Tg treatment and CRT translocation to the cell surface was determined by flow cytometry. In addition, by western blot analyses, we evaluated proteins associated with the CRT translocation pathway, both in A2780 cells and human ovarian samples. We also measured NGF effect on cell apoptosis induced by Mtx. Our results indicate that Mtx and Tg, but not NGF, induce CRT translocation to the cell membrane. NGF, however, inhibited CRT translocation induced by Mtx, while it had no effect on Tg-induced CRT exposure. NGF also diminished cell death induced by Mtx. NGF effect on CRT translocation could have consequences in immunotherapy, potentially lessening the effectiveness of this type of treatment.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection.

PubMed

Xu, Jinxian; Mo, Juan; Liu, Xinglou; Marshall, Brendan; Atherton, Sally S; Dong, Zheng; Smith, Sylvia; Zhang, Ming

2018-05-01

The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection

PubMed Central

Xu, Jinxian; Mo, Juan; Liu, Xinglou; Marshall, Brendan; Atherton, Sally S.; Dong, Zheng; Smith, Sylvia

2018-01-01

Purpose The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3−/−) and Rip3+/+ control mice. Methods Rip3−/− and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3−/− mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3−/− eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3−/− mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3–independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor–κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3−/− eyes. Conclusions Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor–κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Rational development of a cytotoxic peptide to trigger cell death.

PubMed

Boohaker, Rebecca J; Zhang, Ge; Lee, Michael W; Nemec, Kathleen N; Santra, Santimukul; Perez, J Manuel; Khaled, Annette R

2012-07-02

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.

PubMed

McCaig, William D; Patel, Payal S; Sosunov, Sergey A; Shakerley, Nicole L; Smiraglia, Tori A; Craft, Miranda M; Walker, Katharine M; Deragon, Matthew A; Ten, Vadim S; LaRocca, Timothy J

2018-01-01

Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR-Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs in vivo . This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.

Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells.

PubMed

Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

2011-10-01

Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G(1) to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

PubMed

Kaminaga, Kiichi; Noguchi, Miho; Narita, Ayumi; Hattori, Yuya; Usami, Noriko; Yokoya, Akinari

2016-11-01

To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

Preliminary Study of Hyptis pectinata (L.) Poit Extract Biotransformation by Aspergillus niger

NASA Astrophysics Data System (ADS)

Rejeki, D. S.; Aminin, A. L. N.; Suzery, M.

2018-04-01

One alternative approach to increase the content of bioactive compounds is fermentation. Hyptis pectinata (L.) Poit is a plant that can be found in tropical area and potentially as anticancer, anti-inflammatory, insect repellant, antiviral and antioxidant. In this research, efforts have been made to increase bioactive plant capacity of Hyptis pectinata (L.) Poit through submerged fermentation using Aspergillus niger. The study was performed by adding methanol extract of Hyptis pectinata (L.) Poit on two conditions, that was added at the beginning of fermentation and while entering a phase of death. Aspergillus niger growth rate in both conditions was observed by determining the dry weight of cells every 24 hours. The transformation profil of extract was observed after 24 hours of extract addition in early death phase by the TLC method. The results show that the addition of Hyptis pectinata (L.) Poit extract at log phase triggers the cells to growth faster, whereas the addition at the early death phase precisely accelerates cell death. TLC profile shows the emergence of new compounds suspected as the products of transformation of Hyptis pectinata (L.) Poit extract on day 8 after addition of extract.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

PubMed

Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Active Silver Nanoparticles for Wound Healing

PubMed Central

Rigo, Chiara; Ferroni, Letizia; Tocco, Ilaria; Roman, Marco; Munivrana, Ivan; Gardin, Chiara; Cairns, Warren R. L.; Vindigni, Vincenzo; Azzena, Bruno; Barbante, Carlo; Zavan, Barbara

2013-01-01

In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, Acticoat™ Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment. PMID:23455461

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

PubMed Central

Hakansson, Anders P.; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-01-01

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells. PMID:21423701

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

PubMed

Hakansson, Anders P; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-03-10

Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

PubMed

Scarlatti, F; Maffei, R; Beau, I; Codogno, P; Ghidoni, R

2008-08-01

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

PubMed

Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

2017-08-01

Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

PubMed Central

Suzuki, Haruka; Miyashita, Yuri; Choi, Sun Hee; Hisa, Yusuke; Rihei, Shunsuke; Shimada, Ryoko; Jeon, Eun Jin; Abe, Junya; Uyeda, Ichiro

2016-01-01

ABSTRACT Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1. Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV. IMPORTANCE Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses. PMID:27279605

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

PubMed

Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

2014-05-01

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

PubMed

Jiang, Jin-Yi; Cheung, Carmen K M; Wang, Yifang; Tsang, Benjamin K

2003-01-01

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Programmed Cell Death During Caenorhabditis elegans Development

PubMed Central

Conradt, Barbara; Wu, Yi-Chun; Xue, Ding

2016-01-01

Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general. PMID:27516615

Melatonin pre-treatment mitigates SHSY-5Y cells against oxaliplatin induced mitochondrial stress and apoptotic cell death

PubMed Central

Choudhury, Arnab; Kar, Sudeshna; Tabassum, Heena

2017-01-01

Oxaliplatin (Oxa) treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel), could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS) production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm), resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose) polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin’s protective effects may prove successful in eliciting pathways to further alter the neurotoxic pathways of platinum compounds in cancer treatment. PMID:28732061

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells.

PubMed

Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

2017-01-01

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease.

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells

PubMed Central

Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

2017-01-01

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson’s disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3′-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson’s disease. PMID:28250973

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.

Planarian yorkie/YAP functions to integrate adult stem cell proliferation, organ homeostasis and maintenance of axial patterning.

PubMed

Lin, Alexander Y T; Pearson, Bret J

2014-03-01

During adult homeostasis and regeneration, the freshwater planarian must accomplish a constant balance between cell proliferation and cell death, while also maintaining proper tissue and organ size and patterning. How these ordered processes are precisely modulated remains relatively unknown. Here we show that planarians use the downstream effector of the Hippo signaling cascade, yorkie (yki; YAP in vertebrates) to control a diverse set of pleiotropic processes in organ homeostasis, stem cell regulation, regeneration and axial patterning. We show that yki functions to maintain the homeostasis of the planarian excretory (protonephridial) system and to limit stem cell proliferation, but does not affect the differentiation process or cell death. Finally, we show that Yki acts synergistically with WNT/β-catenin signaling to repress head determination by limiting the expression domains of posterior WNT genes and that of the WNT-inhibitor notum. Together, our data show that yki is a key gene in planarians that integrates stem cell proliferation control, organ homeostasis, and the spatial patterning of tissues.

Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

PubMed

Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L; Golstein, Pierre

2003-03-31

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

Molecular Cell Biology of Apoptosis and Necroptosis in Cancer.

PubMed

Dillon, Christopher P; Green, Douglas R

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

Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii

PubMed Central

Yordanova, Zhenya P.; Woltering, Ernst J.; Kapchina-Toteva, Veneta M.; Iakimova, Elena T.

2013-01-01

Background and Aims Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. Methods Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. Key Results MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. Conclusions In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways. PMID:23250917

Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

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

Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp; Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp

Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death.more » - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.« less

Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

PubMed Central

Wang, Lihong; Liu, Liping; Shi, Yan; Cao, Hanwei; Chaturvedi, Rupesh; Calcutt, M. Wade; Hu, Tianhui; Ren, Xiubao; Wilson, Keith T.; Polk, D. Brent; Yan, Fang

2012-01-01

Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apc min mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth. PMID:22574158

The art and science of low-energy applications in medicine: pathology perspectives

NASA Astrophysics Data System (ADS)

Thomsen, Sharon L.

2011-03-01

Applications of low energy non-ionizing irradiation result in non-lethal and lethal effects in cells, tissues and intact individuals. The effects of these applications depend on the physical parameters of the applied energies, the mechanisms of interaction of these energies on the target and the biologic status of the target. Recently, cell death has been found not to be a random accident of situation or age but a range of complicated physiological responses to various extrinsic and intrinsic events some of which are genetically programmed and/ or physiologically regulated. Therefore, cell death has been classified into three general groups: 1) Programmed cell death including apoptosis and necroptosis, cornefication and autophagy; 2) Accidental (traumatic) cell death due to the direct, immediate effects of the lethal event and 3) Necrotic cell death which is, by default, all cell death not associated with programmed or accidental cell death. Lethal low energy non-ionizing application biologic effects involve mechanisms of all three groups as compared to high energy applications that predominantly involve the mechanisms of accidental cell death. Currently, the mechanisms of all these modes of cell death are being vigorously investigated. As research and development of new low energy applications continues, the need to understand the mechanisms of cell death that they produce will be critical to the rational creation of safe, yet effective instruments.

Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress

PubMed Central

Lin, Abraham; Truong, Billy; Patel, Sohil; Kaushik, Nagendra; Choi, Eun Ha; Fridman, Gregory; Fridman, Alexander; Miller, Vandana

2017-01-01

A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy. PMID:28467380

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.

Nonthermal-plasma-mediated animal cell death

NASA Astrophysics Data System (ADS)

Kim, Wanil; Woo, Kyung-Chul; Kim, Gyoo-Cheon; Kim, Kyong-Tai

2011-01-01

Animal cell death comprising necrosis and apoptosis occurred in a well-regulated manner upon specific stimuli. The physiological meanings and detailed molecular mechanisms of cell death have been continuously investigated over several decades. Necrotic cell death has typical morphological changes, such as cell swelling and cell lysis followed by DNA degradation, whereas apoptosis shows blebbing formation and regular DNA fragmentation. Cell death is usually adopted to terminate cancer cells in vivo. The current strategies against tumour are based on the induction of cell death by adopting various methods, including radiotherapy and chemotherapeutics. Among these, radiotherapy is the most frequently used treatment method, but it still has obvious limitations. Recent studies have suggested that the use of nonthermal air plasma can be a prominent method for inducing cancer cell death. Plasma-irradiated cells showed the loss of genomic integrity, mitochondrial dysfunction, plasma membrane damage, etc. Tumour elimination with plasma irradiation is an emerging concept in cancer therapy and can be accelerated by targeting certain tumour-specific proteins with gold nanoparticles. Here, some recent developments are described so that the mechanisms related to plasma-mediated cell death and its perspectives in cancer treatment can be understood.

Resveratrol analogue, HS-1793, induces apoptotic cell death and cell cycle arrest through downregulation of AKT in human colon cancer cells.

PubMed

Kim, Dong Hwan; Kim, Min Jeong; Sung, Bokyung; Suh, Hongsuk; Jung, Jee H; Chung, Hae Young; Kim, Nam Deuk

2017-01-01

Resveratrol, a polyphenolic compound, is a naturally occurring phytochemical and is found in a variety of plants, including grapes, berries and peanuts. It has gained much attention for its potential anticancer activity against various types of human cancer. However, the usefulness of resveratrol as a chemotherapeutic agent is limited by its photosensitivity and metabolic instability. In this study the effects of a synthetic analogue of resveratrol, HS-1793, on the proliferation and apoptotic cell death were investigated using HCT116 human colon cancer cells. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of HS-1793 on human colon cancer and its mechanisms of action have not been extensively studied. HS-1793 inhibited cell growth and induced apoptotic cell death in a concentration-dependent fashion. Induction of apoptosis was determined by morphological changes, cleavage of poly(ADP-ribose) polymerase, alteration of Bax/Bcl-2 expression ratio, and caspase activations. Flow cytometric analysis revealed that HS-1793 induced G2/M arrest in the cell cycle progression in HCT116 cells. Furthermore, HS-1793 showed more potent anticancer effects in several aspects than resveratrol in HCT116 cells. In addition, HS-1793 suppressed Akt and the phosphatidylinositol-3 kinase/Akt inhibitor LY294002 was found to enhance its induction of apoptosis. Thus, these findings suggest that HS-1793 have potential as a candidate chemotherapeutic agent against human colon cancer.

Dictyostelium cell death

PubMed Central

Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L.; Golstein, Pierre

2003-01-01

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of “paddle” cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells. PMID:12654899

[Significance of Hypoxia-related microRNA for Estimating the Cause of Mechanical Asphyxia Death].

PubMed

Zeng, Y; Ma, J L; Chen, L

2017-02-01

Under hypoxia condition, microRNA （miRNA） can interact with transcription factors for regulating the cell metabolism, angiogenesis, erythropoiesis, cellular proliferation, differentiation and apoptosis. The biological processes above may play an important role in mechanical asphyxia death. This article reviews the regulating function of miRNA under hypoxia condition and the influence of hypoxia to biosynthesis of miRNA, which may provide some new ideas to the research of miRNA on determining the cause of mechanical asphyxia death in the field of forensic medicine. Copyright© by the Editorial Department of Journal of Forensic Medicine.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.

PubMed

Knet, Malgorzata; Wartalski, Kamil; Hoja-Lukowicz, Dorota; Tabarowski, Zbigniew; Slomczynska, Maria; Duda, Malgorzata

2015-10-01

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed. Copyright © 2015 Elsevier Inc. All rights reserved.

Ester of Quinoxaline-7-carboxylate 1,4-di-N-oxide as Apoptosis Inductors in K-562 Cell Line: An in vitro, QSAR and DFT Study.

PubMed

Rivera, Gildardo; Andrade-Ochoa, Sergio; Romero, Manolo S Ortega; Palos, Isidro; Monge, Antonio; Sanchez-Torres, Luvia Enid

2017-01-01

Quinoxalines have shown a wide variety of biological activities including as antitumor agents. The aims of this study were to evaluate the activity of quinoxaline 1,4-di-N-oxide derivatives on K562 cells, the establishment of the mechanism of induced cell death, and the construction of predictive QSAR models. Sixteen esters of quinoxaline-7-carboxylate 1,4-di-N-oxide were evaluated for antitumor activity on K562 chronic myelogenous leukemia cells and their IC50 values were determined. The mechanism of induced cell death by the most active molecule was assessed by flow cytometry and an in silico study was conducted to optimize and calculate theoretical descriptors of all quinoxaline 1,4-di-N-oxide derivatives. QSAR and QPAR models were created using genetic algorithms. Our results show that compounds C5, C7, C10, C12 and C15 had the lowest IC50 of the series. C15 was the most active compound (IC50= 3.02 μg/mL), inducing caspase-dependent apoptotic cell death via the intrinsic pathway. QSAR and QPAR studies are discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

PubMed Central

Koshiba, Taichi; Kobayashi, Masaru; Matoh, Toru

2009-01-01

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD. PMID:19054807

Cystine uptake through the cystine/glutamate antiporter xCT triggers glioblastoma cell death under glucose deprivation.

PubMed

Goji, Takeo; Takahara, Kazuhiko; Negishi, Manabu; Katoh, Hironori

2017-12-01

Oncogenic signaling in cancer cells alters glucose uptake and utilization to supply sufficient energy and biosynthetic intermediates for survival and sustained proliferation. Oncogenic signaling also prevents oxidative stress and cell death caused by increased production of reactive oxygen species. However, elevated glucose metabolism in cancer cells, especially in glioblastoma, results in the cells becoming sensitive to glucose deprivation ( i.e. in high glucose dependence), which rapidly induces cell death. However, the precise mechanism of this type of cell death remains unknown. Here, we report that glucose deprivation alone does not trigger glioblastoma cell death. We found that, for cell death to occur in glucose-deprived glioblastoma cells, cystine and glutamine also need to be present in culture media. We observed that cystine uptake through the cystine/glutamate antiporter xCT under glucose deprivation rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death. We conclude that although cystine uptake is crucial for production of antioxidant glutathione in cancer cells its transport through xCT also induces oxidative stress and cell death in glucose-deprived glioblastoma cells. Combining inhibitors targeting cancer-specific glucose metabolism with cystine and glutamine treatment may offer a therapeutic approach for glioblastoma tumors exhibiting high xCT expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death.

PubMed

Kewalramani, Girish; Puthanveetil, Prasanth; Wang, Fang; Kim, Min Suk; Deppe, Sylvia; Abrahani, Ashraf; Luciani, Dan S; Johnson, James D; Rodrigues, Brian

2009-10-01

Although a substantial role for 5' adenosine monophosphate-activated protein kinase (AMPK) has been established in regulating cardiac metabolism, a less studied action of AMPK is its ability to prevent cardiac cell death. Using established AMPK activators like dexamethasone (DEX) or metformin (MET), the objective of the present study was to determine whether AMPK activation prevents tumour necrosis factor-alpha (TNF-alpha) induced apoptosis in adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with DEX, MET, or TNF-alpha for varying durations (0-12 h). TNF-alpha-induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF-alpha-induced cardiomyocyte apoptosis. Incubation of myocytes with TNF-alpha for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF-alpha-induced cytotoxicity. Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells

PubMed Central

2013-01-01

Background Samsoeum (SSE), a traditional herbal formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. Recent studies have demonstrated that SSE retains potent pharmacological efficiency in anti-allergic and anti-inflammatory reactions. However, the anti-cancer activity of SSE and its underlying mechanisms have not been studied. Thus, the present study was designed to determine the effect of SSE on cell death and elucidate its detailed mechanism. Methods Following SSE treatment, cell growth and cell death were measured using an MTT assay and trypan blue exclusion assay, respectively. Cell cycle arrest and YO-PRO-1 uptake were assayed using flow cytometry, and LC3 redistribution was observed using confocal microscope. The mechanisms of anti-cancer effect of SSE were investigated through western blot analysis. Results We initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6–12 h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), which is critical for cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly blocked SSE-induced increases in Beclin-1, LC3-II, and Bax expression and decreases in Bcl-2 expression, indicating that JNK activation plays critical role in cell death caused by SSE. Conclusions These findings suggest that SSE efficiently induces cancer cell death via apoptosis as well as autophagy through modification of the Akt/mTOR and JNK signaling pathways. SSE may be as a potent traditional herbal medicine for treating malignancies. PMID:24053190

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

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

Sun, Hengwen; Yang, Shana; Li, Jianhua

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expressionmore » in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.« 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

Evaluation of Dying Vocal Fold Epithelial Cells by Ultrastructural Features and TUNEL Method

PubMed Central

Novaleski, Carolyn K.; Mizuta, Masanobu; Rousseau, Bernard

2016-01-01

Cell death is a regulated mechanism of eliminating cells to maintain tissue homeostasis. This study described two methodological procedures for evaluating cell death in the epithelium of immobilized, approximated, and vibrated vocal folds from 12 New Zealand white breeder rabbits. The gold standard technique of transmission electron microscopy evaluated high-quality ultrastructural criteria of cell death and a common immunohistochemical marker, terminal deoxynucleotidyl transferase dUTP nick end labeling method, to confirm cell death signaling. Results revealed that ultrastructural characteristics of apoptotic cell death, specifically condensed chromatin and apoptotic bodies, were observed after vocal fold vibration and approximation. Although episodes of necrotic cell death were rare, few enlarged cell nuclei were present after vibration and approximation. The vocal fold expresses an immunohistochemical marker for apoptosis along the apical surface of the epithelium. This study provides a solid foundation for future investigations regarding the role of cell death in vocal fold health and disease. PMID:27537846

Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

PubMed

Solaymani-Mohammadi, Shahram; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Frey, Blake F; Billeskov, Rolf; Singer, Steven M; Berzofsky, Jay A; Eckmann, Lars; Kagnoff, Martin F

2016-03-01

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function. © Society for Leukocyte Biology.

Methods for assessing autophagy and autophagic cell death.

PubMed

Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

2008-01-01

Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

"Falling leaves": a survey of the history of apoptosis.

PubMed

Formigli, L; Conti, A; Lippi, D

2004-04-01

Cell death has long been defined using morphological criteria. A first important concept, "necrosis", was early identified by Areteo from Cappadocia and by Galen. The term apoptosis was introduced by Kerr in 1972 to indicate a particular form of death in which cells commit suicide by chopping themselves into membrane-bounded apoptotic bodies. Apoptosis is distinguished from necrosis, or accidental cell death, which is characterized by nuclear autolysis and cell disintegration. The aim of this study was an evaluation of the concepts of apoptosis and necrosis, starting from the first definition of cell death by Rudolph Virchow in 1859. In recent years substantial progress has been made in the understanding of apoptotic and necrotic cell death. In particular, cell death researchers have evolved a paradigm change, from one in which apoptosis and necrosis were considered distinct forms of cell demise, to one in which the 2 cell deaths share common features, as an integral part of a same cell death process. Since pure apoptosis and necrosis are only extremes in a continuum spectrum of aponecrotic response, a mixture of features associated with both apoptosis and necrosis represents the more typical tissue and cell response to damaging stimuli.

Ferroptosis and Cell Death Analysis by Flow Cytometry.

PubMed

Chen, Daishi; Eyupoglu, Ilker Y; Savaskan, Nicolai

2017-01-01

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

Recurrent hypoinsulinemic hyperglycemia in neonatal rats increases PARP-1 and NF-κB expression and leads to microglial activation in the cerebral cortex.

PubMed

Gisslen, Tate; Ennis, Kathleen; Bhandari, Vineet; Rao, Raghavendra

2015-11-01

Hyperglycemia is a common metabolic problem in extremely low-birth-weight preterm infants. Neonatal hyperglycemia is associated with increased mortality and brain injury. Glucose-mediated oxidative injury may be responsible. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair and cell survival. However, PARP-1 overactivation leads to cell death. NF-κB is coactivated with PARP-1 and regulates microglial activation. The effects of recurrent hyperglycemia on PARP-1/NF-κB expression and microglial activation are not well understood. Rat pups were subjected to recurrent hypoinsulinemic hyperglycemia of 2 h duration twice daily from postnatal (P) day 3-P12 and killed on P13. mRNA and protein expression of PARP-1/NF-κB and their downstream effectors were determined in the cerebral cortex. Microgliosis was determined using CD11 immunohistochemistry. Recurrent hyperglycemia increased PARP-1 expression confined to the nucleus and without causing PARP-1 overactivation and cell death. NF-κB mRNA expression was increased, while IκB mRNA expression was decreased. inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expressions were decreased. Hyperglycemia significantly increased the number of microglia. Recurrent hyperglycemia in neonatal rats is associated with upregulation of PARP-1 and NF-κB expression and subsequent microgliosis but not neuronal cell death in the cerebral cortex.

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

PubMed

Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-04-06

Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

PubMed

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-09

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

PubMed Central

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-01

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

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

TU-F-CAMPUS-T-04: Using Gold Nanoparticles to Target Mitochondria in Radiation Therapy

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

McNamara, A; McMahon, S; Lin, Y

2015-06-15

Purpose: The mitochondrion, like the cell nucleus, contains genetic material and plays several critical roles that determine the cell viability, including neutralization of free radicals within the cell. Studies have shown that irradiated cells with impaired mitochondria will incur more damage to the cell nucleus. This study investigates the potential use of GNPs to enhance radiation-induced damage to the organelle. Methods: The compositions of the organelles of a JURKAT cell were determined experimentally. Using Monte Carlo simulations, we investigate the significance of dose enhancement in a monoenergetic (10–50 keV and 6 MeV) x-ray irradiated cell cytoplasm, consisting of the experimentallymore » determined composition. We also investigate the track structure of secondary electrons in the mitochondria using Geant4-DNA in the presence and absence of GNPs for incident protons and photons. The biological effect was determined using an approach based on the local effect model, assuming the mitochondrial DNA (mtDNA) was the primary target. Results: Adding 0.01% of gold to the cell cytoplasm material can cause substantial dose enhancement, dependent on the incident x-ray energy. Track structure Monte Carlo (MC) simulations show an increased number of ionization events within the mitochondrion structure. The close proximity of GNPs to the mtDNA storing nucleoid may cause the mtDNA to receive doses above ∼100 Gy for keV x-rays, leading to mitochondrial dysfunction. Conclusion: A substantial increase in ionization events can occur in the mitochondria in the presence of GNPs. If GNPs can be delivered to tumors and attached to a sufficient number of mitochondria inside the tumor cells, mitochondrial induced cell death could be a prevalent cause of cell death. The biological structures developed here will be included in the biological MC toolkit, TOPAS-nBio.« less

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

PubMed

Bol, M; Van Geyt, C; Baert, S; Decrock, E; Wang, N; De Bock, M; Gadicherla, A K; Randon, C; Evans, W H; Beele, H; Cornelissen, R; Leybaert, L

2013-04-01

Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability. Copyright © 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Cell death induced by Morarah and Khaltita in hepatoma cancer cells (Huh-7).

PubMed

Baig, Saeeda; Alamgir, Mohiuddin

2009-10-01

To compare the combined and isolated growth inhibitory effects of Morarah and Khaltita (herbs) on hepatoma cell lines (Huh-7), through induction of apoptosis or necrosis. Comparative controlled in-vitro study. The Molecular Biology Laboratory, The Aga Khan University, Karachi, from June to December 2006. The growth of hepatoma cell lines (Huh-7) was checked by adding Khaltita and Morarah to the cells before culture in a 24 well plate. Six wells were selected and labeled for each of the four variables (controls, Khaltita, Morarah and mixture). After 2 days, cells were studied under an inverted phase contrast microscope and fields were recorded. Approximately four fields per slide of higher intensity were selected randomly to determine the dead cell density, and the procedure was repeated 10 or more times. Frequency and percentages were calculated for dead or alive cells in controls, Morarah, Khaltita and their mixture. Chi-square was used to compare the qualitative variables. P-values < 0.05 were considered significant. Morarah and Khaltita were found to induce statistically significant (p < 0.001) cell death in hepatoma cell lines (Huh-7). At a magnification of 40x, the controls showed 1% dead cells compared to 91% in Morarah, 83% in Khaltita and 73% in combined mixture of Khaltita and Morarah. At magnification of 20x, the controls showed 4% dead cells compared to 44% in Morarah, 47% in Khaltita and 49% in the combined mixture of Khaltita and Morarah. Morarah and Khaltita induced cell death in cultured hepatoma cells (Huh-7).

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.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

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

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as amore » plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.« less

Can deaths in police cells be prevented? Experience from Norway and death rates in other countries.

PubMed

Aasebø, Willy; Orskaug, Gunnar; Erikssen, Jan

2016-01-01

To describe the changes in death rates and causes of deaths in Norwegian police cells during the last 2 decades. To review reports on death rates in police cells that have been published in medical journals and elsewhere, and discuss the difficulties of comparing death rates between countries. Data on deaths in Norwegian police cells were collected retrospectively in 2002 and 2012 for two time periods: 1993-2001 (period 1) and 2003-2012 (period 2). Several databases were searched to find reports on deaths in police cells from as many countries as possible. The death rates in Norwegian police cells reduced significantly from 0.83 deaths per year per million inhabitants (DYM) in period 1 to 0.22 DYM in period 2 (p < 0.05). The most common cause of death in period 1 was alcohol intoxication including intracranial bleeding in persons with high blood alcohol levels, and the number declined from 16 persons in period 1 to 1 person in period 2 (p = 0.032). The median death rate in the surveyed Western countries was 0.44 DYM (range: 0.14-1.46 DYM). The number of deaths in Norwegian police cells reduced by about 75% over a period of approximately 10 years. This is probably mainly due to individuals with severe alcohol intoxication no longer being placed in police cells. However, there remain large methodology difficulties in comparing deaths rates between countries. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem1[W

PubMed Central

Pogány, Miklós; von Rad, Uta; Grün, Sebastian; Dongó, Anita; Pintye, Alexandra; Simoneau, Philippe; Bahnweg, Günther; Kiss, Levente; Barna, Balázs; Durner, Jörg

2009-01-01

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels. PMID:19726575

[Methuosis: a novel type of cell death].

PubMed

Cai, Hongbing; Liu, Jinkun; Fan, Qin; Li, Xin

2013-12-01

Cell death is a major physiological or pathological phenomenon in life activities. The classic forms of cell death include apoptosis, necrosis, and autophagy. Recently, a novel type of cell death has been observed and termed as methuosis, in which excessive stimuli can induce cytoplasmic uptake and accumulation of small bubbles that gradually merge into giant vacuoles, eventually leading to decreased cellular metabolic activity, cell membrane rupture and cell death. In this article, we describe the nomenclature, morphological characteristics and underlying mechanisms of methuosis, compare methuosis with autophagy, oncosis and paraptosis, and review the related researches.

Determination of Death: A Scientific Perspective on Biological Integration

PubMed Central

Condic, Maureen L.

2016-01-01

Human life is operationally defined by the onset and cessation of organismal function. At postnatal stages of life, organismal integration critically and uniquely requires a functioning brain. In this article, a distinction is drawn between integrated and coordinated biologic activities. While communication between cells can provide a coordinated biologic response to specific signals, it does not support the integrated function that is characteristic of a living human being. Determining the loss of integrated function can be complicated by medical interventions (i.e., “life support”) that uncouple elements of the natural biologic hierarchy underlying our intuitive understanding of death. Such medical interventions can allow living human beings who are no longer able to function in an integrated manner to be maintained in a living state. In contrast, medical intervention can also allow the cells and tissues of an individual who has died to be maintained in a living state. To distinguish between a living human being and living human cells, two criteria are proposed: either the persistence of any form of brain function or the persistence of autonomous integration of vital functions. Either of these criteria is sufficient to determine a human being is alive. PMID:27075193

Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1

PubMed Central

Yamanaka, K; Urano, Y; Takabe, W; Saito, Y; Noguchi, N

2014-01-01

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography–mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death. PMID:24407243

Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1.

PubMed

Yamanaka, K; Urano, Y; Takabe, W; Saito, Y; Noguchi, N

2014-01-09

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography-mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death.

Irreversible EGFR Inhibitor EKB-569 Targets Low-LET γ-Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma

PubMed Central

Aravindan, Natarajan; Thomas, Charles R.; Aravindan, Sheeja; Mohan, Aswathi S.; Veeraraghavan, Jamunarani; Natarajan, Mohan

2011-01-01

EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted. PMID:22242139

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells.

PubMed

Woo, Seon Min; Seo, Seung Un; Min, Kyoung-Jin; Im, Seung-Soon; Nam, Ju-Ock; Chang, Jong-Soo; Kim, Shin; Park, Jong-Wook; Kwon, Taeg Kyu

2018-04-27

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants ( N -acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage: the neuroprotective effects of adenosine triphosphate against apoptosis

PubMed Central

Lu, Na; Wang, Baoying; Deng, Xiaohui; Zhao, Honggang; Wang, Yong; Li, Dongliang

2014-01-01

After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury. PMID:25368646

Angiopoietin-1 protects the endothelial cells against advanced glycation end product injury by strengthening cell junctions and inhibiting cell apoptosis.

PubMed

Zhao, Jingling; Chen, Lei; Shu, Bin; Tang, Jinming; Zhang, Lijun; Xie, Julin; Liu, Xusheng; Xu, Yingbin; Qi, Shaohai

2015-08-01

Endothelial dysfunction is a major characteristic of diabetic vasculopathy. Protection of the vascular endothelium is an essential aspect of preventing and treating diabetic vascular complications. Although Angiopoietin-1 (Ang-1) is an important endothelial-specific protective factor, whether Ang-1 protects vascular cells undergoing advanced glycation end product (AGE) injury has not been investigated. The aim of the present study was to determine the potential effects of Ang-1 on endothelial cells after exposure to AGE. We show here that Ang-1 prevented AGE-induced vascular leakage by enhancing the adherens junctions between endothelial cells, and this process was mediated by the phosphorylation and membrane localization of VE-cadherin. Furthermore, Ang-1 also protected endothelial cells from AGE-induced death by regulating phosphatidylinositol 3-kinase (PI3K)/Akt-dependent Bad phosphorylation. Our findings suggest that the novel protective mechanisms of Ang-1 on endothelium are achieved by strengthening endothelial cell junctions and reducing endothelial cell death after AGE injury. © 2014 Wiley Periodicals, Inc.

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.

Impaired removal of Vβ8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim).

PubMed

Leucht, K; Caj, M; Fried, M; Rogler, G; Hausmann, M

2013-09-01

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vβ8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation. © 2013 British Society for Immunology.

Rate-equation modelling and ensemble approach to extraction of parameters for viral infection-induced cell apoptosis and necrosis

NASA Astrophysics Data System (ADS)

Domanskyi, Sergii; Schilling, Joshua E.; Gorshkov, Vyacheslav; Libert, Sergiy; Privman, Vladimir

2016-09-01

We develop a theoretical approach that uses physiochemical kinetics modelling to describe cell population dynamics upon progression of viral infection in cell culture, which results in cell apoptosis (programmed cell death) and necrosis (direct cell death). Several model parameters necessary for computer simulation were determined by reviewing and analyzing available published experimental data. By comparing experimental data to computer modelling results, we identify the parameters that are the most sensitive to the measured system properties and allow for the best data fitting. Our model allows extraction of parameters from experimental data and also has predictive power. Using the model we describe interesting time-dependent quantities that were not directly measured in the experiment and identify correlations among the fitted parameter values. Numerical simulation of viral infection progression is done by a rate-equation approach resulting in a system of "stiff" equations, which are solved by using a novel variant of the stochastic ensemble modelling approach. The latter was originally developed for coupled chemical reactions.

Rate-equation modelling and ensemble approach to extraction of parameters for viral infection-induced cell apoptosis and necrosis

NASA Astrophysics Data System (ADS)

Domanskyi, Sergii; Schilling, Joshua; Gorshkov, Vyacheslav; Libert, Sergiy; Privman, Vladimir

We develop a theoretical approach that uses physiochemical kinetics modelling to describe cell population dynamics upon progression of viral infection in cell culture, which results in cell apoptosis (programmed cell death) and necrosis (direct cell death). Several model parameters necessary for computer simulation were determined by reviewing and analyzing available published experimental data. By comparing experimental data to computer modelling results, we identify the parameters that are the most sensitive to the measured system properties and allow for the best data fitting. Our model allows extraction of parameters from experimental data and also has predictive power. Using the model we describe interesting time-dependent quantities that were not directly measured in the experiment and identify correlations among the fitted parameter values. Numerical simulation of viral infection progression is done by a rate-equation approach resulting in a system of ``stiff'' equations, which are solved by using a novel variant of the stochastic ensemble modelling approach. The latter was originally developed for coupled chemical reactions.

Mechanisms of gender-linked ischemic brain injury

PubMed Central

Liu, Mingyue; Dziennis, Suzan; Hurn, Patricia D.; Alkayed, Nabil J.

2010-01-01

Biological sex is an important determinant of stroke risk and outcome. Women are protected from cerebrovascular disease relative to men, an observation commonly attributed to the protective effect of female sex hormones, estrogen and progesterone. However, sex differences in brain injury persist well beyond the menopause and can be found in the pediatric population, suggesting that the effects of reproductive steroids may not completely explain sexual dimorphism in stroke. We review recent advances in our understanding of sex steroids (estradiol, progesterone and testosterone) in the context of ischemic cell death and neuroprotection. Understanding the molecular and cell-based mechanisms underlying sex differences in ischemic brain injury will lead to a better understanding of basic mechanisms of brain cell death and is an important step toward designing more effective therapeutic interventions in stroke. PMID:19531872

Sulphur antioxidants inhibit oxidative stress induced retinal ganglion cell death by scavenging reactive oxygen species but influence nuclear factor (erythroid-derived 2)-like 2 signalling pathway differently.

PubMed

Majid, Aman Shah Abdul; Yin, Zheng Qin; Ji, Dan

2013-01-01

This study aimed to show if two different sulphur containing drugs sulbutiamine and acetylcysteine (NAC) could attenuate the effects of two different insults being serum deprivation and glutamate/buthionine sulfoximine (GB)-induced death to transformed retinal ganglion cell line (RGC-5) in culture. Cells were exposed to either 5 mM of GB for 24 h or serum deprivation for 48 h with inclusion of either NAC or sulbutiamine. Cell viability, microscopic evidence for apoptosis, caspase 3 activity, reactive oxygen species (ROS), glutathione (GSH), catalase and gluthathione-S-transferase (GST) were determined. The effects of NAC and sulbutiamine on the oxidative stress related transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) levels and its dependent phase II enzyme haemeoxygenase-1 (HO-1) were carried out using Western blot and quantitative-polymerase chain reaction (PCR). NAC and sulbutiamine dose-dependently attenuated serum deprivation-induced cell death. However NAC but not sulbutiamine attenuated GB-induced cell death. NAC and sulbutiamine both independently stimulated the GSH and GST production but scavenged different types of ROS with different efficacy. Moreover only sulbutiamine stimulated catalase and significantly increased Nrf-2 and HO-1 levels. In addition, the pan caspase inhibitor, benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) attenuated the negative effect of serum deprivation while the necroptosis inhibitor (necrostatin-1) counteracted solely an insult of GB. The neuroprotective actions of NAC and sulbutiamine in GB or serum-deprivation insult are therefore different.

Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiae.

PubMed

Kemsawasd, Varongsiri; Branco, Patrícia; Almeida, Maria Gabriela; Caldeira, Jorge; Albergaria, Helena; Arneborg, Nils

2015-07-01

The roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed-culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes, i.e. 1000 kDa for mixed- and single-culture fermentations, and 1000 and 3.5-5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed-culture and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed-culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5-5 kDa than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform.

PubMed

Anilkumar, Ujval; Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J; Düssmann, Heiko; Connolly, Niamh M C; Prehn, Jochen H M

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

PubMed Central

Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J.; Düssmann, Heiko; Connolly, Niamh M. C.; Prehn, Jochen H. M.

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis. PMID:29145487

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

PubMed Central

McPhee, C K; Balgley, B M; Nelson, C; Hill, J H; Batlevi, Y; Fang, X; Lee, C S; Baehrecke, E H

2013-01-01

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. PMID:22935612

Effect of medium/ω-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

PubMed Central

Cury-Boaventura, M F; Gorjão, R; Martins de Lima, T; Fiamoncini, J; Godoy, A B P; Deschamphs, F C; Soriano, F G; Curi, R

2011-01-01

Lipid emulsion (LE) containing medium/ω-6 long chain triglyceride-based emulsion (MCT/ω-6 LCT LE) has been recommended in the place of ω-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/ω-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/ω-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/ω-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/ω-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription. PMID:21682721

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.

Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans

PubMed Central

Coburn, Cassandra; Allman, Erik; Mahanti, Parag; Benedetto, Alexandre; Cabreiro, Filipe; Pincus, Zachary; Matthijssens, Filip; Araiz, Caroline; Mandel, Abraham; Vlachos, Manolis; Edwards, Sally-Anne; Fischer, Grahame; Davidson, Alexander; Pryor, Rosina E.; Stevens, Ailsa; Slack, Frank J.; Tavernarakis, Nektarios; Braeckman, Bart P.; Schroeder, Frank C.; Nehrke, Keith; Gems, David

2013-01-01

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death. PMID:23935448

Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

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

Curry, Merril C.; Peters, Amelia A.; Kenny, Paraic A.

Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levelsmore » of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.« less

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice[W][OA

PubMed Central

Steffens, Bianka; Sauter, Margret

2009-01-01

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H2O2). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ-S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Gα subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H2O2 was nearly completely abolished, indicating that signaling through Gα is essential. Ethylene and H2O2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Gα. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Gα activity is regulated posttranscriptionally. PMID:19656904

Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

PubMed

Klein, Johanna C; Wild, Clarissa A; Lang, Stephan; Brandau, Sven

2016-06-01

Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy.

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

PubMed Central

Osawa, T; Davies, D; Hartley, J A

2011-01-01

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death. PMID:21814285

Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries

PubMed Central

Hallow, Daniel M.; Mahajan, Anuj D.; Prausnitz, Mark R.

2007-01-01

This study tested the hypothesis that ultrasound can target intracellular uptake of drugs into vascular endothelial cells (ECs) at low to intermediate energy and into smooth muscle cells (SMCs) at high energy. Ultrasound-enhanced delivery has been shown to enhance and target intracellular drug and gene delivery in the vasculature to treat cardiovascular disease, but quantitative studies of the delivery process are lacking. Viable ex vivo porcine carotid arteries were placed in a solution containing a model drug, TO-PRO®-1, and Optison® microbubbles. Arteries were exposed to ultrasound at 1.1 MHz and acoustic energies of 5.0, 66, or 630 J/cm2. Using confocal microscopy and fluorescent labeling of cells, the artery endothelium and media were imaged to determine the localization and to quantify intracellular uptake and cell death. At low to intermediate ultrasound energy, ultrasound was shown to target intracellular delivery into viable cells that represented 9 – 24% of exposed ECs. These conditions also typically caused 7 – 25% EC death. At high energy, intracellular delivery was targeted to SMCs, which was associated with denuding or death of proximal ECs. This work represents the first known in-depth study to evaluate intracellular uptake into cells in tissue. We conclude that significant intracellular uptake of molecules can be targeted into ECs and SMCs by ultrasound-enhanced delivery suggesting possible applications for treatment of cardivascular diseases and dysfunctions. PMID:17291619

In vitro efficiency and mechanistic role of indocyanine green as photodynamic therapy agent for human melanoma

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

Mamoon, A.M.; Miller, L.; Gamal-Eldeen, A. M.

2009-05-02

Photodynamic therapy (PDT) is a promising treatment for superficial cancer. However, poor therapeutic results have been reported for melanoma, due to the high melanin content. Indocyanine green (ICG) has near infrared absorption (700-800 nm) and melanins do not absorb strongly in this area. This study explores the efficiency of ICG as a PDT agent for human melanoma, and its mechanistic role in the cell death pathway. Human skin melanoma cells (Sk-Mel-28) were incubated with ICG and exposed to a low power Ti:Sapphire laser. Synchrotron-assisted Fourier transform infrared microspectroscopy and hierarchical cluster analysis were used to assess the cell damage andmore » changes in lipid, protein, and nucleic acids. The cell death pathway was determined by analysis of cell viability and apoptosis and necrosis markers. In the cell death pathway, {sup 1}O{sub 2} generation evoked rapid multiple consequences that trigger apoptosis after laser exposure for only 15min including the release of cytochrome c, the activation of total caspases, caspase-3, and caspase-9, the inhibition of NF-{Kappa}B P65, and the enhancement of DNA fragmentation, and histone acetylation. ICG/PDT can efficiently and rapidly induce apoptosis in human melanoma cells and it can be considered as a new therapeutic approach for topical treatment of melanoma.« less

Chrysotoxine, a novel bibenzyl compound selectively antagonizes MPP⁺, but not rotenone, neurotoxicity in dopaminergic SH-SY5Y cells.

PubMed

Song, Ju-Xian; Shaw, Pang-Chui; Wong, Ngok-Shun; Sze, Cho-Wing; Yao, Xin-Sheng; Tang, Chi-Wai; Tong, Yao; Zhang, Yan-Bo

2012-07-11

Chrysotoxine is a naturally occurring bibenzyl compound found in medicinal Dendrobium species. We previously reported that chrysotoxine structure-specifically suppressed 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death. Whether chrysotoxine and other structurally similar bibenzyl compounds could also inhibit the neurotoxicity of 1-methyl-4-phenyl pyridinium (MPP(+)) and rotenone has not been investigated. We showed herein that chrysotoxine inhibited MPP(+), but not rotenone, induced dopaminergic cell death in SH-SY5Y cells. The overproduction of reactive oxygen species (ROS), mitochondrial dysfunction as indexed by the decrease in membrane potential, increase in calcium concentration and NF-κB activation triggered by MPP(+) were blocked by chrysotoxine pretreatment. The imbalance between the pro-apoptotic signals (Bax, caspase-3, ERK and p38 MAPK) and the pro-survival signals (Akt/PI3K/GSK-3β) induced by MPP(+) was partially or totally rectified by chrysotoxine. The results indicated that ROS inhibition, mitochondria protection, NF-κB modulation and regulation of multiple signals determining cell survival and cell death were involved in the protective effects of chrysotoxine against MPP(+) toxicity in SH-SY5Y cells. Given the different toxic profiles of 6-OHDA and MPP(+) as compared to rotenone, our results also indicated that DAT inhibition may partially account for the neuroprotective effects of chrysotoxine. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

Inhibitory effects of mouse bone marrow mesenchymal stem cell soup on staurospurine-induced cell death in MCF-7 and AGS.

PubMed

Zhaleh, M; Azadbakht, M; Bidmeshki Pour, A

2017-01-01

Staurospurine induces apoptosis in cell line. Bone Marrow Mesenchymal stem cells Soup is a promising tool for cell proliferation via a variety of secreted factors. In this study, we examined the effects of BMSCs Soup on Staurospurine induced-cell death in MCF-7 and AGS cells. There were three Groups: Group I: no incubation with BM Soup; Group II: incubated with 24 h BM Soup; Group III: incubation with 48 h BM Soup. There were two treatments in each group. The treatments were 1μM Staurospurine (Treatment 1) and 0.0 μM Staurospurine (Treatment 2). The cells were cultured in culture medium containing 0.2 % BSA. We obtained the cell viability, cell death and NO concentration. Our results showed that BM soup administration for 48 hours protectsed against 1μM staurosporine concentration induced cell death and reduced cell toxicity in MCF-7 and AGS cells. Cell viability and cell toxicity assay showed that BM soup in time dependent manner increased cell viability (p < 0.05) and cell death assay showed that cell death in time dependent manner was decreased(p < 0.05). Our data showed that BM soup with increasing NO concentration reduced staurospurine induced cell death and cell cytotoxicity (p < 0.05). It's concluded that BMSCs soup suppressed staurospurine-induced cytotoxicity activity process in MCF-7 and AGS cells (Fig. 9, Ref. 79).

Determinants of suicide and accidental or violent death in the Australian HIV Observational Database.

PubMed

McManus, Hamish; Petoumenos, Kathy; Franic, Teo; Kelly, Mark D; Watson, Jo; O'Connor, Catherine C; Jeanes, Mark; Hoy, Jennifer; Cooper, David A; Law, Matthew G

2014-01-01

Rates of suicide and accidental or violent death remain high in HIV-positive populations despite significantly improved prognosis since the introduction of cART. We conducted a nested case-control study of suicide and accidental or violent death in the Australian HIV Observational Database (AHOD) between January 1999 and March 2012. For each case, 2 controls were matched by clinic, age, sex, mode of exposure and HIV-positive date to adjust for potential confounding by these covariates. Risk of suicide and accidental or violent death was estimated using conditional logistic regression. We included 27 cases (17 suicide and 10 violent/accidental death) and 54 controls. All cases were men who have sex with men (MSM) or MSM/ injecting drug use (IDU) mode of exposure. Increased risk was associated with unemployment (Odds Ratio (OR) 5.86, 95% CI: 1.69-20.37), living alone (OR 3.26, 95% CI: 1.06-10.07), suicidal ideation (OR 6.55, 95% CI: 1.70-25.21), and >2 psychiatric/cognitive risk factors (OR 4.99, 95% CI: 1.17-30.65). CD4 cell count of >500 cells/µL (OR 0.25, 95% CI: 0.07-0.87) and HIV-positive date ≥1990 (1990-1999 (OR 0.31, 95% CI: 0.11-0.89), post-2000 (OR 0.08, 95% CI: 0.01-0.84)) were associated with decreased risk. CD4 cell count ≥500 cells/µL remained a significant predictor of reduced risk (OR 0.15, 95% CI: 0.03-0.70) in a multivariate model adjusted for employment status, accommodation status and HIV-positive date. After adjustment for psychosocial factors, the immunological status of HIV-positive patients contributed to the risk of suicide and accidental or violent death. The number of psychiatric/cognitive diagnoses contributed to the level of risk but many psychosocial factors were not individually significant. These findings indicate a complex interplay of factors associated with risk of suicide and accidental or violent death.

Die Another Day: Inhibition of Cell Death Pathways by Cytomegalovirus.

PubMed

Brune, Wolfram; Andoniou, Christopher E

2017-09-02

Multicellular organisms have evolved multiple genetically programmed cell death pathways that are essential for homeostasis. The finding that many viruses encode cell death inhibitors suggested that cellular suicide also functions as a first line of defence against invading pathogens. This theory was confirmed by studying viral mutants that lack certain cell death inhibitors. Cytomegaloviruses, a family of species-specific viruses, have proved particularly useful in this respect. Cytomegaloviruses are known to encode multiple death inhibitors that are required for efficient viral replication. Here, we outline the mechanisms used by the host cell to detect cytomegalovirus infection and discuss the methods employed by the cytomegalovirus family to prevent death of the host cell. In addition to enhancing our understanding of cytomegalovirus pathogenesis we detail how this research has provided significant insights into the cross-talk that exists between the various cell death pathways.

Reagents that block neuronal death from Huntington's disease also curb oxidative stress.

PubMed

Valencia, Antonio; Sapp, Ellen; Reeves, Patrick B; Alexander, Jonathan; Masso, Nicholas; Li, Xueyi; Kegel, Kimberly B; DiFiglia, Marian

2012-01-04

Patients with Huntington's disease suffer severe neuronal loss and signs of oxidative damage in the brain. Previously we found that primary neurons from embryonic cortex of mice bearing the Huntington's disease mutation (140 glutamines inserted into exon 1 of huntingtin) showed higher levels of reactive oxygen species before cell death. Here, we treated mutant neurons with known neuroprotective agents and determined the effects on neuronal survival and levels of reactive oxygen species. Primary neurons were exposed to the neurotrophin, brain derived neurotrophic factor, the antioxidant N-acetyl-cysteine or a specific inhibitor of glycogen synthase kinase 3-β, SB216763. Each reagent increased the survival of the mutant neurons compared with untreated mutant neurons and also reduced the levels of reactive oxygen species to levels of wild-type neurons. These results suggest that reducing the levels of reactive oxygen species may be necessary to protect neurons with the Huntington's disease mutation from cell death.

Comparison of Types of Cell Death: Apoptosis and Necrosis.

ERIC Educational Resources Information Center

Manning, Francis; Zuzel, Katherine

2003-01-01

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

ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death

PubMed Central

Kuriakose, Teneema; Malireddi, R.K. Subbarao; Mishra, Ashutosh

2017-01-01

Innate sensing of influenza virus infection induces activation of programmed cell death pathways. We have recently identified Z-DNA–binding protein 1 (ZBP1) as an innate sensor of influenza A virus (IAV). ZBP1-mediated IAV sensing is critical for triggering programmed cell death in the infected lungs. Surprisingly, little is known about the mechanisms regulating ZBP1 activation to induce programmed cell death. Here, we report that the sensing of IAV RNA by retinoic acid inducible gene I (RIG-I) initiates ZBP1-mediated cell death via the RIG-I–MAVS–IFN-β signaling axis. IAV infection induces ubiquitination of ZBP1, suggesting potential regulation of ZBP1 function through posttranslational modifications. We further demonstrate that ZBP1 senses viral ribonucleoprotein (vRNP) complexes of IAV to trigger cell death. These findings collectively indicate that ZBP1 activation requires RIG-I signaling, ubiquitination, and vRNP sensing to trigger activation of programmed cell death pathways during IAV infection. The mechanism of ZBP1 activation described here may have broader implications in the context of virus-induced cell death. PMID:28634194

Apoptotic activities of cardenolide glycosides from Asclepias subulata.

PubMed

Rascón-Valenzuela, L A; Velázquez, C; Garibay-Escobar, A; Vilegas, W; Medina-Juárez, L A; Gámez-Meza, N; Robles-Zepeda, R E

2016-12-04

Asclepias subulata Decne. (Apocynaceae) is a shrub occurring in Sonora-Arizona desert. The ethnic groups of Sonora, Mexico, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. To determine the cell death pathways that the cardenolide glycosides with antiproliferative activity found in the methanol extract of A. subulata are able to activate. The effect of cardenolide glycosides isolated of A. subulata on induction of apoptosis in cancer cells was evaluated through the measuring of several key events of apoptosis. A549 cells were treated for 12h with doses of 3.0, 0.2, 3.0 and 1.0µM of 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively. Apoptotic and necrotic cell levels were measured by double staining with annexin V-FITC/PI. Mitochondrial membrane depolarization was examined through JC-1 staining. Apoptosis cell death and the apoptosis pathways activated by cardenolide glycosides isolated of A. subulata were further characterized by the measurement of caspase-3, caspase-8 and caspase-9 activity. Apoptotic assays showed that the four cardenolide glycosides isolated of A. subulata induced apoptosis in A549 cells, which was evidencing by phosphatidylserine externalization in 18.2%, 17.0%, 23.9% and 22.0% for 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively, compared with 4.6% of control cells. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more than 75% in the treated cultures respect to control. The activation of caspase-3 was observed in all cardenolide glycosides-treated cancer cells indicating the caspase-dependent apoptosis of A549 cells. Extrinsic and intrinsic apoptosis pathways were activated by cardenolide glycosides treatment at the doses tested. In this study was found that cardenolide glycosides, 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, isolated from A. subulata induced the cell death trough caspase-dependent apoptosis, which was activated, preferably, by extrinsic pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

PubMed Central

Pele, Laetitia; Haas, Carolin T; Hewitt, Rachel; Faria, Nuno; Brown, Andy; Powell, Jonathan

2015-01-01

Aim To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. Material & methods The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Results Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. Conclusion In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes. PMID:24991724

THE EFFECT OF TUNGSTATE NANOPARTICLES ON REACTIVE OXYGEN SPECIES AND CYTOTOXICITY IN RAW 264.7 MOUSE MONOCYTE MACROPHAGE CELLS

PubMed Central

Dunnick, Katherine M.; Badding, Melissa A.; Schwegler-Berry, Diane; Patete, Jonathan M.; Koenigsmann, Christopher; Wong, Stanislaus S.; Leonard, Stephen S.

2015-01-01

Due to their unique size, surface area, and chemical characteristics, nanoparticles’ use in consumer products has increased. However, the toxicity of nanoparticle (NP) exposure during the manufacturing process has not been fully assessed. Tungstate NP are used in numerous products, including but not limited to scintillator detectors and fluorescent lighting. As with many NP, no apparent toxicity studies have been completed with tungstate NP. The hypothesis that tungstate NP in vitro exposure results in reactive oxygen species (ROS) formation and cytotoxicity was examined. Differences in toxicity based on tungstate NP size, shape (sphere vs. wire), and chemical characteristics were determined. RAW 264.7 mouse monocyte macrophages were exposed to tungstate NP, and ROS formation was assessed via electron spin resonance (ESR), and several assays including hydrogen peroxide, intracellular ROS, and Comet. Results showed ROS production induced by tungstate nanowire exposure, but this exposure did not result in oxidative DNA damage. Nanospheres showed neither ROS nor DNA damage following cellular exposure. Cells were exposed over 72 h to assess cytotoxicity using an MTT (tetrazolium compound) assay. Results showed that differences in cell death between wires and spheres occurred at 24 h but were minimal at both 48 and 72 h. The present results indicate that tungstate nanowires are more reactive and produce cell death within 24 h of exposure, whereas nanospheres are less reactive and did not produce cell death. Results suggest that differences in shape may affect reactivity. However, regardless of the differences in reactivity, in general both shapes produced mild ROS and resulted in minimal cell death at 48 and 72 h in RAW 264.7 cells. PMID:25208664

Hepatitis B Vaccine Antibody Response and the Risk of Clinical AIDS or Death

PubMed Central

Landrum, Michael L.; Hullsiek, Katherine Huppler; O'Connell, Robert J.; Chun, Helen M.; Ganesan, Anuradha; Okulicz, Jason F.; Lalani, Tahaniyat; Weintrob, Amy C.; Crum-Cianflone, Nancy F.; Agan, Brian K.

2012-01-01

Background Whether seroresponse to a vaccine such as hepatitis B virus (HBV) vaccine can provide a measure of the functional immune status of HIV-infected persons is unknown.This study evaluated the relationship between HBV vaccine seroresponses and progression to clinical AIDS or death. Methods and Findings From a large HIV cohort, we evaluated those who received HBV vaccine only after HIV diagnosis and had anti-HBs determination 1–12 months after the last vaccine dose. Non-response and positive response were defined as anti-HBs <10 and ≥10 IU/L, respectively. Participants were followed from date of last vaccination to clinical AIDS, death, or last visit. Univariate and multivariable risk of progression to clinical AIDS or death were evaluated with Cox regression models. A total of 795 participants vaccinated from 1986–2010 were included, of which 41% were responders. During 3,872 person-years of observation, 122 AIDS or death events occurred (53% after 1995). Twenty-two percent of non-responders experienced clinical AIDS or death compared with 5% of responders (p<0.001). Non-response to HBV vaccine was associated with a greater than 2-fold increased risk of clinical AIDS or death (HR 2.47; 95% CI, 1.38–4.43) compared with a positive response, after adjusting for CD4 count, HIV viral load, HAART use, and delayed type hypersensitivity skin test responses (an in vivo marker of cell-mediated immunity). This association remained evident among those with CD4 count ≥500 cells/mm3 (HR 3.40; 95% CI, 1.39–8.32). Conclusions HBV vaccine responses may have utility in assessing functional immune status and risk stratificating HIV-infected individuals, including those with CD4 count ≥500 cells/mm3. PMID:22457767

Cell Size Influences the Reproductive Potential and Total Lifespan of the Saccharomyces cerevisiae Yeast as Revealed by the Analysis of Polyploid Strains.

PubMed

Zadrag-Tecza, Renata; Kwolek-Mirek, Magdalena; Alabrudzińska, Małgorzata; Skoneczna, Adrianna

2018-01-01

The total lifespan of the yeast Saccharomyces cerevisiae may be divided into two phases: the reproductive phase, during which the cell undergoes mitosis cycles to produce successive buds, and the postreproductive phase, which extends from the last division to cell death. These phases may be regulated by a common mechanism or by distinct ones. In this paper, we proposed a more comprehensive approach to reveal the mechanisms that regulate both reproductive potential and total lifespan in cell size context. Our study was based on yeast cells, whose size was determined by increased genome copy number, ranging from haploid to tetraploid. Such experiments enabled us to test the hypertrophy hypothesis, which postulates that excessive size achieved by the cell-the hypertrophy state-is the reason preventing the cell from further proliferation. This hypothesis defines the reproductive potential value as the difference between the maximal size that a cell can reach and the threshold value, which allows a cell to undergo its first cell cycle and the rate of the cell size to increase per generation. Here, we showed that cell size has an important impact on not only the reproductive potential but also the total lifespan of this cell. Moreover, the maximal cell size value, which limits its reproduction capacity, can be regulated by different factors and differs depending on the strain ploidy. The achievement of excessive size by the cell (hypertrophic state) may lead to two distinct phenomena: the cessation of reproduction without "mother" cell death and the cessation of reproduction with cell death by bursting, which has not been shown before.

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

PubMed

Gröbner, Sabine; Fritz, Evelyn; Schoch, Friederike; Schaller, Martin; Berger, Alexander C; Bitzer, Michael; Autenrieth, Ingo B

2010-10-01

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential DeltaPsi(m). Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.

Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells.

PubMed

Morimoto, Satoshi; Tanaka, Yumi; Sasaki, Kaori; Tanaka, Hiroyuki; Fukamizu, Tomohide; Shoyama, Yoshinari; Shoyama, Yukihiro; Taura, Futoshi

2007-07-13

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Delta(1)-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca(2+) and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

Inhibition of ERK activity enhances the cytotoxic effect of peroxisome proliferator-activated receptor γ (PPARγ) agonists in HeLa cells

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

Chang, Ha Kyun; Kim, Dae Seong; Chae, Jung Jun

In this study, we examined whether the peroxisome proliferator-activated receptor γ (PPARγ) agonists, ciglitazone (CGZ) and troglitazone (TGZ), induce cell death in human cervical cancer HeLa cells. The cells were treated with a range of CGZ or TGZ doses for 24 or 48 h. Low concentrations of CGZ (≤10 μM) or TGZ (≤20 μM) had no effect on cell viability whereas higher doses induced cell death in a time- and dose-dependent manner as evidenced by the detection of activated caspase-3 and PARP cleavage. Treatment with the PPARγ antagonist GW9662 followed by PPARγ agonists did not increase CGZ- or TGZ-induced cell death, indicating thatmore » PPARγ agonists induced HeLa cell death independently of PPARγ. Moreover, ERK1/2 activation was observed at a CGZ concentration of 25 μM and a TGZ concentration of 35 μM, both of which induced cell death. To elucidate the role of ERK1/2 activated by the two PPARγ agonists, the effect of U0126, an inhibitor of ERK1/2, on PPARγ-agonist-induced cell death was examined. Treatment with 10 or 20 μM U0126 followed by CGZ or TGZ induced the down-regulation of ERK1/2 activity and a decrease in Bcl-2 expression accompanied by the collapse of mitochondrial membrane potential, which in turn significantly enhanced CGZ- or TGZ-induced apoptotic cell death. Our results suggest that PPARγ agonists are capable of inducing apoptotic cell death in HeLa cells independently of PPARγ and that inhibition of ERK1/2 activity offers a strategy to enhance the cytotoxicity of PPARγ agonists in the treatment of cervical cancer. - Highlights: • The PPARγ agonists CGZ and TGZ induce apoptotic cell death in HeLa cells. • CGZ or TGZ induces apoptotic cell death independently of PPARγ in HeLa cells. • Inhibition of ERK1/2 enhances CGZ- or TGZ-induced cell death via the collapse of MMP.« less

Regulation of apoptosis by low serum in cells of different stages of neoplastic progression: enhanced susceptibility after loss of a senescence gene and decreased susceptibility after loss of a tumor suppressor gene.

PubMed

Preston, G A; Lang, J E; Maronpot, R R; Barrett, J C

1994-08-01

A cell culture model system has been used to study the susceptibility of cells to apoptotic cell death during different stages of neoplastic progression. This system consists of normal diploid Syrian hamster embryo (SHE) cells, two preneoplastic cell lines [tumor suppressor stage I (sup +I) and non-tumor suppressor stage II (sup -II)], and hamster tumor cell lines. Stage I preneoplastic cells are nontumorigenic immortal clones that suppress tumorigenicity when hybridized to tumor cells, whereas stage II cells have lost the ability to suppress tumorigenicity in cell hybrids. We refer to these two types of preneoplastic cells as sup +I and sup -II, respectively. Neoplastic progression is generally associated with cellular alterations in growth factor responsiveness. Therefore, to study the regulation of apoptosis in the system described above, cells were cultured in low serum (0.2%) as a means of withdrawing growth factors. In low serum, normal SHE cells were quiescent (labeling index of 0.2%), with little cell death. The sup +I cells showed a relatively low labeling index (1.6%) but, in contrast to the normal cells, died at a high rate (55% cell loss after 48 h) by apoptosis, as evidenced by morphology, DNA fragmentation, and in situ end-labeling of fragmented DNA. The apoptotic cells did not go through a replicative cycle while in low serum, implying that apoptosis was initiated in the G0/G1 phase of the cell cycle. The sup -II cell line showed a high labeling index (40%) after 48 h, but cell growth was balanced by cell death that occurred at approximately the same rate. The cells died, however, predominantly by necrosis. The tumor cell lines continued to proliferate in low serum, with high labeling indices (ranging from 27% to 43%) and a low level of apoptotic or necrotic cell death. To determine the relative ability of these cells to survive in vivo, normal SHE cells, sup +I cells, and sup -II cells were injected s.c. into nude mice. At 5 or 21 days after injection, the normal SHE cells were readily retrieved from the mice and grew well in culture. In contrast, few sup +I cells were retrieved 5 days after injection and no viable cells were retrieved after 21 days. Sup -II cells were not retrieved at either the 5-day or 21-day harvest, and histological examinations of the sites of injection showed the presence of macrophages, eosinophils, and neutrophils, indicating an inflammatory response associated with necrotic cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Detrimental effects of discectomy on intervertebral disc biology can be decelerated by growth factor treatment during surgery: a large animal organ culture model.

PubMed

Illien-Jünger, Svenja; Lu, Young; Purmessur, Devina; Mayer, Jillian E; Walter, Benjamin A; Roughley, Peter J; Qureshi, Sheeraz A; Hecht, Andrew C; Iatridis, James C

2014-11-01

Lumbar discectomies are common surgical interventions that treat radiculopathy by removing herniated and loose intervertebral disc (IVD) tissues. However, remaining IVD tissue can continue to degenerate resulting in long-term clinical problems. Little information is available on the effects of discectomy on IVD biology. Currently, no treatments exist that can suspend or reverse the degeneration of the remaining IVD. To improve the knowledge on how discectomy procedures influence IVD physiology and to assess the potential of growth factor treatment as an augmentation during surgery. To determine effects of discectomy on IVDs with and without transforming growth factor beta 3 (TGFβ3) augmentation using bovine IVD organ culture. This study determined effects of discectomy with and without TGFβ3 injection using 1-, 6-, and 19-day organ culture experiments. Treated IVDs were injected with 0.2 μg TGFβ3 in 20 μL phosphate-buffered saline+bovine serum albumin into several locations of the discectomy site. Cell viability, gene expression, nitric oxide (NO) release, IVD height, aggrecan degradation, and proteoglycan content were determined. Discectomy significantly increased cell death, aggrecan degradation, and NO release in healthy IVDs. Transforming growth factor beta 3 injection treatment prevented or mitigated these effects for the 19-day culture period. Discectomy procedures induced cell death, catabolism, and NO production in healthy IVDs, and we conclude that post-discectomy degeneration is likely to be associated with cell death and matrix degradation. Transforming growth factor beta 3 injection augmented discectomy procedures by acting to protect IVD tissues by maintaining cell viability, limiting matrix degradation, and suppressing NO. We conclude that discectomy procedures can be improved with injectable therapies at the time of surgery although further in vivo and human studies are required. Copyright © 2014 Elsevier Inc. All rights reserved.

Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary.

PubMed

Tanner, Elizabeth A; Blute, Todd A; Brachmann, Carrie Baker; McCall, Kimberly

2011-01-01

The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.

Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome

PubMed Central

Takemura, Naoki; Kawasaki, Takumi; Kunisawa, Jun; Sato, Shintaro; Lamichhane, Aayam; Kobiyama, Kouji; Aoshi, Taiki; Ito, Junichi; Mizuguchi, Kenji; Karuppuchamy, Thangaraj; Matsunaga, Kouta; Miyatake, Shoichiro; Mori, Nobuko; Tsujimura, Tohru; Satoh, Takashi; Kumagai, Yutaro; Kawai, Taro; Standley, Daron M.; Ishii, Ken J.; Kiyono, Hiroshi; Akira, Shizuo; Uematsu, Satoshi

2014-01-01

High-dose ionizing radiation induces severe DNA damage in the epithelial stem cells in small intestinal crypts and causes gastrointestinal syndrome (GIS). Although the tumour suppressor p53 is a primary factor inducing death of crypt cells with DNA damage, its essential role in maintaining genome stability means inhibiting p53 to prevent GIS is not a viable strategy. Here we show that the innate immune receptor Toll-like receptor 3 (TLR3) is critical for the pathogenesis of GIS. Tlr3−/− mice show substantial resistance to GIS owing to significantly reduced radiation-induced crypt cell death. Despite showing reduced crypt cell death, p53-dependent crypt cell death is not impaired in Tlr3−/− mice. p53-dependent crypt cell death causes leakage of cellular RNA, which induces extensive cell death via TLR3. An inhibitor of TLR3–RNA binding ameliorates GIS by reducing crypt cell death. Thus, we propose blocking TLR3 activation as a novel approach to treat GIS. PMID:24637670

Drug-induced cellular death dynamics monitored by a highly sensitive organic electrochemical system.

PubMed

Romeo, Agostino; Tarabella, Giuseppe; D'Angelo, Pasquale; Caffarra, Cristina; Cretella, Daniele; Alfieri, Roberta; Petronini, Pier Giorgio; Iannotta, Salvatore

2015-06-15

We propose and demonstrate a sensitive diagnostic device based on an Organic Electrochemical Transistor (OECT) for direct in-vitro monitoring cell death. The system efficiently monitors cell death dynamics, being able to detect signals related to specific death mechanisms, namely necrosis or early/late apoptosis, demonstrating a reproducible correlation between the OECT electrical response and the trends of standard cell death assays. The innovative design of the Twell-OECT system has been modeled to better correlate electrical signals with cell death dynamics. To qualify the device, we used a human lung adenocarcinoma cell line (A549) that was cultivated on the micro-porous membrane of a Transwell (Twell) support, and exposed to the anticancer drug doxorubicin. Time-dependent and dose-dependent dynamics of A549 cells exposed to doxorubicin are evaluated by monitoring cell death upon exposure to a range of doses and times that fully covers the protocols used in cancer treatment. The demonstrated ability to directly monitor cell stress and death dynamics upon drug exposure using simple electronic devices and, possibly, achieving selectivity to different cell dynamics is of great interest for several application fields, including toxicology, pharmacology, and therapeutics. Copyright © 2015 Elsevier B.V. All rights reserved.

Programming stress-induced altruistic death in engineered bacteria

PubMed Central

Tanouchi, Yu; Pai, Anand; Buchler, Nicolas E; You, Lingchong

2012-01-01

Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is ‘altruistic': the killing of some cells can benefit the survivors through release of ‘public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, we determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. We further predicted that altruistic death could generate the ‘Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, we experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. Our findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment. PMID:23169002

The engulfment receptor Draper is required for autophagy during cell death.

PubMed

McPhee, Christina K; Baehrecke, Eric H

2010-11-01

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction: during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.

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.

Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells.

PubMed

Matsuo, Taisuke; Sadzuka, Yasuyuki

2018-02-19

In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Exosomal microRNA Signatures in the Diagnosis and Prognosis of Ovarian Cancer

DTIC Science & Technology

2012-04-01

released exosomes modulate lymphocyte functions by mimicking “activation induced cell death” (AICD).27,28 Lymphoid cells appear to release exosomes... advantage of defining concentration. The disadvantage of SPA and DLS is that they are unable to determine the phenotype of the vesicles. Since biological...related apoptosis- inducing ligand-carrying microvesicles during activation- induced death of human T cells . J. Immunol. 167:6736-6744. 18. Raposo, G., D

Apoptotic effect of the selective PPARβ/δ agonist GW501516 in invasive bladder cancer cells.

PubMed

Péchery, Adeline; Fauconnet, Sylvie; Bittard, Hugues; Lascombe, Isabelle

2016-11-01

GW501516 is a selective and high-affinity synthetic agonist of peroxisome proliferator-activated receptor β/δ (PPARβ/δ). This molecule promoted the inhibition of proliferation and apoptosis in few cancer cell lines, but its anticancer action has never been investigated in bladder tumor cells. Thus, this study was undertaken to determine whether GW501516 had antiproliferative and/or apoptotic effects on RT4 and T24 urothelial cancer cells and to explore the molecular mechanisms involved. Our results indicated that, in RT4 cells (derived from a low-grade papillary tumor), GW501516 did not induce cell death. On the other hand, in T24 cells (derived from an undifferentiated high-grade carcinoma), this PPARβ/δ agonist induced cytotoxic effects including cell morphological changes, a decrease of cell viability, a G2/M cell cycle arrest, and the cell death as evidenced by the increase of the sub-G1 cell population. Furthermore, GW501516 triggered T24 cell apoptosis in a caspase-dependent manner including both extrinsic and intrinsic apoptotic pathways through Bid cleavage. In addition, the drug led to an increase of the Bax/Bcl-2 ratio, a mitochondrial dysfunction associated with the dissipation of ΔΨm, and the release of cytochrome c from the mitochondria to the cytosol. GW501516 induced also ROS generation which was not responsible for T24 cell death since NAC did not rescue cells upon PPARβ/δ agonist exposure. For the first time, our data highlight the capacity of GW501516 to induce apoptosis in invasive bladder cancer cells. This molecule could be relevant as a therapeutic drug for high-grade urothelial cancers.

Cell Size Influences the Reproductive Potential and Total Lifespan of the Saccharomyces cerevisiae Yeast as Revealed by the Analysis of Polyploid Strains

PubMed Central

Kwolek-Mirek, Magdalena; Alabrudzińska, Małgorzata

2018-01-01

The total lifespan of the yeast Saccharomyces cerevisiae may be divided into two phases: the reproductive phase, during which the cell undergoes mitosis cycles to produce successive buds, and the postreproductive phase, which extends from the last division to cell death. These phases may be regulated by a common mechanism or by distinct ones. In this paper, we proposed a more comprehensive approach to reveal the mechanisms that regulate both reproductive potential and total lifespan in cell size context. Our study was based on yeast cells, whose size was determined by increased genome copy number, ranging from haploid to tetraploid. Such experiments enabled us to test the hypertrophy hypothesis, which postulates that excessive size achieved by the cell—the hypertrophy state—is the reason preventing the cell from further proliferation. This hypothesis defines the reproductive potential value as the difference between the maximal size that a cell can reach and the threshold value, which allows a cell to undergo its first cell cycle and the rate of the cell size to increase per generation. Here, we showed that cell size has an important impact on not only the reproductive potential but also the total lifespan of this cell. Moreover, the maximal cell size value, which limits its reproduction capacity, can be regulated by different factors and differs depending on the strain ploidy. The achievement of excessive size by the cell (hypertrophic state) may lead to two distinct phenomena: the cessation of reproduction without “mother” cell death and the cessation of reproduction with cell death by bursting, which has not been shown before. PMID:29743970

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii.

PubMed

Du, Zhi-Qiang; Lan, Jiang-Feng; Weng, Yu-Ding; Zhao, Xiao-Fan; Wang, Jin-Xing

2013-07-01

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish. Copyright © 2013 Elsevier Ltd. All rights reserved.

Opioid Use after TBI

DTIC Science & Technology

2013-07-01

hippocampal formation. b. Cresyl violet histochemistry Cresyl violet histological processing of tissue stains Nissl substance, which is composed mostly of...for:  Reactive glial response is being determined by measuring the luminance intensity of GFAP staining  Necrotic and apoptotic cell death by

15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} induces renal epithelial cell death through NF-{kappa}B-dependent and MAPK-independent mechanism

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

Kang, Dae Sik; Kwon, Chae Hwa; Park, Ji Yeon

2006-11-01

The peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) ligand 15d-PGJ{sub 2} induces cell death in renal proximal tubular cells. However, the underlying molecular mechanism(s) remains unidentified. The present study was undertaken to examine the roles of reactive oxygen species (ROS), mitogen-activated protein kinase, and NF-{kappa}B in opossum kidney (OK) cell death induced by 15d-PGJ{sub 2}. Treatment of OK cells with 15d-PGJ{sub 2} resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. 15d-PGJ{sub 2} increased ROS production and the effect was inhibited by catalase and N-acetylcysteine. The 15d-PGJ{sub 2}-induced cell death was also prevented by these antioxidants, suggesting thatmore » the cell death was associated with ROS generation. The PPAR{gamma} antagonist GW9662 did not prevent the 15d-PGJ{sub 2}-induced cell death. 15d-PGJ{sub 2} caused a transient activation of extracellular signal-regulated kinase (ERK). However, inhibitors (PD98059 and U0126) of MEK, an ERK upstream kinase, did not alter the 15d-PGJ{sub 2}-induced cell death. Transfection with constitutively active MEK and dominant-negative MEK had no effect on the cell death. 15d-PGJ{sub 2} inhibited the NF-{kappa}B transcriptional activity, which was accompanied by an inhibition of nuclear translocation of the NF-{kappa}B subunit p65 and impairment in DNA binding. Inhibition of NF-{kappa}B with a NF-{kappa}B specific inhibitor pyrrolidinecarbodithioate and transfection with I{kappa}B{alpha} (S32A/36A) caused cell death. These results suggest that the 5d-PGJ{sub 2}-induced OK cell death was associated with ROS production and NF-{kappa}B inhibition, but not with MAPK activation.« less

Sorafenib-induced defective autophagy promotes cell death by necroptosis.

PubMed

Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Björklund, Ann-Charlotte; Zhivotovsky, Boris; Grandér, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

2015-11-10

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5.

PubMed

Chen, Linjie; Wolff, Dennis W; Xie, Yan; Lin, Ming-Fong; Tu, Yaping

2017-03-07

Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported. Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay. Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells. Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

Early Events in Retinal Degeneration Caused by Rhodopsin Mutation or Pigment Epithelium Malfunction: Differences and Similarities

PubMed Central

Di Pierdomenico, Johnny; García-Ayuso, Diego; Pinilla, Isabel; Cuenca, Nicolás; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta; Villegas-Pérez, María P.

2017-01-01

To study the course of photoreceptor cell death and macro and microglial reactivity in two rat models of retinal degeneration with different etiologies. Retinas from P23H-1 (rhodopsin mutation) and Royal College of Surgeon (RCS, pigment epithelium malfunction) rats and age-matched control animals (Sprague-Dawley and Pievald Viro Glaxo, respectively) were cross-sectioned at different postnatal ages (from P10 to P60) and rhodopsin, L/M- and S-opsin, ionized calcium-binding adapter molecule 1 (Iba1), glial fibrillary acid protein (GFAP), and proliferating cell nuclear antigen (PCNA) proteins were immunodetected. Photoreceptor nuclei rows and microglial cells in the different retinal layers were quantified. Photoreceptor degeneration starts earlier and progresses quicker in P23H-1 than in RCS rats. In both models, microglial cell activation occurs simultaneously with the initiation of photoreceptor death while GFAP over-expression starts later. As degeneration progresses, the numbers of microglial cells increase in the retina, but decreasing in the inner retina and increasing in the outer retina, more markedly in RCS rats. Interestingly, and in contrast with healthy animals, microglial cells reach the outer nuclei and outer segment layers. The higher number of microglial cells in dystrophic retinas cannot be fully accounted by intraretinal migration and PCNA immunodetection revealed microglial proliferation in both models but more importantly in RCS rats. The etiology of retinal degeneration determines the initiation and pattern of photoreceptor cell death and simultaneously there is microglial activation and migration, while the macroglial response is delayed. The actions of microglial cells in the degeneration cannot be explained only in the basis of photoreceptor death because they participate more actively in the RCS model. Thus, the retinal degeneration caused by pigment epithelium malfunction is more inflammatory and would probably respond better to interventions by inhibiting microglial cells. PMID:28321183

Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.

PubMed

Xue, Kai; Gu, Juan J; Zhang, Qunling; Mavis, Cory; Hernandez-Ilizaliturri, Francisco J; Czuczman, Myron S; Guo, Ye

2016-02-01

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48 h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo(®) luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Mouse embryonic stem cells undergo Charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment

PubMed Central

Tichy, Elisia D.; Stephan, Zachary A.; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J.

2013-01-01

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term Charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs. PMID:23500643

Necroptosis in neurodegenerative diseases: a potential therapeutic target

PubMed Central

Zhang, Shuo; Tang, Mi-bo; Luo, Hai-yang; Shi, Chang-he; Xu, Yu-ming

2017-01-01

Neurodegenerative diseases are a group of chronic progressive disorders characterized by neuronal loss. Necroptosis, a recently discovered form of programmed cell death, is a cell death mechanism that has necrosis-like morphological characteristics. Necroptosis activation relies on the receptor-interacting protein (RIP) homology interaction motif (RHIM). A variety of RHIM-containing proteins transduce necroptotic signals from the cell trigger to the cell death mediators RIP3 and mixed lineage kinase domain-like protein (MLKL). RIP1 plays a particularly important and complex role in necroptotic cell death regulation ranging from cell death activation to inhibition, and these functions are often cell type and context dependent. Increasing evidence suggests that necroptosis plays an important role in the pathogenesis of neurodegenerative diseases. Moreover, small molecules such as necrostatin-1 are thought inhibit necroptotic signaling pathway. Understanding the precise mechanisms underlying necroptosis and its interactions with other cell death pathways in neurodegenerative diseases could provide significant therapeutic insights. The present review is aimed at summarizing the molecular mechanisms of necroptosis and highlighting the emerging evidence on necroptosis as a major driver of neuron cell death in neurodegenerative diseases. PMID:28661482

Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

PubMed Central

Fricker, M; O'Prey, J; Tolkovsky, A M; Ryan, K M

2010-01-01

Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death. PMID:21364664

Memory B cell dysregulation in HIV-1-infected individuals.

PubMed

Carrillo, Jorge; Negredo, Eugènia; Puig, Jordi; Molinos-Albert, Luis Manuel; Rodríguez de la Concepción, Maria Luisa; Curriu, Marta; Massanella, Marta; Navarro, Jordi; Crespo, Manel; Viñets, Ester; Millá, Fuensanta; Clotet, Bonaventura; Blanco, Julià

2018-01-14

To characterize the effect of the HIV-1 infection and antiretroviral treatment (ART) in the human memory B (MEB)-cell compartment. A cross-sectional study was designed to analyze MEB cells of HIV-1 ART treated and ART-naive study participants, and uninfected individuals. Frequency and absolute counts of MEB cell subsets in blood were determined by multicolor flow cytometry. Spontaneous cell death and B-cell proliferative capacity was evaluated in vitro by cell culture and flow cytometry. Splenic function was determined by pitted erythrocytes quantification in HIV-1 ART-treated study participants. HIV-1 ART-treated individuals did not show functional hyposplenism despite the lack of recovery IgMIgDCD27 marginal zone-like B cells. Moreover, two germinal center-dependent MEB cells subsets were also dysregulated in HIV-1 individuals: IgMIgDCD27 (IgM only) cells were increased, whereas the switched subset (IgMIgD) was reduced in viremic individuals. Althought ART restored the numbers of these populations; the switched MEB cells were enriched in CD27 cells, which showed the highest susceptibility to spontaneous cell death ex vivo. In addition, B cells from viremic individuals showed a poor response to B-cell receptor and toll-like receptor 9 stimulation that was circumvented when both stimuli were used simultaneously. B cells from HIV-1 study participants show a poor stimulation capacity, that may be bypassed by the proper combination of stimuli, and a dysregulated MEB cell pool that suggest an affectation of the germinal center reaction, only partially normalized by ART. Interestingly, hyposplenism does not explain the lack of recovery of the marginal zone-like B cells in ART-treated HIV-1 individuals.

Proton induces apoptosis of hypoxic tumor cells by the p53-dependent and p38/JNK MAPK signaling pathways.

PubMed

Lee, Kheun Byeol; Kim, Kye-Ryung; Huh, Tae-Lin; Lee, You Mie

2008-12-01

Tumor hypoxia is a main obstacle for radiation therapy. To investigate whether exposure to a proton beam can overcome radioresistance in hypoxic tumor cells, three kinds of cancer cells, Lewis lung carcinoma (LLC) cells, hepatoma HepG2 and Molt-4 leukemia cells, were treated with a proton beam (35 MeV, 1, 2, 5, 10 Gy) in the presence or absence of hypoxia. Cell death rates were determined 72 h after irradiation. Hypoxic cells exposed to the proton beam underwent a typical apoptotic program, showing condensed nuclei, fragmented DNA ladders, and poly-ADP-ribose polymerase (PARP) cleavage. Fluorescence-activated cell sorter analysis revealed a significant increase in Annexin-V-positive cells. Cells treated with the proton beam and hypoxia displayed increased expression of p53, p21 and Bax, but decreased levels of phospho-Rb, Bcl-2 and XIAP, as well as activated caspase-9 and -3. The proton beam with hypoxia induced cell death in wild-type HCT116 cells, but not in a p53 knockout cell line, demonstrating a requirement for p53. As reactive oxygen species (ROS) were also significantly increased, apoptosis could also be abolished by treatment with the anti-oxidant N-acetyl cysteine (NAC). P38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) were activated by the treatment, and their respective DN mutants restored the cell death induced by either proton therapy alone or with hypoxia. In conclusion, proton beam treatment did not differently regulate cancer cell apoptosis either in normoxic or hypoxic conditions via a p53-dependent mechanism and by the activation of p38/JNK MAPK pathways through ROS.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

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

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila.

PubMed

Nezis, Ioannis P; Shravage, Bhupendra V; Sagona, Antonia P; Johansen, Terje; Baehrecke, Eric H; Stenmark, Harald

2010-11-01

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.

Cell death at the intestinal epithelial front line.

PubMed

Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

2016-07-01

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

Death penalty for keratinocytes: apoptosis versus cornification.

PubMed

Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

2005-11-01

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

The intersection between DNA damage response and cell death pathways.

PubMed

Nowsheen, S; Yang, E S

2012-10-01

Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

PubMed

Gilloteaux, J; Jamison, J M; Arnold, D; Taper, H S; Summers, J L

2001-01-01

Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

Dynamic metabolic exchange governs a marine algal-bacterial interaction

PubMed Central

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

2016-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.17473.001 PMID:27855786

Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells.

PubMed

Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

2016-10-25

Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

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

PubMed

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

2006-02-01

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

Cell type-dependent variation in paracrine potency determines therapeutic efficacy against neonatal hyperoxic lung injury.

PubMed

Ahn, So Yoon; Chang, Yun Sil; Sung, Dong Kyung; Yoo, Hye Soo; Sung, Se In; Choi, Soo Jin; Park, Won Soon

2015-08-01

The aim of this study was to determine the optimal cell type for transplantation to protect against neonatal hyperoxic lung injury. To this end, the in vitro and in vivo therapeutic efficacies and paracrine potencies of human umbilical cord blood-derived mesenchymal stromal cells (HUMs), human adipose tissue-derived mesenchymal stromal cells (HAMs) and human umbilical cord blood mononuclear cells (HMNs) were compared. Hyperoxic injury was induced in vitro in A549 cells by challenge with H2O2. Alternatively, hyperoxic injury was induced in newborn Sprague-Dawley rats in vivo by exposure to hyperoxia (90% oxygen) for 14 days. HUMs, HAMs or HMNs (5 × 10(5) cells) were given intratracheally at postnatal day 5. Hyperoxia-induced increases in in vitro cell death and in vivo impaired alveolarization were significantly attenuated in both the HUM and HAM groups but not in the HMN group. Hyperoxia impaired angiogenesis, increased the cell death and pulmonary macrophages and elevated inflammatory cytokine levels. These effects were significantly decreased in the HUM group but not in the HAM or HMN groups. The levels of human vascular endothelial growth factor and hepatocyte growth factor produced by donor cells were highest in HUM group, followed by HAM group and then HMN group. HUMs exhibited the best therapeutic efficacy and paracrine potency than HAMs or HMNs in protecting against neonatal hyperoxic lung injury. These cell type-dependent variations in therapeutic efficacy might be associated or mediated with the paracrine potency of the transplanted donor cells. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas.

PubMed

Atsumi, Go; Suzuki, Haruka; Miyashita, Yuri; Choi, Sun Hee; Hisa, Yusuke; Rihei, Shunsuke; Shimada, Ryoko; Jeon, Eun Jin; Abe, Junya; Nakahara, Kenji S; Uyeda, Ichiro

2016-08-15

Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1 Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV. Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells.

PubMed

Reshi, Latif; Wang, Hua-Ven; Hui, Cho-Fat; Su, Yu-Chin; Hong, Jiann-Ruey

2017-02-01

Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells

PubMed Central

Kim, Keun-Young; Lindsey, James D.; Angert, Mila; Patel, Ankur; Scott, Ray T.; Liu, Quan; Crowston, Jonathan G.; Ellisman, Mark H.; Perkins, Guy A.; Weinreb, Robert N.

2009-01-01

Purpose This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. Methods Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimenstional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Results Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35±14% on day 2, but reduced expression by 36±4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. Conclusions Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria. PMID:19169378

Investigations of the toxic effects of glycans-based silver nanoparticles on different types of human cells

NASA Astrophysics Data System (ADS)

Panzarini, E.; Mariano, S.; Dini, L.

2017-08-01

The effects of glycans-capped AgNPs (30±5 nm average diameter, spherical shape) on biocompatibility and uptake was studied in relation to the glycan capping (glucose AgNPs-G, glucose/sucrose AgNPs-GS, glucose/fructose AgNPs-GF), and to the cell types (HeLa cells, lymphocytes, and HepG2 cells). Glycan capping and type of cells drive morphological changes, viability loss and type and extent of cell death induction; in addition cells response is largely influenced by the AgNPs amount. The MTT photometric method to determine cell metabolism and the analysis of the membrane integrity by Annexin V-Propidium Iodide labelling were used to quantify cell viability and cell death with different concentrations of NPs. It turns out that i) AgNPs-GF are the most toxic, whereas ii) AgNPs-GS are the less toxic NPs, probably due to the stability of glucose/sucrose capping up to 5 days in culture medium; iii) HepG2 cells are the most sensitive to the presence of NPs. A deeper investigation is necessary to explain the interesting PBLs proliferation increase observed in the presence of AgNPs-GS.

Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

PubMed

Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

2014-01-01

Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.