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

Detecting Anastasis In Vivo by CaspaseTracker Biosensor.

PubMed

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

2018-02-01

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

Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms.

PubMed

Horita, Henrick; Frankel, Arthur E; Thorburn, Andrew

2008-01-01

Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

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

PubMed Central

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

2009-01-01

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

5 CFR 1651.11 - Simultaneous death.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Simultaneous death. 1651.11 Section 1651.11 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD DEATH BENEFITS § 1651.11 Simultaneous death. If a beneficiary dies at the same time as the participant, the beneficiary will be treated...

5 CFR 1651.11 - Simultaneous death.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 5 Administrative Personnel 3 2012-01-01 2012-01-01 false Simultaneous death. 1651.11 Section 1651.11 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD DEATH BENEFITS § 1651.11 Simultaneous death. If a beneficiary dies at the same time as the participant, the beneficiary will be treated...

5 CFR 1651.11 - Simultaneous death.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 5 Administrative Personnel 3 2013-01-01 2013-01-01 false Simultaneous death. 1651.11 Section 1651.11 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD DEATH BENEFITS § 1651.11 Simultaneous death. If a beneficiary dies at the same time as the participant, the beneficiary will be treated...

5 CFR 1651.11 - Simultaneous death.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 5 Administrative Personnel 3 2014-01-01 2014-01-01 false Simultaneous death. 1651.11 Section 1651.11 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD DEATH BENEFITS § 1651.11 Simultaneous death. If a beneficiary dies at the same time as the participant, the beneficiary will be treated...

5 CFR 1651.11 - Simultaneous death.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 5 Administrative Personnel 3 2011-01-01 2011-01-01 false Simultaneous death. 1651.11 Section 1651.11 Administrative Personnel FEDERAL RETIREMENT THRIFT INVESTMENT BOARD DEATH BENEFITS § 1651.11 Simultaneous death. If a beneficiary dies at the same time as the participant, the beneficiary will be treated...

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

PubMed

Milutinovic, Snezana; Kashyap, Arun K; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O'Neil, Ryann; Kurtzman, Aaron L; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H; Diaz, Paul W; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R; Reed, John C

2016-01-01

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. ©2015 American Association for Cancer Research.

Dual agonist Surrobody™ simultaneously activates death receptors DR4 and DR5 to induce cancer cell death

PubMed Central

Milutinovic, Snezana; Kashyap, Arun K.; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O' Neil, Ryann; Kurtzman, Aaron L.; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H.; Diaz, Paul W.; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R.; Reed, John C.

2015-01-01

Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, TRAIL (Apo2L). Since most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody™ technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing pro-apoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 mono-specific antibodies. Taken together, Surrobody shows promising preclinical pro-apoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. PMID:26516157

Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy

PubMed Central

El Kaffas, Ahmed; Gangeh, Mehrdad J.; Farhat, Golnaz; Tran, William Tyler; Hashim, Amr; Giles, Anoja; Czarnota, Gregory J.

2018-01-01

High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to characterize vascular and tumour response effects simultaneously, as well as a therapeutic modality to complement radiation therapy. PMID:29290810

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

PubMed

Massimini, M; Palmieri, C; De Maria, R; Romanucci, M; Malatesta, D; De Martinis, M; Maniscalco, L; Ciccarelli, A; Ginaldi, L; Buracco, P; Bongiovanni, L; Della Salda, L

2017-05-01

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 μM, 1 μM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

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

Hemendinger, Richelle A., E-mail: richelle.hemendinger@carolinashealthcare.org; Armstrong, Edward J.; Brooks, Benjamin Rix

Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC{sub 50} (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolatemore » (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC{sub 50} (concentration at which 50% of maximal cell death is inhibited) of 0.6 {mu}M and 0.4 {mu}M, respectively. In contrast, MTHF (up to 10 {mu}M) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.« less

Theranostic Gold Nanoantennas for Simultaneous Multiplexed Raman Imaging of Immunomarkers and Photothermal Therapy.

PubMed

Webb, Joseph A; Ou, Yu-Chuan; Faley, Shannon; Paul, Eden P; Hittinger, Joseph P; Cutright, Camden C; Lin, Eugene C; Bellan, Leon M; Bardhan, Rizia

2017-07-31

In this study, we demonstrate the theranostic capability of actively targeted, site-specific multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. By utilizing multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled by the narrow peak widths of Raman signatures, we simultaneously targeted immune checkpoint receptor programmed death ligand 1 (PDL1) and the epidermal growth factor receptor (EGFR) overexpressed in TNBC cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs functionalized with anti-EGFR antibodies and Raman tag para -mercaptobenzoic acid ( p MBA) were incubated with the cells. SERS imaging revealed a cellular traffic map of MGN localization by surface binding and receptor-mediated endocytosis, enabling targeted diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR- p MBA-MGNs and illuminated with an 808 nm laser for 15 min at 4.7 W/cm 2 exhibited photothermal cell death only within the laser spot (indicated by live/dead cell fluorescence assay). Therefore, this study not only provides an optical imaging platform that can track immunomarkers with spatiotemporal control but also demonstrates an externally controlled light-triggered therapeutic approach enabling receptor-specific treatment with biocompatible theranostic nanoprobes.

Theranostic Gold Nanoantennas for Simultaneous Multiplexed Raman Imaging of Immunomarkers and Photothermal Therapy

PubMed Central

2017-01-01

In this study, we demonstrate the theranostic capability of actively targeted, site-specific multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. By utilizing multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled by the narrow peak widths of Raman signatures, we simultaneously targeted immune checkpoint receptor programmed death ligand 1 (PDL1) and the epidermal growth factor receptor (EGFR) overexpressed in TNBC cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs functionalized with anti-EGFR antibodies and Raman tag para-mercaptobenzoic acid (pMBA) were incubated with the cells. SERS imaging revealed a cellular traffic map of MGN localization by surface binding and receptor-mediated endocytosis, enabling targeted diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR–pMBA–MGNs and illuminated with an 808 nm laser for 15 min at 4.7 W/cm2 exhibited photothermal cell death only within the laser spot (indicated by live/dead cell fluorescence assay). Therefore, this study not only provides an optical imaging platform that can track immunomarkers with spatiotemporal control but also demonstrates an externally controlled light-triggered therapeutic approach enabling receptor-specific treatment with biocompatible theranostic nanoprobes. PMID:28782050

Accelerated lipid catabolism and autophagy are cancer survival mechanisms under inhibited glutaminolysis.

PubMed

Halama, Anna; Kulinski, Michal; Dib, Shaima S; Zaghlool, Shaza B; Siveen, Kodappully S; Iskandarani, Ahmad; Zierer, Jonas; Prabhu, Kirti S; Satheesh, Noothan J; Bhagwat, Aditya M; Uddin, Shahab; Kastenmüller, Gabi; Elemento, Olivier; Gross, Steven S; Suhre, Karsten

2018-08-28

Suppressing glutaminolysis does not always induce cancer cell death in glutamine dependent tumors because cells may switch to alternative energy sources. To reveal compensatory metabolic pathways, we investigated the metabolome-wide cellular response to inhibited glutaminolysis in cancer cells. Glutaminolysis inhibition with C.968 suppressed cell proliferation but was insufficient to induce cancer cell death. We found that lipid catabolism was activated as a compensation for glutaminolysis inhibition. Accelerated lipid catabolism, together with oxidative stress induced by glutaminolysis inhibition, triggered autophagy. Simultaneously inhibiting glutaminolysis and either beta oxidation with trimetazidine or autophagy with chloroquine both induced cancer cell death. Here we identified metabolic escape mechanisms contributing to cancer cell survival under treatment and we suggest potentially translational strategy for combined cancer therapy, given that chloroquine is an FDA approved drug. Our findings are first to show efficiency of combined inhibition of glutaminolysis and beta oxidation as potential anti-cancer strategy as well as add to the evidence that combined inhibition of glutaminolysis and autophagy may be effective in glutamine-addicted cancers. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

PubMed Central

Cheng, Yu-Chi; Chang, Jer-Ming; Chen, Chien-An

2015-01-01

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage. PMID:25322957

Ketorolac Administration Attenuates Retinal Ganglion Cell Death After Axonal Injury.

PubMed

Nadal-Nicolás, Francisco M; Rodriguez-Villagra, Esther; Bravo-Osuna, Irene; Sobrado-Calvo, Paloma; Molina-Martínez, Irene; Villegas-Pérez, Maria Paz; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta; Herrero-Vanrell, Rocío

2016-03-01

To assess the neuroprotective effects of ketorolac administration, in solution or delivered from biodegradable microspheres, on the survival of axotomized retinal ganglion cells (RGCs). Retinas were treated intravitreally with a single injection of tromethamine ketorolac solution and/or with ketorolac-loaded poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Ketorolac treatments were administered either 1 week before optic nerve crush (pre-ONC) or right after the ONC (simultaneous). In all cases, animals were euthanized 7 days after the ONC. As control, nonloaded microspheres or vehicle (balanced salt solution, BSS) were administered in parallel groups. All retinas were dissected as flat mounts; RGCs were immunodetected with brain-specific homeobox/POU domain protein 3A (Brn3a), and their number was automatically quantified. The percentage of Brn3a+RGCs was 36% to 41% in all control groups (ONC with or without BSS or nonloaded microparticles). Ketorolac solution administered pre-ONC resulted in 63% survival of RGCs, while simultaneous administration promoted a 53% survival. Ketorolac-loaded microspheres were not as efficient as ketorolac solution (43% and 42% of RGC survival pre-ONC or simultaneous, respectively). The combination of ketorolac solution and ketorolac-loaded microspheres did not have an additive effect (54% and 55% survival pre-ONC and simultaneous delivery, respectively). Treatment with the nonsteroidal anti-inflammatory drug ketorolac delays RGC death triggered by a traumatic axonal insult. Pretreatment seems to elicit a better output than simultaneous administration of ketorolac solution. This may be taken into account when performing procedures resulting in RGC axonal injury.

A cell impedance measurement device for the cytotoxicity assay dependent on the velocity of supplied toxic fluid

NASA Astrophysics Data System (ADS)

Kang, Yoon-Tae; Kim, Min-Ji; Cho, Young-Ho

2018-04-01

We present a cell impedance measurement chip capable of characterizing the toxic response of cells depending on the velocity of the supplied toxic fluid. Previous impedance-based devices using a single open-top chamber have been limited to maintaining a constant supply velocity, and devices with a single closed-top chamber present difficulties in simultaneous cytotoxicity assay for varying levels of supply velocities. The present device, capable of generating constant and multiple levels of toxic fluid velocity simultaneously within a single stepwise microchannel, performs a cytotoxicity assay dependent on toxic fluid velocity, in order to find the effective velocity of toxic fluid to cells for maximizing the cytotoxic effect. We analyze the cellular toxic response of 5% ethanol media supplied to cancer cells within a toxic fluid velocity range of 0-8.3 mm s-1. We observe the velocity-dependent cell detachment rate, impedance, and death rate. We find that the cell detachment rate decreased suddenly to 2.4% at a velocity of 4.4 mm s-1, and that the change rates of cell resistance and cell capacitance showed steep decreases to 8% and 41%, respectively, at a velocity of 5.7 mm s-1. The cell death rate and impedance fell steeply to 32% at a velocity of 5.7 mm s-1. We conclude that: (1) the present device is useful in deciding on the toxic fluid velocity effective to cytotoxicity assay, since the cellular toxic response is dependent on the velocity of toxic fluid, and; (2) the cell impedance analysis facilitates a finer cellular response analysis, showing better correlation with the cell death rate, compared to conventional visual observation. The present device, capable of performing the combinational analysis of toxic fluid velocity and cell impedance, has potential for application to the fine cellular toxicity assay of drugs with proper toxic fluid velocity.

Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)*

PubMed Central

Coultrap, Steven J.; Bayer, K. Ulrich

2014-01-01

Both signaling by nitric oxide (NO) and by the Ca2+/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca2+-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca2+/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca2+. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death. PMID:24855644

Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury

PubMed Central

Matylevitch, Natalia P.; Schuschereba, Steven T.; Mata, Jennifer R.; Gilligan, George R.; Lawlor, David F.; Goodwin, Cleon W.; Bowman, Phillip D.

1998-01-01

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72°C for 1 second. After exposure to temperatures of 58 to 59°C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66°C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72°C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59°C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation. PMID:9708816

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

PubMed

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

1998-08-01

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

Anti-cell death engineering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction.

PubMed

Lee, Jae Seong; Ha, Tae Kwang; Park, Jin Hyoung; Lee, Gyun Min

2013-08-01

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti-apoptosis engineering. Recently, autophagy has received attention as a new anti-cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti-apoptosis and pro-autophagy in CHO cells (DG44) was attempted by co-overexpressing an anti-apoptotic protein, Bcl-2, and a key regulator of autophagy pathway, Beclin-1, respectively. Co-overexpression of Bcl-2 and Beclin-1 exhibited a longer culture period as well as higher viability during serum-free suspension culture, compared with the control (without co-overexpression of Bcl-2 and Beclin-1) and Bcl-2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl-2 overexpression, Beclin-1 overexpression successfully induced the increase in the autophagic marker protein, LC3-II, and autophagosome formation with the decrease in mTOR activity. Co-immunoprecipitation and qRT-PCR experiments revealed that the enforced expression of Beclin-1 increased Ulk1 expression and level of free-Beclin-1 that did not bind to the Bcl-2 despite the Bcl-2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co-overexpression of Bcl-2 and Beclin-1 also protected the cells from cell death more efficiently than Bcl-2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro-autophagy engineering together with anti-apoptosis engineering yields a synergistic effect and successfully enhances the anti-cell death engineering of CHO cells. Copyright © 2013 Wiley Periodicals, Inc.

Regulation of programmed cell death or apoptosis in atherosclerosis.

PubMed

Geng, Y J

1997-01-01

Intimal thickening caused by accumulation of cells, lipids, and connective tissue characterizes atherosclerosis, an arterial disease that leads to cardiac and cerebral infarction. Apoptosis, or genetically programmed cell death, is important for the development and morphogenesis of organs and tissues. As in other tissues, cells of cardiovascular tissues can undergo apoptosis. Increased apoptosis has been found in both human and animal atherosclerotic lesions, mediating tissue turnover and lesion development. In addition to vascular cells, many activated immune cells, mainly macrophages and T cells, are present in atherosclerotic lesions, where these cells produce biologically active substances such as the proinflammatory cytokines tumor necrosis factor, interleukin-1 (IL-1), and interferon-gamma. Simultaneous exposure to these cytokines may trigger apoptosis of vascular smooth muscle cells. The products of death-regulating genes including Fas/Fas ligand, members of IL-1 beta cysteinyl protease (caspase) family, the tumor suppressive gene p53, and the protooncogene c-myc have been found in vascular cells and may participate in the regulation of vascular apoptosis during the development of atherosclerosis. Abnormal occurrence of apoptosis may take place in atherosclerotic lesions, including attenuation or acceleration of the apoptotic death process. The former may cause an increase in the cellularity of the lesions, and the latter can reduce cellular components important for maintaining the integrity and stability of the plaques. Clarification of the molecular mechanism that regulates apoptosis may help design a new strategy for treatment of patients with atherosclerosis and its major complications, heart attack and stroke.

Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells.

PubMed

Lengwehasatit, Idsada; Dickson, Alan J

2002-12-30

Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment. Copyright 2002 Wiley Periodicals, Inc.

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout.

PubMed

Ehlken, H; Krishna-Subramanian, S; Ochoa-Callejero, L; Kondylis, V; Nadi, N E; Straub, B K; Schirmacher, P; Walczak, H; Kollias, G; Pasparakis, M

2014-11-01

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.

Normal and leukaemic human haemopoietic cells in diffusion chamber. A morphological and functional CFU-C study.

PubMed

Laurent, M; Clémancey-Marcille, G; Hollard, D

1980-03-01

Leukaemic human bone marrow and peripheral blood cells were cultured for 25 d in diffusion chambers implanted into cyclophosphamide treated mice. Normal bone marrow cells were cultured simultaneously. These cells were studied both morphologically and functionally (CFU-C). The leukaemic cells behaved heterogeneously, 2 groups being distinguishable in accordance with their initial in vitro growth pattern (1: no growth or microcluster growth. 2: macrocluster growth). Group I showed progressive cellular death with a diminution of granulocytic progenitors and the appearance of a predominantly macrophagic population. This behaviour resembled that of the control group. The initial microcluster growth pattern remained identical throughout the entire culture period. Group 2, after considerable cellular death up to d 5, showed an explosive proliferation of the granulocytic progenitors and incomplete differentiation (up to myelocyte). The initial macrocluster growth pattern remained identical.

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

PubMed

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

2017-03-04

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

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.

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.

Simultaneous homicide-suicide: a case report of double drowning.

PubMed

Melez, İpek Esen; Avşar, Abdullah; Başpınar, Bünyamin; Melez, Deniz Oğuzhan; Şahin, Fatih; Özdeş, Taşkın

2014-09-01

Homicide-suicide is a tragic phenomenon which typically does not result in a criminal charge or trial. However, correct diagnosis and classification of homicide-suicide cases are important to determine the perpetrators and dynamics of each category properly. The deaths in the homicide-suicide acts can be divided into two categories with respect to the number of involved individuals: dyadic deaths and triple or multiple deaths. These two categories can also be divided into two subgroups according to the chronology of the incidents: simultaneous deaths and consecutive deaths. Herein, a simultaneous homicide-suicide case of a father and daughter where both deaths occurred through drowning which was not found in the selected literature review and where the victim was a child is presented. The article aims to clarify the term discrepancies about multiple death cases in the literature and to discuss the pathological and psychosocial characteristics of the simultaneous dyadic death cases. © 2014 American Academy of Forensic Sciences.

TUBERCULOSIS AND LETHAL AS WELL AS SUBLETHAL WHOLE-BODY X-RAY IRRADIATION OF GUINEA PIGS (in German)

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

Gabler, E.

1964-02-01

Lethally total-body-x-ray-irradiated (550 r) and simultaneously Tb- infected guinea pigs died earlier (1.5 to 3.2 days) than lethally irradiated control animals. A tuberculous focus formation could not be found microscopically or macroscopically in these guinea pigs or in sublethally irradiated and simultaneously infected animals. However, in tubcrculous control animals, which were killed at this time, specific foci could be found in liver, spleen, and lungs. Using sublethal irradiation (300 r) and simultaneous Tb inoculation half of the animals died a radiation death and the rest died of tuberculosis. It was found that 86.4% of the animals die a radiation deathmore » and 13.5% because of tuberculosis when irradiated sublethally 30 days after infection. The greatest tuberculosis foci in these animais appeared in lungs, spleen, and especially in the liver ( destroyed iiver''). Tuberculous giant cells of the Langhans-type were missing in case of irradiation and simultaneous tuberculosis. They appeared again about 20 to 30 days after irradiation. The native resistance to tuberculosis was very reduced in cases of simultaneous exposure; radioinduced cell shortage and cell damage permit tuberculous focus formation only after overcoming the acute radiation syndrome in case of sublethal irradiations. (auth)« less

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

Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

PubMed Central

Li, Xiaoming; Bai, Jing; Li, Jianchun; Li, Shenghao; Wang, Zeming; Zhou, Mingrui

2017-01-01

Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC. PMID:28526807

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.

Synergistic administration of photothermal therapy and chemotherapy to cancer cells using polypeptide-based degradable plasmonic matrices

PubMed Central

Huang, Huang-Chiao; Yang, Yoonsun; Nanda, Alisha; Koria, Piyush; Rege, Kaushal

2012-01-01

Aim Resistance of cancer cells to hyperthermic temperatures and spatial limitations of nanoparticle-induced hyperthermia necessitates the identification of effective combination treatments that can enhance the efficacy of this treatment. Here we show that novel polypeptide-based degradable plasmonic matrices can be employed for simultaneous administration of hyperthermia and chemotherapeutic drugs as an effective combination treatment that can overcome cancer cell resistance to hyperthermia. Method Novel gold nanorod elastin-like polypeptide matrices were generated and characterized. The matrices were also loaded with the heat-shock protein (HSP)90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), currently in clinical trials for different malignancies, in order to deliver a combination of hyperthermia and chemotherapy. Results Laser irradiation of cells cultured over the plasmonic matrices (without 17-AAG) resulted in the death of cells directly in the path of the laser, while cells outside the laser path did not show any loss of viability. Such spatial limitations, in concert with expression of prosurvival HSPs, reduce the efficacy of hyperthermia treatment. 17-AAG–gold nanorod–polypeptide matrices demonstrated minimal leaching of the drug to surrounding media. The combination of hyperthermic temperatures and the release of 17-AAG from the matrix, both induced by laser irradiation, resulted in significant (>90%) death of cancer cells, while ‘single treatments’ (i.e., hyperthermia alone and 17-AAG alone) demonstrated minimal loss of cancer cell viability (<10%). Conclusion Simultaneous administration of hyperthermia and HSP inhibitor release from plasmonic matrices is a powerful approach for the ablation of malignant cells and can be extended to different combinations of nanoparticles and chemotherapeutic drugs for a variety of malignancies. PMID:21542685

Calibrating the imaging and therapy performance of magneto-fluorescent gold nanoshells for breast cancer

NASA Astrophysics Data System (ADS)

Dowell, Adam; Chen, Wenxue; Biswal, Nrusingh; Ayala-Orozco, Ciceron; Giuliano, Mario; Schiff, Rachel; Halas, Naomi J.; Joshi, Amit

2012-03-01

Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, p<0.001). For MCF-7/HER2-18 cells, death was greater with HER2-targeted nanoshells and was independent of laser power. This study demonstrates the capability of magneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells, and the advantages of targeting receptors unique to cancer cells.

Cleavage and shedding of E-cadherin after induction of apoptosis.

PubMed

Steinhusen, U; Weiske, J; Badock, V; Tauber, R; Bommert, K; Huber, O

2001-02-16

Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca(2+)-dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium.

Hetero-bivalent Imaging Agents for Simultaneous Targeting Prostate-Specific Membrane Antigen (PSMA) and Hepsin

DTIC Science & Technology

2013-09-01

Suzuki cross-coupling reaction. The effects of solvent and base on the synthesis of 3 were studied using Pd(PPh3)4 as catalyst . DMF, ethanol, and DMSO...PSMA/hepsin for in vitro cell uptake and in vivo imaging studies . Compound 13 showed a low but detectable increased cell uptake into the developed...have been comprehensive clinical studies whether PSA testing is an efficient biomarker in diagnosing PCa and reducing PCa deaths. Two European studies

Cell death induced by ozone and various non-thermal plasmas: therapeutic perspectives and limitations

PubMed Central

Lunov, Oleg; Zablotskii, Vitalii; Churpita, Olexander; Chánová, Eliška; Syková, Eva; Dejneka, Alexandr; Kubinová, Šárka

2014-01-01

Non-thermal plasma has been recognized as a promising tool across a vast variety of biomedical applications, with the potential to create novel therapeutic methods. However, the understanding of the molecular mechanisms behind non-thermal plasma cellular effects remains a significant challenge. In this study, we show how two types of different non-thermal plasmas induce cell death in mammalian cell cultures via the formation of multiple intracellular reactive oxygen/nitrogen species. Our results showed a discrepancy in the superoxide accumulation and lysosomal activity in response to air and helium plasma, suggesting that triggered signalling cascades might be grossly different between different plasmas. In addition, the effects of ozone, a considerable component of non-thermal plasma, have been simultaneously evaluated and have revealed much faster and higher cytotoxic effects. Our findings offer novel insight into plasma-induced cellular responses, and provide a basis for better controlled biomedical applications. PMID:25410636

The pro-apoptotic protein Bmf co-operates with Bim and Puma in neuron death induced by β-amyloid or NGF deprivation.

PubMed

Akhter, Rumana; Saleem, Suraiya; Saha, Akash; Biswas, Subhas Chandra

2018-04-01

The pro-apoptotic Bcl-2 homology 3 domain only (BH3-only) proteins are central regulators of cell death in various physiological and pathological conditions, including Alzheimer's disease (AD). Bcl-2 modifying factor (Bmf) is one such BH3-only protein that is implicated in various death paradigms such as anoikis, seizures, cancer and autoimmunity. It also co-operates with other BH3-only proteins such as Bim in various death paradigms. However, its role in neurodegeneration is under-investigated. Here, we report for the first time the essential role of Bmf and its co-operativity with direct activator BH3-only proteins Bim and Puma in neuron death induced by beta-amyloid (Aβ) toxicity or NGF deprivation. Oligomeric Aβ is main pathologic species in AD and NGF deprivation is relevant for both developmental as well as pathologic neuron death. We find that Bmf over-expression causes cell death and Bmf knockdown protects neurons against death evoked by Aβ or NGF deprivation. We also find that Bmf co-operates with other important BH3-only proteins such as Bim and Puma in neuron death induced by Aβ or NGF deprivation. Simultaneous knocking down of these molecules by their respective shRNAs provide enhanced protection against Aβ. Taken together, our results elucidate the essential role of Bmf and its co-operative effects with already known neuron death inducers, Bim and Puma, in neuron death evoked by Aβ treatment or NGF deprivation. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of the Function of Apoptosis during Imaginal Wing Disc Regeneration in Drosophila melanogaster.

PubMed

Diaz-Garcia, Sandra; Ahmed, Sara; Baonza, Antonio

2016-01-01

Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.

Spirulina maxima extract prevents cell death through BDNF activation against amyloid beta 1-42 (Aβ1-42) induced neurotoxicity in PC12 cells.

PubMed

Koh, Eun-Jeong; Kim, Kui-Jin; Choi, Jia; Kang, Do-Hyung; Lee, Boo-Yong

2018-04-23

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ 1-42 -induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ 1-42 -induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ 1-42 -induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ 1-42 -induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ 1-42 -treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ 1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ 1-42 -induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ 1-42 -induced neurotoxicity via the activation of BDNF signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis.

PubMed

Ping, Suning; Liu, Shuying; Zhou, Yuhuan; Li, Ziqing; Li, Yuhuang; Liu, Kefeng; Bardeesi, Adham Sa; Wang, Linli; Chen, Jingbo; Deng, Lie; Wang, Jingjing; Wang, Hong; Chen, Dadi; Zhang, Zhengyu; Sheng, Puyi; Li, Chaohong

2017-05-25

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

PubMed Central

Ping, Suning; Liu, Shuying; Zhou, Yuhuan; Li, Ziqing; Li, Yuhuang; Liu, Kefeng; Bardeesi, Adham SA; Wang, Linli; Chen, Jingbo; Deng, Lie; Wang, Jingjing; Wang, Hong; Chen, Dadi; Zhang, Zhengyu; Sheng, Puyi; Li, Chaohong

2017-01-01

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases. PMID:28542133

Protective Effects of a Rhodiola Crenulata Extract and Salidroside on Hippocampal Neurogenesis against Streptozotocin-Induced Neural Injury in the Rat

PubMed Central

Qu, Ze-qiang; Zhou, Yan; Zeng, Yuan-shan; Lin, Yu-kun; Li, Yan; Zhong, Zhi-qiang; Chan, Wood Yee

2012-01-01

Previously we have demonstrated that a Rhodiola crenulata extract (RCE), containing a potent antioxidant salidroside, promotes neurogenesis in the hippocampus of depressive rats. The current study was designed to further investigate the protective effect of the RCE on neurogenesis in a rat model of Alzheimer's disease (AD) induced by an intracerebroventricular injection of streptozotocin (STZ), and to determine whether this neuroprotective effect is induced by the antioxidative activity of salidroside. Our results showed that pretreatment with the RCE significantly improved the impaired neurogenesis and simultaneously reduced the oxidative stress in the hippocampus of AD rats. In vitro studies revealed that (1) exposure of neural stem cells (NSCs) from the hippocampus to STZ strikingly increased intracellular reactive oxygen species (ROS) levels, induced cell death and perturbed cell proliferation and differentiation, (2) hydrogen peroxide induced similar cellular activities as STZ, (3) pre-incubation of STZ-treated NSCs with catalase, an antioxidant, suppressed all these cellular activities induced by STZ, and (4) likewise, pre-incubation of STZ-treated NSCs with salidroside, also an antioxidant, suppressed all these activities as catalase: reduction of ROS levels and NSC death with simultaneous increases in proliferation and differentiation. Our findings indicated that the RCE improved the impaired hippocampal neurogenesis in the rat model of AD through protecting NSCs by its main ingredient salidroside which scavenged intracellular ROS. PMID:22235318

Hepatoprotective properties of kombucha tea against TBHP-induced oxidative stress via suppression of mitochondria dependent apoptosis.

PubMed

Bhattacharya, Semantee; Gachhui, Ratan; Sil, Parames C

2011-06-01

Kombucha, a fermented tea (KT) is claimed to possess many beneficial properties. Recent studies have suggested that KT prevents paracetamol and carbon tetrachloride-induced hepatotoxicity. We investigated the beneficial role of KT was against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity and cell death in murine hepatocytes. TBHP is a well known reactive oxygen species (ROS) inducer, and it induces oxidative stress in organ pathophysiology. In our experiments, TBHP caused a reduction in cell viability, enhanced the membrane leakage and disturbed the intra-cellular antioxidant machineries while simultaneous treatment of the cells with KT and this ROS inducer maintained membrane integrity and prevented the alterations in the cellular antioxidant status. These findings led us to explore the detailed molecular mechanisms involved in the protective effect of KT. TBHP introduced apoptosis as the primary phenomena of cell death as evidenced by flow cytometric analyses. In addition, ROS generation, changes in the mitochondrial membrane potential, cytochrome c release, activation of caspases (3 and 9) and Apaf-1 were detected confirming involvement of mitochondrial pathway in this pathophysiology. Simultaneous treatment of KT with TBHP, on the other hand, protected the cells against oxidative injury and maintained their normal physiology. In conclusion, KT was found to modulate the oxidative stress induced apoptosis in murine hepatocytes probably due to its antioxidant activity and functioning via mitochondria dependent pathways and could be beneficial against liver diseases, where oxidative stress is known to play a crucial role. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

PubMed

Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

2015-11-26

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.

Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

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

Li Ping; Zhang Qing; Torossian, Artour

2012-07-01

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used tomore » investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.« less

Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.

PubMed

Ankri, Chen; Shamalov, Katerina; Horovitz-Fried, Miryam; Mauer, Shmuel; Cohen, Cyrille J

2013-10-15

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

Mangifera indica L. extract protects T cells from activation-induced cell death.

PubMed

Hernández, Patricia; Delgado, Rene; Walczak, Henning

2006-09-01

The aqueous stem bark extract of Mangifera indica L. (Vimang) has been reported to have antioxidant properties. AIDS is characterized by up-regulation of CD95 ligand (CD95L) expression and enhancement of activation-induced cell death (AICD). Recent studies demonstrate oxidative signals combined with simultaneous calcium (Ca(2+)) influx into the cytosol are required for induction of CD95L expression. In this study we show that M. indica extract attenuated anti-CD3-induced accumulation of reactive oxygen species (ROS) and intracellular free Ca(2+) and consequently, downregulates CD95L mRNA expression and CD95-mediated AICD. In addition, TCR triggering caused an elevation in the antioxidant enzyme manganous superoxide dismutase (Mn-SOD) and the increase in c-Jun N-terminal kinase (JNK) phosphorylation, both effects being prevented by M. indica extract. We provide a number of evidences regarding how M. indica extract enhance T-cell survival by inhibiting AICD, a finding associated with a decrease in oxidative stress generated through the TCR signaling pathway in activated T cells.

Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix

PubMed Central

Su, Yuan; Shi, Yufang; Stolow, Melissa A.; Shi, Yun-Bo

1997-01-01

Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. PMID:9396758

Simultaneous saccharification and cofermentation of lignocellulosic residues from commercial furfural production and corn kernels using different nutrient media

PubMed Central

2011-01-01

Background As the supply of starch grain and sugar cane, currently the main feedstocks for bioethanol production, become limited, lignocelluloses will be sought as alternative materials for bioethanol production. Production of cellulosic ethanol is still cost-inefficient because of the low final ethanol concentration and the addition of nutrients. We report the use of simultaneous saccharification and cofermentation (SSCF) of lignocellulosic residues from commercial furfural production (furfural residue, FR) and corn kernels to compare different nutritional media. The final ethanol concentration, yield, number of live yeast cells, and yeast-cell death ratio were investigated to evaluate the effectiveness of integrating cellulosic and starch ethanol. Results Both the ethanol yield and number of live yeast cells increased with increasing corn-kernel concentration, whereas the yeast-cell death ratio decreased in SSCF of FR and corn kernels. An ethanol concentration of 73.1 g/L at 120 h, which corresponded to a 101.1% ethanol yield based on FR cellulose and corn starch, was obtained in SSCF of 7.5% FR and 14.5% corn kernels with mineral-salt medium. SSCF could simultaneously convert cellulose into ethanol from both corn kernels and FR, and SSCF ethanol yield was similar between the organic and mineral-salt media. Conclusions Starch ethanol promotes cellulosic ethanol by providing important nutrients for fermentative organisms, and in turn cellulosic ethanol promotes starch ethanol by providing cellulosic enzymes that convert the cellulosic polysaccharides in starch materials into additional ethanol. It is feasible to produce ethanol in SSCF of FR and corn kernels with mineral-salt medium. It would be cost-efficient to produce ethanol in SSCF of high concentrations of water-insoluble solids of lignocellulosic materials and corn kernels. Compared with prehydrolysis and fed-batch strategy using lignocellulosic materials, addition of starch hydrolysates to cellulosic ethanol production is a more suitable method to improve the final ethanol concentration. PMID:21801455

S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson’s disease

PubMed Central

2013-01-01

Background Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson’s disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. Results We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. Conclusions Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD. PMID:23985028

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

PubMed Central

Cinti, Alessandro; De Giorgi, Marco; Chisci, Elisa; Arena, Claudia; Galimberti, Gloria; Farina, Laura; Bugarin, Cristina; Rivolta, Ilaria; Gaipa, Giuseppe; Smolenski, Ryszard Tom; Cerrito, Maria Grazia; Lavitrano, Marialuisa; Giovannoni, Roberto

2015-01-01

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings. PMID:26513260

Noninvasive optical monitoring multiple physiological parameters response to cytokine storm

NASA Astrophysics Data System (ADS)

Li, Zebin; Li, Ting

2018-02-01

Cancer and other disease originated by immune or genetic problems have become a main cause of death. Gene/cell therapy is a highlighted potential method for the treatment of these diseases. However, during the treatment, it always causes cytokine storm, which probably trigger acute respiratory distress syndrome and multiple organ failure. Here we developed a point-of-care device for noninvasive monitoring cytokine storm induced multiple physiological parameters simultaneously. Oxy-hemoglobin, deoxy-hemoglobin, water concentration and deep-tissue/tumor temperature variations were simultaneously measured by extended near infrared spectroscopy. Detection algorithms of symptoms such as shock, edema, deep-tissue fever and tissue fibrosis were developed and included. Based on these measurements, modeling of patient tolerance and cytokine storm intensity were carried out. This custom device was tested on patients experiencing cytokine storm in intensive care unit. The preliminary data indicated the potential of our device in popular and milestone gene/cell therapy, especially, chimeric antigen receptor T-cell immunotherapy (CAR-T).

Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling.

PubMed

Nikolova, Teodora; Roos, Wynand P; Krämer, Oliver H; Strik, Herwig M; Kaina, Bernd

2017-08-01

Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O 6 -chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Lee, Te-Chang; Institute of Pharmacology, Yang Min University, Taipei, Taiwan; Cheng, I-Cheng

Arsenic trioxide (ATO) treatment is a useful therapy against human acute promyelocytic leukemia (APL), however, it concomitantly brings potential adverse consequences including serious side effect, human carcinogenicity and possible development of resistance. This investigation revealed that those problems might be relaxed by simultaneous application with (-)-epigallocatechin-3-gallate (EGCG), one of the major components from green tea. EGCG significantly lowered down the ATO concentration required for an effective control of APL cells, HL-60. The simultaneous treatment of ATO with EGCG induced a mitochondria-dependent apoptosis in HL-60 cells significantly, which accounted for more than 70% of the cell death in the treatment. Themore » mechanism of apoptosis induction was elucidated. EGCG in HL-60 cells acted as a pro-oxidant enhancing intracellular hydrogen peroxide significantly. ATO, on the other hand, induced heme oxygenase-1 (HO-1) to catalyze heme degradation, thereby provided ferrous iron for EGCG-induced hydrogen peroxide to precede Fenton reaction, which in turn generated deleterious reactive oxygen species to damage cell. In addition, EGCG inhibited expression of ferritin, which supposedly to sequester harmful ferrous iron, thereby augmented the occurrence of Fenton reaction. This investigation also provided evidence that ATO, since mainly acted to induce HO-1 in simultaneous treatment with EGCG, could be replaced by other HO-1 inducer with much less human toxicity. Furthermore, several of our preliminary investigations revealed that the enhanced cytotoxicity induced by combining heme degradation and Fenton reaction is selectively toxic to malignant but not non-malignant cells.« less

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose*

PubMed Central

Du, Yatao; Zhang, Huihui; Lu, Jun; Holmgren, Arne

2012-01-01

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin. PMID:22977247

ALA-Butyrate prodrugs for Photo-Dynamic Therapy

NASA Astrophysics Data System (ADS)

Berkovitch, G.; Nudelman, A.; Ehenberg, B.; Rephaeli, A.; Malik, Z.

2010-05-01

The use of 5-aminolevulinic acid (ALA) administration has led to many applications of photodynamic therapy (PDT) in cancer. However, the hydrophilic nature of ALA limits its ability to penetrate the cells and tissues, and therefore the need for ALA derivatives became an urgent research target. In this study we investigated the activity of novel multifunctional acyloxyalkyl ester prodrugs of ALA that upon metabolic hydrolysis release active components such as, formaldehyde, and the histone deacetylase inhibitory moiety, butyric acid. Evaluation of these prodrugs under photo-irradiation conditions showed that butyryloxyethyl 5-amino-4-oxopentanoate (ALA-BAC) generated the most efficient photodynamic destruction compared to ALA. ALA-BAC stimulated a rapid biosynthesis of protoporphyrin IX (PpIX) in human glioblastoma U-251 cells which resulted in generation of intracellular ROS, reduction of mitochondrial activity, leading to apoptotic and necrotic death of the cells. The apoptotic cell death induced by ALA / ALA-BAC followed by PDT equally activate intrinsic and extrinsic apoptotic signals and both pathways may occur simultaneously. The main advantage of ALA-BAC over ALA stems from its ability to induce photo-damage at a significantly lower dose than ALA.

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

PubMed Central

Rojas, Cristina; Pan-Castillo, Belén; Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Arola, Lluis; Mulero, Miquel

2014-01-01

Background Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines. Principal Findings We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity. Conclusions Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy. PMID:25436452

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells.

PubMed

Mlejnek, Petr

2013-10-01

N(6)-benzyladenine (BA) and N(6)-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723-1735, 2003, Plant Sci 168:389-395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N(6)-benzyladenine 7-β-D-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.

Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

PubMed

Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

2009-07-31

Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

PubMed Central

Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

2009-01-01

Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

Effect of ellagic acid on proliferation, cell adhesion and apoptosis in SH-SY5Y human neuroblastoma cells.

PubMed

Fjaeraa, Christina; Nånberg, Eewa

2009-05-01

Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.

Ameliorating Effects of Green Synthesized Silver Nanoparticles on Glycated End Product Induced Reactive Oxygen Species Production and Cellular Toxicity in Osteogenic Saos-2 Cells.

PubMed

Ashe, Sarbani; Nayak, Debasis; Kumari, Manisha; Nayak, Bismita

2016-11-09

Advanced glycation end-products (AGEs) that result from nonenzymatic glycation are one of the major factors involved in diabetes and its secondary complications and diseases. This necessitates our urge to discover new compounds that may be used as potential AGEs inhibitors without affecting the normal structure and function of biomolecules. In the present study, we investigated the inhibitory effects of AgNP (silver nanoparticles) on AGEs formation as well as their inhibitory effects on glycation mediated cell toxicity via reactive oxygen species (ROS) production and DNA damage. The excitation-emission fluorescence spectroscopy was employed to investigate the interaction of AgNP during glycation. The values of conditional stability constant (log K a = 4.44) derived from the Stern-Volmer equation indicate that AgNP have strong binding capacity for glycated protein. UV-vis, fluorescence, and Fourier transform infrared spectral data reveal complexation of AgNP with glycated bovine serum albumin, which significantly inhibits AGEs formation in a concentration-dependent manner. Cytotoxic evaluations suggest that simultaneous administration of AgNP and glycated product reduces cell death (42.82% ± 3.54) as compared to the glycated product alone. Similarly, ROS production in AgNP treated cells is significantly less compared to only glycated product treated cells. Although DNA damage studies show DNA damage in both GP and GP-AgNP treated cells, fluorescence activated cell sorting analysis demonstrates that glycated products induce cell death by necrosis, while AgNP cause cell death via apoptotic pathways. AgNP have a positive effect on restoring native protein structure deduced from spectral studies, and hence, inferences can be drawn that AgNP have ameliorating effects on glycated induced cytotoxicity observed in osteogenic Saos-2 cells.

Autophagy prevention sensitizes AKTi-1/2-induced anti-hepatocellular carcinoma cell activity in vitro and in vivo

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

Zhang, Qi; Yang, Manyi; Qu, Zhan

Molecule-targeted therapy has become the research focus for hepatocellular carcinoma (HCC). Persistent PI3K-AKT activation is often detected in HCC, representing a valuable oncotarget for treatment. Here, we tested the anti-HCC activity by a potent AKT inhibitor: AKT inhibitor 1/2 (AKTi-1/2). In both established (HepG2 and Huh-7) and primary human HCC cells, treatment with AKTi-1/2 inhibited cell survival and proliferation, but induced cell apoptosis. AKTi-1/2 blocked AKT-mTOR activation, yet simultaneously provoked cytoprotective autophagy in HCC cells. The latter was evidenced by ATG-5 and Beclin-1 upregulation, p62 downregulation as well as LC3B-GFP puncta formation. Autophagy inhibition, via pharmacological inhibitors (3-methyladenine, ammonium chloride,more » and bafilomycin A1) or Beclin-1 siRNA knockdown, significantly potentiated AKTi-1/2-induced HepG2 cell death and apoptosis. In nude mice, AKTi-1/2 intraperitoneal injection inhibited HepG2 tumor growth. Significantly, its anti-tumor activity in vivo was further sensitized when combined with Beclin-1 shRNA knockdown in HepG2 tumors. Together, these results demonstrate that autophagy activation serves as a main resistance factor of AKTi-1/2 in HCC cells. Autophagy prevention therefore sensitizes AKTi-1/2-induced anti-HCC activity in vitro and in vivo. - Highlights: • AKTi-1/2 inhibits human HCC cells in vitro. • Autophagy inhibitors sensitize AKTi-1/2-induced HCC cell death and apoptosis. • Beclin-1 siRNA potentiates AKTi-1/2-induced HepG2 cell death and apoptosis. • Beclin-1 knockdown augments AKTi-1/2-induced anti-HepG2 tumor activity in vivo.« less

DNA Repair Domain Modeling Can Predict Cell Death and Mutation Frequency for Wide Range Spectrum of Radiation

NASA Technical Reports Server (NTRS)

Viger, Louise; Ponomarev, Artem L.; Plante, Ianik; Evain, Trevor; Penninckx, Sebastien; Blattnig, Steve R.; Costes, Sylvain V.

2017-01-01

Exploration missions to Mars and other destinations raise many questions about the health of astronauts. The continuous exposure of astronauts to galactic cosmic rays is one of the main concerns for long-term missions. Cosmic ionizing radiations are composed of different ions of various charges and energies notably, highly charged energy (HZE) particles. The HZE particles have been shown to be more carcinogenic than low-LET radiation, suggesting the severity of chromosomal aberrations induced by HZE particles is one possible explanation. However, most mathematical models predicting cell death and mutation frequency are based on directly fitting various HZE dose response and are in essence empirical approaches. In this work, we assume a simple biological mechanism to model DNA repair and use it to simultaneously explain the low- and high-LET response using the exact same fitting parameters. Our work shows that the geometrical position of DNA repair along tracks of heavy ions are sufficient to explain why high-LET particles can induce more death and mutations. Our model is based on assuming DNA double strand breaks (DSBs) are repaired within repair domain, and that any DSBs located within the same repair domain cluster into one repair unit, facilitating chromosomal rearrangements and increasing the probability of cell death. We introduced this model in 2014 using simplified microdosimetry profiles to predict cell death. In this work, we collaborated with NASA Johnson Space Center to generate more accurate microdosimetry profiles derived by Monte Carlo techniques, taking into account track structure of HZE particles and simulating DSBs in realistic cell geometry. We simulated 224 data points (D, A, Z, E) with the BDSTRACKS model, leading to a large coverage of LET from 10 to 2,400 keV/µm. This model was used to generate theoretical RBE for various particles and energies for both cell death and mutation frequencies. The RBE LET dependence is in agreement with experimental data known in human and murine cells. It suggests that cell shape and its orientation with respect to the HZE particle beam can modify the biological response to radiation. Such discovery will be tested experimentally and, if proven accurate, will be another strong supporting evidence for DNA repair domains and their critical role in interpreting cosmic radiation sensitivity.

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

Role of the metabotropic P2Y(4) receptor during hypoglycemia: cross talk with the ionotropic NMDAR1 receptor.

PubMed

Cavaliere, Fabio; Amadio, Susanna; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2004-10-15

It is well established that both extracellular ATP and glutamate exert a critical role during metabolic impairment, that several P2 receptor subunits are directly involved in this action and that a strong relationship exists between glutamatergic and purinergic signals. Therefore, here we studied the molecular behavior of the purinergic metabotropic P2Y(4) and the glutamatergic ionotropic NMDAR1 receptors during hypoglycemic cell death. We find that these proteins are oppositely modulated during glucose starvation (P2Y(4) is induced, whereas NMDAR1 is inhibited) and that both P2 and NMDA antagonists can restore basal protein expression levels. Moreover, double immunofluorescence experiments with confocal laser microscopy reveal co-localization at the membrane level between the P2Y(4) and NMDAR1 receptors, in both homologous (cerebellar granule neurons) and heterologous (Hek-293) cellular systems. This is furthermore confirmed by co-immunoprecipitation experiments. Finally, when we express the P2Y(4) receptor in the heterologous SH-SY5Y neuronal cell line, hypoglycemia then causes severe cell death and simultaneous downregulation of the NMDAR1 protein. In summary, our work establishes a potential molecular interplay between P2Y(4) and NMDAR1 receptors during glucose deprivation and the causative role of the P2Y(4) during cell death.

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

Bacterial Pore-Forming Toxins Promote the Activation of Caspases in Parallel to Necroptosis to Enhance Alarmin Release and Inflammation During Pneumonia.

PubMed

Gonzalez-Juarbe, Norberto; Bradley, Kelley M; Riegler, Ashleigh N; Reyes, Luis F; Brissac, Terry; Park, Sang-Sang; Restrepo, Marcos I; Orihuela, Carlos J

2018-04-11

Pore-forming toxins are the most common virulence factor in pathogenic bacteria. They lead to membrane permeabilization and cell death. Herein, we show that respiratory epithelial cells (REC) undergoing bacterial pore-forming toxin (PFT)-induced necroptosis simultaneously experienced caspase activation independently of RIPK3. MLKL deficient REC treated with a pan-caspase inhibitor were protected in an additive manner against PFT-induced death. Subsequently, cleaved versions of caspases-2, -4 and -10 were detected within REC undergoing necroptosis by immunoblots and monoclonal antibody staining. Caspase activation was observed in lung samples from mice and non-human primates experiencing Gram-negative and Gram-positive bacterial pneumonia, respectively. During apoptosis, caspase activation normally leads to cell shrinkage, nuclear condensation, and immunoquiescent death. In contrast, caspase activity during PFT-induced necroptosis increased the release of alarmins to the extracellular milieu. Caspase-mediated alarmin release was found sufficient to activate resting macrophages, leading to Interleukin-6 production. In a mouse model of Gram-negative pneumonia, deletion of caspases -2 and -11, the mouse orthologue of caspase-4, reduced pulmonary inflammation, immune cell infiltration and lung damage. Thus, our study describes a previously unrecognized role for caspase activation in parallel to necroptosis, and indicates that their activity plays a critical pro-inflammatory role during bacterial pneumonia.

Targeting Cancer Cells with Reactive Oxygen and Nitrogen Species Generated by Atmospheric-Pressure Air Plasma

PubMed Central

Hoan, Nguyen Ngoc; Kim, Churl Ho; Moon, Eunpyo; Choi, Kyeong Sook; Yang, Sang Sik; Lee, Jong-Soo

2014-01-01

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH−, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells. PMID:24465942

Synergistic Anti-Cancer Effect of Phenformin and Oxamate

PubMed Central

Miskimins, W. Keith; Ahn, Hyun Joo; Kim, Ji Yeon; Ryu, Sun; Jung, Yuh-Seog; Choi, Joon Young

2014-01-01

Phenformin (phenethylbiguanide; an anti-diabetic agent) plus oxamate [lactate dehydrogenase (LDH) inhibitor] was tested as a potential anti-cancer therapeutic combination. In in vitro studies, phenformin was more potent than metformin, another biguanide, recently recognized to have anti-cancer effects, in promoting cancer cell death in the range of 25 times to 15 million times in various cancer cell lines. The anti-cancer effect of phenformin was related to complex I inhibition in the mitochondria and subsequent overproduction of reactive oxygen species (ROS). Addition of oxamate inhibited LDH activity and lactate production by cells, which is a major side effect of biguanides, and induced more rapid cancer cell death by decreasing ATP production and accelerating ROS production. Phenformin plus oxamate was more effective than phenformin combined with LDH knockdown. In a syngeneic mouse model, phenformin with oxamate increased tumor apoptosis, reduced tumor size and 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography compared to control. We conclude that phenformin is more cytotoxic towards cancer cells than metformin. Furthermore, phenformin and oxamate have synergistic anti-cancer effects through simultaneous inhibition of complex I in the mitochondria and LDH in the cytosol, respectively. PMID:24465604

Synergistic anti-cancer effect of phenformin and oxamate.

PubMed

Miskimins, W Keith; Ahn, Hyun Joo; Kim, Ji Yeon; Ryu, Sun; Jung, Yuh-Seog; Choi, Joon Young

2014-01-01

Phenformin (phenethylbiguanide; an anti-diabetic agent) plus oxamate [lactate dehydrogenase (LDH) inhibitor] was tested as a potential anti-cancer therapeutic combination. In in vitro studies, phenformin was more potent than metformin, another biguanide, recently recognized to have anti-cancer effects, in promoting cancer cell death in the range of 25 times to 15 million times in various cancer cell lines. The anti-cancer effect of phenformin was related to complex I inhibition in the mitochondria and subsequent overproduction of reactive oxygen species (ROS). Addition of oxamate inhibited LDH activity and lactate production by cells, which is a major side effect of biguanides, and induced more rapid cancer cell death by decreasing ATP production and accelerating ROS production. Phenformin plus oxamate was more effective than phenformin combined with LDH knockdown. In a syngeneic mouse model, phenformin with oxamate increased tumor apoptosis, reduced tumor size and (18)F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography compared to control. We conclude that phenformin is more cytotoxic towards cancer cells than metformin. Furthermore, phenformin and oxamate have synergistic anti-cancer effects through simultaneous inhibition of complex I in the mitochondria and LDH in the cytosol, respectively.

A kinetic theory for age-structured stochastic birth-death processes

NASA Astrophysics Data System (ADS)

Chou, Tom; Greenman, Chris

Classical age-structured mass-action models such as the McKendrick-von Foerster equation have been extensively studied but they are structurally unable to describe stochastic fluctuations or population-size-dependent birth and death rates. Conversely, current theories that include size-dependent population dynamics (e.g., carrying capacity) cannot be easily extended to take into account age-dependent birth and death rates. In this paper, we present a systematic derivation of a new fully stochastic kinetic theory for interacting age-structured populations. By defining multiparticle probability density functions, we derive a hierarchy of kinetic equations for the stochastic evolution of an aging population undergoing birth and death. We show that the fully stochastic age-dependent birth-death process precludes factorization of the corresponding probability densities, which then must be solved by using a BBGKY-like hierarchy. Our results generalize both deterministic models and existing master equation approaches by providing an intuitive and efficient way to simultaneously model age- and population-dependent stochastic dynamics applicable to the study of demography, stem cell dynamics, and disease evolution. NSF.

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe

PubMed Central

Rello-Varona, S; Kepp, O; Vitale, I; Michaud, M; Senovilla, L; Jemaà, M; Joza, N; Galluzzi, L; Castedo, M; Kroemer, G

2010-01-01

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and – in this context – revealed an important role of p53 in the control of centrosome number. PMID:21364633

Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle

PubMed Central

Agol, Vadim I.; Belov, George A.; Bienz, Kurt; Egger, Denise; Kolesnikova, Marina S.; Romanova, Lyudmila I.; Sladkova, Larissa V.; Tolskaya, Elena A.

2000-01-01

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors. PMID:10823859

Responses of Solid Tumor Cells in DMEM to Reactive Oxygen Species Generated by Non-Thermal Plasma and Chemically Induced ROS Systems

PubMed Central

Kaushik, Neha; Uddin, Nizam; Sim, Geon Bo; Hong, Young June; Baik, Ku Youn; Kim, Chung Hyeok; Lee, Su Jae; Kaushik, Nagendra Kumar; Choi, Eun Ha

2015-01-01

In this study, we assessed the role of different reactive oxygen species (ROS) generated by soft jet plasma and chemical-induced ROS systems with regard to cell death in T98G, A549, HEK293 and MRC5 cell lines. For a comparison with plasma, we generated superoxide anion (O2−), hydroxyl radical (HO·), and hydrogen peroxide (H2O2) with chemicals inside an in vitro cell culture. Our data revealed that plasma decreased the viability and intracellular ATP values of cells and increased the apoptotic population via a caspase activation mechanism. Plasma altered the mitochondrial membrane potential and eventually up-regulated the mRNA expression levels of BAX, BAK1 and H2AX gene but simultaneously down-regulated the levels of Bcl-2 in solid tumor cells. Moreover, a western blot analysis confirmed that plasma also altered phosphorylated ERK1/2/MAPK protein levels. At the same time, using ROS scavengers with plasma, we observed that scavengers of HO· (mannitol) and H2O2 (catalase and sodium pyruvate) attenuated the activity of plasma on cells to a large extent. In contrast, radicals generated by specific chemical systems enhanced cell death drastically in cancer as well as normal cell lines in a dose-dependent fashion but not specific with regard to the cell type as compared to plasma. PMID:25715710

Responses of Solid Tumor Cells in DMEM to Reactive Oxygen Species Generated by Non-Thermal Plasma and Chemically Induced ROS Systems

NASA Astrophysics Data System (ADS)

Kaushik, Neha; Uddin, Nizam; Sim, Geon Bo; Hong, Young June; Baik, Ku Youn; Kim, Chung Hyeok; Lee, Su Jae; Kaushik, Nagendra Kumar; Choi, Eun Ha

2015-02-01

In this study, we assessed the role of different reactive oxygen species (ROS) generated by soft jet plasma and chemical-induced ROS systems with regard to cell death in T98G, A549, HEK293 and MRC5 cell lines. For a comparison with plasma, we generated superoxide anion (O2-), hydroxyl radical (HO.), and hydrogen peroxide (H2O2) with chemicals inside an in vitro cell culture. Our data revealed that plasma decreased the viability and intracellular ATP values of cells and increased the apoptotic population via a caspase activation mechanism. Plasma altered the mitochondrial membrane potential and eventually up-regulated the mRNA expression levels of BAX, BAK1 and H2AX gene but simultaneously down-regulated the levels of Bcl-2 in solid tumor cells. Moreover, a western blot analysis confirmed that plasma also altered phosphorylated ERK1/2/MAPK protein levels. At the same time, using ROS scavengers with plasma, we observed that scavengers of HO. (mannitol) and H2O2 (catalase and sodium pyruvate) attenuated the activity of plasma on cells to a large extent. In contrast, radicals generated by specific chemical systems enhanced cell death drastically in cancer as well as normal cell lines in a dose-dependent fashion but not specific with regard to the cell type as compared to plasma.

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

PubMed

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

2014-07-25

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

The interruption of PKC-ι signaling and TRAIL combination therapy against glioblastoma cells.

PubMed

McCray, Andrea N; Desai, Shraddha; Acevedo-Duncan, Mildred

2014-09-01

Glioblastoma is a highly aggressive type of brain cancer which currently has limited options for treatment. It is imperative to develop combination therapies that could cause apoptosis in glioblastoma. The aim of this study was to characterize the affect of modified ICA-1, a PKC-iota inhibitor, on the growth pattern of various glioblastoma cell lines. T98G and U87 glioblastoma cells were treated with ICA-1 alone and the absolute cell numbers of each group were determined for cell growth expansion analysis, cell viability analysis, and cell death analysis. Low dose ICA-1 treatment alone significantly inhibited cell growth expansion of high density glioblastoma cells without inducing cell death. However, the high dose ICA-1 treatment regimen provided significant apoptosis for glioblastoma cells. Furthermore, this study was conducted to use a two layer molecular level approach for treating glioblastoma cells with ICA-1 plus an apoptosis agent, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), to induce apoptosis in such chemo-refractory cancer cells. Following ICA-1 plus TRAIL treatment, apoptosis was detected in glioblastoma cells via the TUNEL assay and via flow cytometric analysis using Annexin-V FITC/PI. This study offers the first evidence for ICA-1 alone to inhibit glioblastoma cell proliferation as well as the novel combination of ICA-1 with TRAIL to cause robust apoptosis in a caspase-3 mediated mechanism. Furthermore, ICA-1 plus TRAIL simultaneously modulates down-regulation of PKC-iota and c-Jun.

miR-203 inhibits cell proliferation and promotes cisplatin induced cell death in tongue squamous cancer

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

Lin, Jiong; Lin, Yao; Fan, Li

Oral squamous cell carcinoma (OSCC) is one of the most common types of the head and neck cancer. Chemo resistance of OSCC has been identified as a substantial therapeutic hurdle. In this study, we analyzed the role of miR-203 in the OSCC and its effects on cisplatin-induced cell death in an OSCC cell line, Tca8113. There was a significant decrease of miR-203 expression in OSCC samples, compared with the adjacent normal, non-cancerous tissue. After 3 days cisplatin treatment, the survived Tca8113 cells had a lower expression of miR-203 than that in the untreated control group. In contrast, PIK3CA showed an inversemore » expression in cancer and cisplatin survived Tca8113 cells. Transfection of Tca8113 cells with miR-203 mimics greatly reduced PIK3CA expression and Akt activation. Furthermore, miR-203 repressed PIK3CA expression through targeting the 3′UTR. Restoration of miR-203 not only suppressed cell proliferation, but also sensitized cells to cisplatin induced cell apoptosis. This effect was absent in cells that were simultaneously treated with PIK3CA RNAi. In summary, these findings suggest miR-203 plays an important role in cisplatin resistance in OSCC, and furthermore delivery of miR-203 analogs may serve as an adjuvant therapy for OSCC. - Highlights: • Much lower miR-203 expression in cisplatin resistant Tca8113 cells is discovered. • Delivery of miR-203 can sensitize the Tca8113 cells to cisplatin induced cell death. • MiR-203 can downregulate PIK3CA through the 3′UTR. • The effects of miR-203 on cisplatin sensitivity is mainly through PIK3CA pathway.« less

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

Scott, F.; Stec, B; Pop, C

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis1, 2, 3. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation4, 5. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed andmore » isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.« less

Interplay between autophagy and apoptosis in lead(II)-induced cytotoxicity of primary rat proximal tubular cells.

PubMed

Chu, Bing-Xin; Fan, Rui-Feng; Lin, Shu-Qian; Yang, Du-Bao; Wang, Zhen-Yong; Wang, Lin

2018-05-01

Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity. Copyright © 2018. Published by Elsevier Inc.

Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production.

PubMed

Singh, Shuchi; Agarwal, Mayank; Sarma, Shyamali; Goyal, Arun; Moholkar, Vijayanand S

2015-09-01

This paper presents investigations into mechanism of ultrasound assisted bioethanol synthesis using Parthenium hysterophorus biomass through simultaneous saccharification and fermentation (SSF) mode. Approach of coupling experimental results to mathematical model for SSF using Genetic Algorithm based optimization has been adopted. Comparison of model parameters for experiments with mechanical shaking and sonication (10% duty cycle) give an interesting mechanistic account of influence of ultrasound on SSF system. A 4-fold rise in ethanol and cell mass productivity is seen with ultrasound. The analysis reveals following facets of influence of ultrasound on SSF: increase in Monod constant for glucose for cell growth, maximal specific growth rate and inhibition constant of cell growth by glucose and reduction in specific cell death rate. Values of inhibition constant of cell growth by ethanol (K3E), and constants for growth associated (a) and non-growth associated (b) ethanol production remained unaltered with sonication. Beneficial effects of ultrasound are attributed to enhanced cellulose hydrolysis, enhanced trans-membrane transport of substrate and products as well as dilution of the toxic substances due to micro-convection induced by ultrasound. Intrinsic physiological functioning of cells remained unaffected by ultrasound as indicated by unaltered values of K3E, a and b. Copyright © 2015 Elsevier B.V. All rights reserved.

Lethal cellular changes induced by near ultraviolet radiation.

PubMed

Tyrrell, R M

1979-01-01

There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths. No particular DNA lesion can be linked to cell death in wild type strains. However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair. There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general. Some important specific questions remain. For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested. In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model.

Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

PubMed

Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

2012-10-01

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF).

Activation of concurrent apoptosis and necroptosis by SMAC mimetics for the treatment of refractory and relapsed ALL.

PubMed

McComb, Scott; Aguadé-Gorgorió, Júlia; Harder, Lena; Marovca, Blerim; Cario, Gunnar; Eckert, Cornelia; Schrappe, Martin; Stanulla, Martin; von Stackelberg, Arend; Bourquin, Jean-Pierre; Bornhauser, Beat C

2016-05-18

More precise treatment strategies are urgently needed to decrease toxicity and improve outcomes for treatment-refractory leukemia. We used ex vivo drug response profiling of high-risk, relapsed, or refractory acute lymphoblastic leukemia (ALL) cases and identified a subset with exquisite sensitivity to small-molecule mimetics of the second mitochondria-derived activator of caspases (SMAC) protein. Potent ex vivo activity of the SMAC mimetic (SM) birinapant correlated with marked in vivo antileukemic effects, as indicated by delayed engraftment, decreased leukemia burden, and prolonged survival of xenografted mice. Antileukemic activity was dependent on simultaneous execution of apoptosis and necroptosis, as demonstrated by functional genomic dissection with a multicolored lentiCRISPR approach to simultaneously disrupt multiple genes in patient-derived ALL. SM specifically targeted receptor-interacting protein kinase 1 (RIP1)-dependent death, and CRISPR-mediated disruption of RIP1 completely blocked SM-induced death yet had no impact on the response to standard antileukemic agents. Thus, SM compounds such as birinapant circumvent escape from apoptosis in leukemia by activating a potent dual RIP1-dependent apoptotic and necroptotic cell death, which is not exploited by current therapy. Ex vivo drug activity profiling could provide important functional diagnostic information to identify patients who may benefit from targeted treatment with birinapant in early clinical trials. Copyright © 2016, American Association for the Advancement of Science.

Light acclimation, retrograde signalling, cell death and immune defences in plants.

PubMed

Karpiński, Stanisław; Szechyńska-Hebda, Magdalena; Wituszyńska, Weronika; Burdiak, Paweł

2013-04-01

This review confronts the classical view of plant immune defence and light acclimation with recently published data. Earlier findings have linked plant immune defences to nucleotide-binding site leucine-rich repeat (NBS-LRR)-dependent recognition of pathogen effectors and to the role of plasma membrane-localized NADPH-dependent oxidoreductase (AtRbohD), reactive oxygen species (ROS) and salicylic acid (SA). However, recent results suggest that plant immune defence also depends on the absorption of excessive light energy and photorespiration. Rapid changes in light intensity and quality often cause the absorption of energy, which is in excess of that required for photosynthesis. Such excessive light energy is considered to be a factor triggering photoinhibition and disturbance in ROS/hormonal homeostasis, which leads to cell death in foliar tissues. We highlight here the tight crosstalk between ROS- and SA-dependent pathways leading to light acclimation, and defence responses leading to pathogen resistance. We also show that LESION SIMULATING DISEASE 1 (LSD1) regulates and integrates these processes. Moreover, we discuss the role of plastid-nucleus signal transduction, photorespiration, photoelectrochemical signalling and 'light memory' in the regulation of acclimation and immune defence responses. All of these results suggest that plants have evolved a genetic system that simultaneously regulates systemic acquired resistance (SAR), cell death and systemic acquired acclimation (SAA). © 2012 Blackwell Publishing Ltd.

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

PubMed Central

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

2014-01-01

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

Gold nanorod embedded reduction responsive block copolymer micelle-triggered drug delivery combined with photothermal ablation for targeted cancer therapy.

PubMed

Parida, Sheetal; Maiti, Chiranjit; Rajesh, Y; Dey, Kaushik K; Pal, Ipsita; Parekh, Aditya; Patra, Rusha; Dhara, Dibakar; Dutta, Pranab Kumar; Mandal, Mahitosh

2017-01-01

Gold nanorods, by virtue of surface plasmon resonance, convert incident light energy (NIR) into heat energy which induces hyperthermia. We designed unique, multifunctional, gold nanorod embedded block copolymer micelle loaded with GW627368X for targeted drug delivery and photothermal therapy. Glutathione responsive diblock co-polymer was synthesized by RAFT process forming self-assembled micelle on gold nanorods prepared by seed mediated method and GW627368X was loaded on to the reduction responsive gold nanorod embedded micelle. Photothermal therapy was administered using cwNIR laser (808nm; 4W/cm 2 ). Efficacy of nanoformulated GW627368X, photothermal therapy and combination of both were evaluated in vitro and in vivo. In response to photothermal treatment, cells undergo regulated, patterned cell death by necroptosis. Combining GW627368X with photothermal treatment using single nanoparticle enhanced therapeutic outcome. In addition, these nanoparticles are effective X-ray CT contrast agents, thus, can help in monitoring treatment. Reduction responsive nanorod embedded micelle containing folic acid and lipoic acid when treated on cervical cancer cells or tumour bearing mice, aggregate in and around cancer cells. Due to high glutathione concentration, micelles degrade releasing drug which binds surface receptors inducing apoptosis. When incident with 808nm cwNIR lasers, gold nanorods bring about photothermal effect leading to hyperthermic cell death by necroptosis. Combination of the two modalities enhances therapeutic efficacy by inducing both forms of cell death. Our proposed treatment strategy achieves photothermal therapy and targeted drug delivery simultaneously. It can prove useful in overcoming general toxicities associated with chemotherapeutics and intrinsic/acquired resistance to chemo and radiotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

NASA Technical Reports Server (NTRS)

Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

1999-01-01

BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

Extra-virgin olive oil phenols block cell cycle progression and modulate chemotherapeutic toxicity in bladder cancer cells

PubMed Central

Coccia, Andrea; Mosca, Luciana; Puca, Rosa; Mangino, Giorgio; Rossi, Alessandro; Lendaro, Eugenio

2016-01-01

Epidemiological data indicate that the daily consumption of extra-virgin olive oil (EVOO), a common dietary habit of the Mediterranean area, lowers the incidence of certain types of cancer, in particular bladder neoplasm. The aim of the present study was to evaluate the antiproliferative activity of polyphenols extracted from EVOO on bladder cancer (BCa), and to clarify the biological mechanisms that trigger cell death. Furthermore, we also evaluated the ability of low doses of extra-virgin olive oil extract (EVOOE) to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer. Our results showed that EVOOE significantly inhibited the proliferation and clonogenic ability of T24 and 5637 BCa cells in a dose-dependent manner. Furthermore, cell cycle analysis after EVOOE treatment showed a marked growth arrest prior to mitosis in the G2/M phase for both cell lines, with the subsequent induction of apoptosis only in the T24 cells. Notably, simultaneous treatment of mitomycin C and EVOOE reduced the drug cytotoxicity due to inhibition of ROS production. Conversely, the co-treatment of T24 cells with paclitaxel and the polyphenol extract strongly increased the apoptotic cell death at each tested concentration compared to paclitaxel alone. Our results support the epidemiological evidence indicating that olive oil consumption exerts health benefits and may represent a starting point for the development of new anticancer strategies. PMID:27748855

The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2

PubMed Central

Chen, Bo; Xu, Ming; Zhang, Hui; Xu, Ming-zheng; Wang, Xu-jing; Tang, Qing-he

2015-01-01

In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK–mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer. PMID:26284306

The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2.

PubMed

Chen, Bo; Xu, Ming; Zhang, Hui; Xu, Ming-zheng; Wang, Xu-jing; Tang, Qing-he; Tang, Jian-ying

2015-10-01

In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK-mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy.

PubMed

Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo . Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy

PubMed Central

Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

2014-01-01

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities. PMID:25954597

Primary hepatocytes and their cultures in liver apoptosis research

PubMed Central

Vinken, Mathieu; Maes, Michaël; Oliveira, André G.; Cogliati, Bruno; Marques, Pedro E.; Menezes, Gustavo B.; Dagli, Maria Lúcia Zaidan; Vanhaecke, Tamara; Rogiers, Vera

2014-01-01

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed. PMID:24013573

Curcumin nanoformulations: a future nanomedicine for cancer

PubMed Central

Yallapu, Murali M; Jaggi, Meena; Chauhan, Subhash C

2011-01-01

Curcumin, a natural diphenolic compound derived from turmeric Curcuma longa, has proven to be a modulator of intracellular signaling pathways that control cancer cell growth, inflammation, invasion, apoptosis and cell death, revealing its anticancer potential. In this review, we focus on the design and development of nanoparticles, self-assemblies, nanogels, liposomes and complex fabrication for sustained and efficient curcumin delivery. We also discuss the anticancer applications and clinical benefits of nanocurcumin formulations. Only a few novel multifunctional and composite nanosystem strategies offer simultaneous therapy as well as imaging characteristics. We also summarize the challenges to developing curcumin delivery platforms and up-to-date solutions for improving curcumin bioavailability and anticancer potential for therapy. PMID:21959306

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.

Intracellular localization analysis of npAu-PpIX in HeLa cells using specific dyes and confocal microscopy

NASA Astrophysics Data System (ADS)

Roblero-Bartolón, Victoria Gabriela; Maldonado-Alvarado, Elizabeth; Galván-Mendoza, José Iván; Ramón-Gallegos, Eva

2012-10-01

Cervical carcinoma (CC) represents the second leading cause of cancer death in Mexican women. No conventional treatments are being developed such as photodynamic therapy (PDT), involving the simultaneous presence of a photosensitizer (Ps), light of a specific wavelength and tissue oxygen. On the other hand, it has seen that the use of gold nanoparticles coupled to protoporphyrin IX increases the effectiveness of PDT. The aim of this study was to determine the site of accumulation of the conjugate npAu-PpIX in cells of cervical cancer by the use of specific dyes and confocal microscopy. The results indicate that the gold nanoparticles coupled to protoporphyrin IX are accumulated in both the cytoplasm and nucleus of HeLa cells.

Cancer: shift of the paradigm.

PubMed

Lichtenstein, Anatoly V

2008-12-01

Cancer is usually considered to be a by-product of design limitations of a multicellular organism and its intrinsic fallibility. However, recent data prompt a revision of some established notions about carcinogenesis and form a new paradigm of carcinogenesis as a highly conserved biological phenomenon - a programmed death of an organism. This altruistic program, which is unleashed when mutagenesis surpasses a certain critical threshold, gives a population the important benefit acting as a guardian of the gene pool against the spread of certain mutant genes. A growing body of evidence supports this point of view: (i) epigenetic changes leading to cancer arise early, simultaneously in many cells and look like deterministic regulation; (ii) concept of cancer stem cell suggests a view of carcinogenesis not as vague transformation but as well known differentiation; (iii) tumor/host relations usually perceived as antagonistic are, in reality, synergistic; (iv) death of an individual from cancer is predetermined and results apparently from a specific activity (killer function) of cancer cell and (v) evolutionary conservation indicates that cancer comes with a general advantage that explains its evolutionary success. A holistic approach to carcinogenesis suggests new avenues of research and new therapeutic strategy.

Antibacterial effect of silver nanoparticles and the modeling of bacterial growth kinetics using a modified Gompertz model.

PubMed

Chatterjee, Tanaya; Chatterjee, Barun K; Majumdar, Dipanwita; Chakrabarti, Pinak

2015-02-01

An alternative to conventional antibiotics is needed to fight against emerging multiple drug resistant pathogenic bacteria. In this endeavor, the effect of silver nanoparticle (Ag-NP) has been studied quantitatively on two common pathogenic bacteria Escherichia coli and Staphylococcus aureus, and the growth curves were modeled. The effect of Ag-NP on bacterial growth kinetics was studied by measuring the optical density, and was fitted by non-linear regression using the Logistic and modified Gompertz models. Scanning Electron Microscopy and fluorescence microscopy were used to study the morphological changes of the bacterial cells. Generation of reactive oxygen species for Ag-NP treated cells were measured by fluorescence emission spectra. The modified Gompertz model, incorporating cell death, fits the observed data better than the Logistic model. With increasing concentration of Ag-NP, the growth kinetics of both bacteria shows a decline in growth rate with simultaneous enhancement of death rate constants. The duration of the lag phase was found to increase with Ag-NP concentration. SEM showed morphological changes, while fluorescence microscopy using DAPI showed compaction of DNA for Ag-NP-treated bacterial cells. E. coli was found to be more susceptible to Ag-NP as compared to S. aureus. The modified Gompertz model, using a death term, was found to be useful in explaining the non-monotonic nature of the growth curve. The modified Gompertz model derived here is of general nature and can be used to study any microbial growth kinetics under the influence of antimicrobial agents. Copyright © 2014 Elsevier B.V. All rights reserved.

CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons.

PubMed

Valencia-Olvera, Ana Carolina; Morán, Julio; Camacho-Carranza, Rafael; Prospéro-García, Oscar; Espinosa-Aguirre, Jesús Javier

2014-10-01

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. In the liver, some xenobiotics can be metabolized by CYPs into more reactive products that can damage hepatocytes and induce cell death. In addition, normal CYP activity may produce reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS leading to cytotoxicity in multiple tissue types. The aim of this study was to determine whether CYP2E1 induction may lead to significant brain cell impairment. Immunological analysis revealed that exposure of primary cerebellar granule neuronal cultures to the CYP inducer isoniazid, increased CYP2E1 expression. In the presence of buthionine sulfoximine, an agent that reduces glutathione levels, isoniazid treatment also resulted in reactive oxygen species (ROS) production, DNA oxidation and cell death. These effects were attenuated by simultaneous exposure to diallyl sulfide, a CYP2E1 inhibitor, or to a mimetic of superoxide dismutase/catalase, (Euka). These results suggest that in cases of reduced antioxidant levels, the induction of brain CYP2E1 could represent a risk of in situ neuronal damage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

PubMed Central

Vardi, Assaf; Haramaty, Liti; Van Mooy, Benjamin A. S.; Fredricks, Helen F.; Kimmance, Susan A.; Larsen, Aud; Bidle, Kay D.

2012-01-01

Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host–virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the “arms race” between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi–EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host–virus interactions in natural systems. PMID:23134731

Lansoprazole protects and heals gastric mucosa from non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy by inhibiting mitochondrial as well as Fas-mediated death pathways with concurrent induction of mucosal cell renewal.

PubMed

Maity, Pallab; Bindu, Samik; Choubey, Vinay; Alam, Athar; Mitra, Kalyan; Goyal, Manish; Dey, Sumanta; Guha, Mithu; Pal, Chinmay; Bandyopadhyay, Uday

2008-05-23

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl(xL) to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells.

PubMed

Cavaliere, Fabio; Nestola, Valeria; Amadio, Susanna; D'Ambrosi, Nadia; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2005-02-01

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X1,2,4,5,6,7 and P2Y1,2,4,6, but not the P2X3 and P2Y12 receptors. We then investigated which biological effects were mediated by the P2Y4 subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y4 protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y4 also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP also induced cell death, both in naive, differentiated, and P2Y4-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y4 receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.

Hippo circuitry and the redox modulation of hippo components in cancer cell fate decisions.

PubMed

Ashraf, Asma; Pervaiz, Shazib

2015-12-01

Meticulous and precise control of organ size is undoubtedly one of the most pivotal processes in mammalian development and regeneration along with cell differentiation, morphogenesis and programmed cell death. These processes are strictly regulated by complex and highly coordinated mechanisms to maintain a steady growth state. There are a number of extrinsic and intrinsic factors that dictate the total number and/or size of cells by influencing growth, proliferation, differentiation and cell death. Multiple pathways, such as those involved in promoting organ size and others that restrict disproportionate tissue growth act simultaneously to maintain cellular and tissue homeostasis. Aberrations at any level in these organ size-regulating processes can lead to various pathological states with cancers being the most formidable one (Yin and Zhang, 2011). Extensive research in the realm of growth control has led to the identification of the Hippo-signaling pathway as a critical network in modulating tissue growth via its effect on multiple signaling pathways and through intricate crosstalk with proteins that regulate cell polarity, adhesion and cell-cell interactions (Zhao et al., 2011b). The Hippo pathway controls cell number and organ size by transducing signals from the plasma membrane to the nucleus to regulate the expression of genes involved in cell fate determination (Shi et al., 2015). In this review, we summarize the recent discoveries concerning Hippo pathway, its diversiform regulation in mammals as well as its implications in cancers, and highlight the possible role of oxidative stress in Hippo pathway regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Angiotensin II type 1 receptor blocker telmisartan induces apoptosis and autophagy in adult T-cell leukemia cells.

PubMed

Kozako, Tomohiro; Soeda, Shuhei; Yoshimitsu, Makoto; Arima, Naomichi; Kuroki, Ayako; Hirata, Shinya; Tanaka, Hiroaki; Imakyure, Osamu; Tone, Nanako; Honda, Shin-Ichiro; Soeda, Shinji

2016-05-01

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

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.

Multifunctional hybrid materials for combined photo and chemotherapy of cancer.

PubMed

Botella, Pablo; Ortega, Ilida; Quesada, Manuel; Madrigal, Roque F; Muniesa, Carlos; Fimia, Antonio; Fernández, Eduardo; Corma, Avelino

2012-08-21

Combined chemo and photothermal therapy in in vitro testing has been achieved by means of multifunctional nanoparticles formed by plasmonic gold nanoclusters with a protecting shell of porous silica that contains an antitumor drug. We propose a therapeutic nanoplatform that associates the optical activity of small gold nanoparticles aggregates with the cytotoxic activity of 20(S)-camptothecin simultaneously released for the efficient destruction of cancer cells. For this purpose, a method was used for the controlled assembly of gold nanoparticles into stable clusters with a tailored absorption cross-section in the vis/NIR spectrum, which involves aggregation in alkaline medium of 15 nm diameter gold colloids protected with a thin silica layer. Clusters were further encapsulated in an ordered homogeneous mesoporous silica coating that provides biocompatibility and stability in physiological fluids. After internalization in 42-MG-BA human glioma cells, these protected gold nanoclusters were able to produce effective photothermolysis under femtosecond pulse laser irradiation of 790 nm. Cell death occurred by combination of a thermal mechanism and mechanical disruption of the membrane cell due to induced generation of micrometer-scale bubbles by vaporizing the water inside the channels of the mesoporous silica coating. Moreover, the incorporation of 20(S)-camptothecin within the pores of the external shell, which was released during the process, provoked significant cell death increase. This therapeutic model could be of interest for application in the treatment and suppression of non-solid tumors.

Pathways of tumor development and progression in drug-induced nonmelanoma skin cancer: a new hope or the next great confusion?

PubMed

Tchernev, Georgi; Wollina, Uwe

2014-07-01

The factors that lead to the clinical manifestation of the nonmelanocytic skin tumors are different. Ultraviolet radiation, infections with human papillomaviruses, and inherited or iatrogenic-induced immunosuppression (in cases of autoimmune diseases and organ transplant recipients) are considered to be some of the most important generators and/or costimulating factors supporting the appearance of "de-novo" mutations and obstruct, in one or another way, the cell cycle arrest, the programmed cell death (apoptosis), and the immunosurveillance. Preconditions are thus created for the initial persistence and subsequent proliferation of the malignant cell branch in the genome, with the simultaneous increase of the risk of nonmelanocytic skin tumor manifestation.A number of medical drugs that possess a currently well-known selective, targeting, and immunomodulating effect, like the TNF-alpha inhibitors for example, most probably possess an additional blocking action on the death receptors within the framework of the extrinsic apoptotic pathway. In this way, they seem to be one of the major factors for the clinical manifestation not only of nonmelanocytic skin but also of a number of other type of tumors with a dependency on the genetic predisposition of each separate patient.This article focuses the attention on the basic exogenic and endogenic factors that affect the regulatory processes of the cellular cycle, apoptosis, immunosurveillance, and the human inflammasome in patients with nonmelanocytic skin tumors. These processes are interwoven in a complex network and are controlled by (1) the genome regulator p53, (2) its interaction with the proapoptotic acting proteins Bak and Bax, (3) as well as the interaction with the key regulatory protein of the inflammasome-ASC/TMS1.As a process, the malignant transformation is exceptionally dynamic, plastic, and adaptive. The exterior "interferences", on the part of the clinician, in the form of a planned therapy should be targeted at the simultaneous impact on the various pathogenetic chains with the objective of bringing the tumor cells to their total collapse. This can be made possible only after the careful and simultaneous-or parallel-examination of a much greater number of markers that serve to characterize the process of the malignant transformation-a fact, which is currently being disregarded by many researchers.

Influence of pulsed electromagnetic and pulsed vector magnetic potential field on the growth of tumor cells.

PubMed

Loja, Tomas; Stehlikova, Olga; Palko, Lukas; Vrba, Kamil; Rampl, Ivan; Klabusay, Martin

2014-09-01

Tumor diseases cause 20% of deaths in Europe and they are the second most common cause of death and morbidity after cardiovascular diseases. Thus, tumor cells are target of many therapeutic strategies and tumor research is focused on searching more efficient and specific drugs as well as new therapeutic approaches. One of the areas of tumor research is an issue of external fields. In our work, we tested influence of a pulsed electromagnetic field (PEMF) and a hypothetic field of the pulsed vector magnetic potential (PVMP) on the growth of tumor cells; and further the possible growth inhibition effect of the PVMP. Both unipolar and bipolar PEMF fields of 5 mT and PVMP fields of 0 mT at frequencies of 15 Hz, 125 Hz and 625 Hz were tested on cancer cell lines derived from various types of tumors: CEM/C2 (acute lymphoblastic leukemia), SU-DHL-4 (B-cell lymphoma), COLO-320DM (colorectal adenocarcinoma), MDA-BM-468 (breast adenocarcinoma), and ZR-75-1 (ductal carcinoma). Cell morphology was observed, proliferation activity using WST assay was measured and simultaneous proportion of live, early apoptotic and dead cells was detected using flow cytometry. A PEMF of 125 Hz and 625 Hz for 24 h-48 h increased proliferation activity in the 2 types of cancer cell lines used, i.e. COLO-320DM and ZR-75-1. In contrast, any of employed methods did not confirm a significant inhibitory effect of hypothetic PVMP field on tumor cells.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

A mechanistic model for the evolution of multicellularity

NASA Astrophysics Data System (ADS)

Amado, André; Batista, Carlos; Campos, Paulo R. A.

2018-02-01

Through a mechanistic approach we investigate the formation of aggregates of variable sizes, accounting mechanisms of aggregation, dissociation, death and reproduction. In our model, cells can produce two metabolites, but the simultaneous production of both metabolites is costly in terms of fitness. Thus, the formation of larger groups can favor the aggregates to evolve to a configuration where division of labor arises. It is assumed that the states of the cells in a group are those that maximize organismal fitness. In the model it is considered that the groups can grow linearly, forming a chain, or compactly keeping a roughly spherical shape. Starting from a population consisting of single-celled organisms, we observe the formation of groups with variable sizes and usually much larger than two-cell aggregates. Natural selection can favor the formation of large groups, which allows the system to achieve new and larger fitness maxima.

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2

PubMed Central

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E.; Chen, Shuang; Bodie, Wesley W.; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A.; Kramer, Lora B.; Dent, Paul

2011-01-01

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition. PMID:21911831

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

PubMed

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E; Chen, Shuang; Bodie, Wesley W; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A; Kramer, Lora B; Dent, Paul; Grant, Steven

2011-11-10

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Changes in antioxidants are critical in determining cell responses to short- and long-term heat stress.

PubMed

Sgobba, Alessandra; Paradiso, Annalisa; Dipierro, Silvio; De Gara, Laura; de Pinto, Maria Concetta

2015-01-01

Heat stress can have deleterious effects on plant growth by impairing several physiological processes. Plants have several defense mechanisms that enable them to cope with high temperatures. The synthesis and accumulation of heat shock proteins (HSPs), as well as the maintenance of an opportune redox balance play key roles in conferring thermotolerance to plants. In this study changes in redox parameters, the activity and/or expression of reactive oxygen species (ROS) scavenging enzymes and the expression of two HSPs were studied in tobacco Bright Yellow-2 (TBY-2) cells subjected to moderate short-term heat stress (SHS) and long-term heat stress (LHS). The results indicate that TBY-2 cells subjected to SHS suddenly and transiently enhance antioxidant systems, thus maintaining redox homeostasis and avoiding oxidative damage. The simultaneous increase in HSPs overcomes the SHS and maintains the metabolic functionality of cells. In contrast the exposure of cells to LHS significantly reduces cell growth and increases cell death. In the first phase of LHS, cells enhance antioxidant systems to prevent the formation of an oxidizing environment. Under prolonged heat stress, the antioxidant systems, and particularly the enzymatic ones, are inactivated. As a consequence, an increase in H2 O2 , lipid peroxidation and protein oxidation occurs. This establishment of oxidative stress could be responsible for the increased cell death. The rescue of cell growth and cell viability, observed when TBY-2 cells were pretreated with galactone-γ-lactone, the last precursor of ascorbate, and glutathione before exposure to LHS, highlights the crucial role of antioxidants in the acquisition of basal thermotolerance. © 2014 Scandinavian Plant Physiology Society.

Autophagy: not good OR bad, but good AND bad.

PubMed

Altman, Brian J; Rathmell, Jeffrey C

2009-05-01

Autophagy is a well-established mechanism to degrade intracellular components and provide a nutrient source to promote survival of cells in metabolic distress. Such stress can be caused by a lack of available nutrients or by insufficient rates of nutrient uptake. Indeed, growth factor deprivation leads to internalization and degradation of nutrient transporters, leaving cells with limited means to access extracellular nutrients even when plentiful.This loss of growth factor signaling and extracellular nutrients ultimately leads to apoptosis, but also activates autophagy, which may degrade intracellular components and provide fuel for mitochondrial bioenergetics. The precise metabolic role of autophagy and how it intersects with the apoptotic pathways in growth factor withdrawal, however, has been uncertain. Our recent findings ingrowth factor-deprived hematopoietic cells show that autophagy can simultaneously contribute to cell metabolism and initiate a pathway to sensitize cells to apoptotic death. This pathway may promote tissue homeostasis by ensuring that only cells with high resistance to apoptosis may utilize autophagy as a survival mechanism when growth factors are limiting and nutrient uptake decreases.

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Schober, Wendy D.; Koller, Erich; Pinilla, Clemencia; Vassilev, Lyubomir T.; Reed, John C.

2010-01-01

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a–induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic. PMID:19897582

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

PubMed

Grassi, Elisa Stellaria; Vezzoli, Valeria; Negri, Irene; Lábadi, Árpád; Fugazzola, Laura; Vitale, Giovanni; Persani, Luca

2015-11-03

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

Effects of Nosema ceranae and thiametoxam in Apis mellifera: A comparative study in Africanized and Carniolan honey bees.

PubMed

Gregorc, Ales; Silva-Zacarin, Elaine C M; Carvalho, Stephan Malfitano; Kramberger, Doris; Teixeira, Erica W; Malaspina, Osmar

2016-03-01

Multiple stressors, such as chemicals and pathogens, are likely to be detrimental for the health and lifespan of Apis mellifera, a bee species frequently exposed to both factors in the field and inside hives. The main objective of the present study was to evaluate comparatively the health of Carniolan and Africanized honey bees (AHB) co-exposed to thiamethoxam and Nosema ceranae (N. ceranae) spores. Newly-emerged worker honey bees were exposed solely with different sublethal doses of thiamethoxam (2% and 0.2% of LD50 for AHB), which could be consumed by bees under field conditions. Toxicity tests for the Carniolan bees were performed, and the LD50 of thiamethoxam for Carniolan honey bees was 7.86 ng bee(-1). Immunohistological analyses were also performed to detect cell death in the midgut of thiamethoxam and/or N. ceranae treated bees. Thiamethoxam exposure had no negative impact on Nosema development in experimental conditions, but it clearly inhibited cell death in the midgut of thiamethoxam and Nosema-exposed bees, as demonstrated by immunohistochemical data. Indeed, thiamethoxam exposure only had a minor synergistic toxic effect on midgut tissue when applied as a low dose simultaneously with N. ceranae to AHB and Carniolan honey bees, in comparison with the effect caused by both stressors separately. Our data provides insights into the effects of the neonicotenoid thiamethoxam on the AHB and Carniolan honey bee life span, as well as the effects of simultaneous application of thiamethoxam and N. ceranae spores to honey bees. Copyright © 2016. Published by Elsevier Ltd.

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

PubMed

San José-Enériz, Edurne; Agirre, Xabier; Rabal, Obdulia; Vilas-Zornoza, Amaia; Sanchez-Arias, Juan A; Miranda, Estibaliz; Ugarte, Ana; Roa, Sergio; Paiva, Bruno; Estella-Hermoso de Mendoza, Ander; Alvarez, Rosa María; Casares, Noelia; Segura, Victor; Martín-Subero, José I; Ogi, François-Xavier; Soule, Pierre; Santiveri, Clara M; Campos-Olivas, Ramón; Castellano, Giancarlo; de Barrena, Maite Garcia Fernandez; Rodriguez-Madoz, Juan Roberto; García-Barchino, Maria José; Lasarte, Juan Jose; Avila, Matias A; Martinez-Climent, Jose Angel; Oyarzabal, Julen; Prosper, Felipe

2017-05-26

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival.

PubMed

Kishton, Rigel J; Barnes, Carson E; Nichols, Amanda G; Cohen, Sivan; Gerriets, Valerie A; Siska, Peter J; Macintyre, Andrew N; Goraksha-Hicks, Pankuri; de Cubas, Aguirre A; Liu, Tingyu; Warmoes, Marc O; Abel, E Dale; Yeoh, Allen Eng Juh; Gershon, Timothy R; Rathmell, W Kimryn; Richards, Kristy L; Locasale, Jason W; Rathmell, Jeffrey C

2016-04-12

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5' AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

PubMed Central

Singh, Melissa M.; Manton, Christa A.; Bhat, Krishna P.; Tsai, Wen-Wei; Aldape, Kenneth; Barton, Michelle C.; Chandra, Joya

2011-01-01

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM. PMID:21653597

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids.

PubMed

Murphy, Kaitlin C; Whitehead, Jacklyn; Zhou, Dejie; Ho, Steve S; Leach, J Kent

2017-12-01

Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE 2 ) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE 2 ). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE 2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE 2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE 2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE 2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE 2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs. Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Reversal of intramyocellular lipid accumulation by lipophagy and a p62-mediated pathway.

PubMed

Lam, T; Harmancey, R; Vasquez, H; Gilbert, B; Patel, N; Hariharan, V; Lee, A; Covey, M; Taegtmeyer, H

2016-01-01

We have previously observed the reversal of lipid droplet deposition in skeletal muscle of morbidly obese patients following bariatric surgery. We now investigated whether activation of autophagy is the mechanism underlying this observation. For this purpose, we incubated rat L6 myocytes over a period of 6 days with long-chain fatty acids (an equimolar, 1.0 mM, mixture of oleate and palmitate in the incubation medium). At day 6, the autophagic inhibitor (bafilomycin A1, 200 nM) and the autophagic activator (rapamycin, 1 μM) were added separately or in combination for 48 h. Intracellular triglyceride (TG) accumulation was visualized and quantified colorimetrically. Protein markers of autophagic flux (LC3 and p62) and cell death (caspase-3 cleavage) were measured by immunoblotting. Inhibition of autophagy by bafilomycin increased TG accumulation and also increased lipid-mediated cell death. Conversely, activation of autophagy by rapamycin reduced both intracellular lipid accumulation and cell death. Unexpectedly, treatment with both drugs added simultaneously resulted in decreased lipid accumulation. In this treatment group, immunoblotting revealed p62 degradation (autophagic flux), immunofluorescence revealed the colocalization of p62 with lipid droplets, and co-immunoprecipitation confirmed the interaction of p62 with ADRP (adipose differentiation-related protein), a lipid droplet membrane protein. Thus the association of p62 with lipid droplet turnover suggests a novel pathway for the breakdown of lipid droplets in muscle cells. In addition, treatment with rapamycin and bafilomycin together also suggested the export of TG into the extracellular space. We conclude that lipophagy promotes the clearance of lipids from myocytes and switches to an alternative, p62-mediated, lysosomal-independent pathway in the context of chronic lipid overload (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

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

Attitudes toward Stillbirth and Death Threat Level in a Sample of Obstetricians.

ERIC Educational Resources Information Center

Kirkley-Best, Elizabeth; And Others

1985-01-01

Obstetricians and obstetrical residents (N=70) were polled on their attitudes regarding stillbirth and simultaneously were assessed for their level of death threat as measured by the Threat Index. No effect of death threat level on attitudes toward stillbirth was observed, even when extreme scores were isolated and compared. (Author/NRB)

[Sudden death of twins: botulism because of contamination by pap vegetables].

PubMed

Fischer, D; Freislederer, A; Jorch, G

2004-01-01

Botulism is caused by the blockage of the neural transmission in the cholinergic synapses by botulinum neurotoxin (BoNT) which is produced by Clostridium botulinum or other Clostridia. The classic form of botulism occurs after the ingestion of food contaminated by BoNT. The course of the infection can be asymptomatic, mild with subtle paralysis ("failure to thrive") oder severe with generalized paralysis ("floppy infant"). Infected infants can also die sudden and unexpectedly. These deaths often are attributed to Sudden Infant Death Syndrome (SIDS), unless a thorough postmortem examination reveals Botulism. The rate of fatal Botulism falsely attributed to SIDS is not known, because it is difficult in most cases to show the causal relationship between contamination, disease and death. We report the sudden and unexpected simultaneous death of twins of 22 months which could be attributed to Botulism. Contamination of food, colonization of the gut by Clostridia and infection with specific pathomorphological changes could be proven. The initial suspicion of infanticide could be excluded. lt could be shown, that Botulism is a potential cause of simultaneous unexpected deaths in twins.

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

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

PubMed

Cremers, Niels A J; Lundvig, Ditte M S; van Dalen, Stephanie C M; Schelbergen, Rik F; van Lent, Peter L E M; Szarek, Walter A; Regan, Raymond F; Carels, Carine E; Wagener, Frank A D T G

2014-10-08

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

PubMed Central

Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

2015-01-01

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.

PubMed

Fonseca, Nuno A; Rodrigues, Ana S; Rodrigues-Santos, Paulo; Alves, Vera; Gregório, Ana C; Valério-Fernandes, Ângela; Gomes-da-Silva, Lígia C; Rosa, Manuel Santos; Moura, Vera; Ramalho-Santos, João; Simões, Sérgio; Moreira, João Nuno

2015-11-01

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Melatonin alleviates hyperthyroidism induced oxidative stress and neuronal cell death in hippocampus of aged female golden hamster, Mesocricetus auratus.

PubMed

Rao, Geeta; Verma, Rakesh; Mukherjee, Arun; Haldar, Chandana; Agrawal, Neeraj Kumar

2016-09-01

Oxidative stress is a well known phenomenon under hyperthyroid condition that induces various physiological and neural problems with a higher prevalence in females. We, therefore investigated the antioxidant potential of melatonin (Mel) on hyperthyroidism-induced oxidative stress and neuronal cell death in the hippocampus region of brain (cognition and memory centre) of aged female golden hamster, Mesocricetus auratus. Aged female hamsters were randomly divided into four experimental groups (n=7); group-I: control, group-II: Melatonin (5mgkg(-1)day(-1), i.p., for one week), group-III: Hyperthyroid (100μg kg(-1)day(-1), i.p., for two weeks) and group-IV- Hyper+Mel. Hormonal profiles (thyroid and melatonin), activity of antioxidant enzymes (SOD, CAT and GPX), lipid peroxidation level (TBARS) and the specific apoptotic markers (Bax/Bcl-2 ratio and Caspase-3) expression were evaluated. A significant increase in the profile of total thyroid hormone (tT3 and tT4) in hyperthyroidic group as compared to control while tT3 significantly decreased in melatonin treated hyperthyroidic group. However, Mel level significantly decreased in hyperthyroidic group but increased in melatonin treated hyperthyroidic group. Further, the number of immune-positive cells for thyroid hormone receptor-alpha (TR-α) decreased in the hippocampus of hyperthyroidic group and increased in melatonin treated hyperthyroidic group. Profiles of antioxidant enzymes showed a significant decrease in hyperthyroidic group with a simultaneous increase in lipid peroxidation (TBARS). Melatonin treatment to hyperthyroidic group lead to decreased TBARS level with a concomitant increase in antioxidant enzyme activity. Moreover, increased expression of Bax/Bcl-2 ratio and Caspase-3, in hyperthyroidic group had elevated neuronal cell death in hippocampal area and melatonin treatment reduced its expression in hyperthyroidic group. Our findings thus indicate that melatonin reduced the hyperthyroidism-induced oxidative stress and neuronal cell death in the hippocampus region of brain, suggesting a novel therapeutic approach of melatonin for management of cognition and memory function in females under hyperthyroid condition. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

MicroRNA-124 expression counteracts pro-survival stress responses in glioblastoma.

PubMed

Mucaj, V; Lee, S S; Skuli, N; Giannoukos, D N; Qiu, B; Eisinger-Mathason, T S K; Nakazawa, M S; Shay, J E S; Gopal, P P; Venneti, S; Lal, P; Minn, A J; Simon, M C; Mathew, L K

2015-04-23

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. As miR-124 expression is repressed in various cancer types (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared with regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 reintroduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

MicroRNA-124 expression counteracts pro-survival stress responses in glioblastoma

PubMed Central

Mucaj, Vera; Lee, Samuel S.; Skuli, Nicolas; Giannoukos, Dionysios N.; Qiu, Bo; Eisinger-Mathason, T.S. Karin; Nakazawa, Michael S.; Shay, Jessica E.S.; Gopal, Pallavi P.; Venneti, Sriram; Lal, Priti; Minn, Andy J.; Simon, M. Celeste; Mathew, Lijoy K.

2014-01-01

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression, and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. Since miR-124 expression is repressed in various cancers (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival, during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared to regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo, and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 re-introduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression. PMID:24954504

Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells.

PubMed

Lin, C-Y; Chang, T-W; Hsieh, W-H; Hung, M-C; Lin, I-H; Lai, S-C; Tzeng, Y-J

Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIP S ) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIP S is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIP S , consequently causes FLIP S to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIP S levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIP S regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.

E-cadherin genetic variants predict survival outcome in breast cancer patients.

PubMed

Memni, Hager; Macherki, Yosra; Klayech, Zahra; Ben-Haj-Ayed, Ahlem; Farhat, Karim; Remadi, Yassmine; Gabbouj, Sallouha; Mahfoudh, Wijden; Bouzid, Nadia; Bouaouina, Noureddine; Chouchane, Lotfi; Zakhama, Abdelfattah; Hassen, Elham

2016-11-16

E-cadherin is a major component of adherens junctions that regulates cell shape and maintains tissue integrity. A complete loss or any decrease in cell surface expression of E-cadherin will interfere with the cell-to-cell junctions' strength and leads to cell detachment and escape from the primary tumor site. In this prospective study, three functional single nucleotide polymorphisms (-347G/GA, rs5030625; -160C/A, rs16260; +54C/T, rs1801026), were found to modulate E-cadherin expression. 577 DNA samples from breast cancer (BC) cases were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We detected no significant correlations between each polymorphism and the clinical parameters of the patients whereas the GACC haplotype was significantly associated with low SBR grading. Overall survival analysis showed that both -347G/G and +54C/C wild (wt) genotypes had a significantly worse effect compared to the other genotypes (non-wt). Moreover, carrying simultaneously both the -347 and +54 wt genotypes confers a significantly higher risk of death. However, with metastatic recurrence, the death-rate was null in patients carrying the non-wt genotypes, and attained 37% in those carrying the wt genotype. A multivariate analysis showed that these two polymorphisms are independent prognostic factors for overall survival in BC patients. Our results support the fact that E-cadherin genetic variants control disease severity and progression and could be a marker of disease outcome. These findings could be useful in selecting patients that should be monitored differently.

Kinetic theory of age-structured stochastic birth-death processes

NASA Astrophysics Data System (ADS)

Greenman, Chris D.; Chou, Tom

2016-01-01

Classical age-structured mass-action models such as the McKendrick-von Foerster equation have been extensively studied but are unable to describe stochastic fluctuations or population-size-dependent birth and death rates. Stochastic theories that treat semi-Markov age-dependent processes using, e.g., the Bellman-Harris equation do not resolve a population's age structure and are unable to quantify population-size dependencies. Conversely, current theories that include size-dependent population dynamics (e.g., mathematical models that include carrying capacity such as the logistic equation) cannot be easily extended to take into account age-dependent birth and death rates. In this paper, we present a systematic derivation of a new, fully stochastic kinetic theory for interacting age-structured populations. By defining multiparticle probability density functions, we derive a hierarchy of kinetic equations for the stochastic evolution of an aging population undergoing birth and death. We show that the fully stochastic age-dependent birth-death process precludes factorization of the corresponding probability densities, which then must be solved by using a Bogoliubov--Born--Green--Kirkwood--Yvon-like hierarchy. Explicit solutions are derived in three limits: no birth, no death, and steady state. These are then compared with their corresponding mean-field results. Our results generalize both deterministic models and existing master equation approaches by providing an intuitive and efficient way to simultaneously model age- and population-dependent stochastic dynamics applicable to the study of demography, stem cell dynamics, and disease evolution.

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.

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.

Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

PubMed Central

Matter, A

1979-01-01

A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell. Images Figure 3 Figures 4-7 Figures 8-11 Figure 12 Figures 13-14 Figure 15 PMID:312256

Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells.

PubMed

Menezes, Ana Catarina; Carvalheiro, Manuela; Ferreira de Oliveira, José Miguel P; Ascenso, Andreia; Oliveira, Helena

2018-03-01

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Concerted suppression of STAT3 and GSK3β is involved in growth inhibition of non-small cell lung cancer by Xanthatin.

PubMed

Tao, Li; Fan, Fangtian; Liu, Yuping; Li, Weidong; Zhang, Lei; Ruan, Junshan; Shen, Cunsi; Sheng, Xiaobo; Zhu, Zhijie; Wang, Aiyun; Chen, Wenxing; Huang, Shile; Lu, Yin

2013-01-01

Xanthatin, a sesquiterpene lactone purified from Xanthium strumarium L., possesses prominent anticancer activity. We found that disruption of GSK3β activity was essential for xanthatin to exert its anticancer properties in non-small cell lung cancer (NSCLC), concurrent with preferable suppression of constitutive activation of STAT3. Interestingly, inactivation of the two signals are two mutually exclusive events in xanthatin-induced cell death. Moreover, we surprisingly found that exposure of xanthatin failed to trigger the presumable side effect of canonical Wnt/β-Catenin followed by GSK3β inactivation. We further observed that the downregulation of STAT3 was required for xanthatin to fine-tune the risk. Thus, the discovery of xanthatin, which has ability to simultaneously orchestrate two independent signaling cascades, may have important implications for screening promising drugs in cancer therapies.

Concerted Suppression of STAT3 and GSK3β Is Involved in Growth Inhibition of Non-Small Cell Lung Cancer by Xanthatin

PubMed Central

Tao, Li; Fan, Fangtian; Liu, Yuping; Li, Weidong; Zhang, Lei; Ruan, Junshan; Shen, Cunsi; Sheng, Xiaobo; Zhu, Zhijie; Wang, Aiyun; Chen, Wenxing; Huang, Shile; Lu, Yin

2013-01-01

Xanthatin, a sesquiterpene lactone purified from Xanthium strumarium L., possesses prominent anticancer activity. We found that disruption of GSK3β activity was essential for xanthatin to exert its anticancer properties in non-small cell lung cancer (NSCLC), concurrent with preferable suppression of constitutive activation of STAT3. Interestingly, inactivation of the two signals are two mutually exclusive events in xanthatin-induced cell death. Moreover, we surprisingly found that exposure of xanthatin failed to trigger the presumable side effect of canonical Wnt/β-Catenin followed by GSK3β inactivation. We further observed that the downregulation of STAT3 was required for xanthatin to fine-tune the risk. Thus, the discovery of xanthatin, which has ability to simultaneously orchestrate two independent signaling cascades, may have important implications for screening promising drugs in cancer therapies. PMID:24312384

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.

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.

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

Consensus guidelines for the detection of immunogenic cell death

PubMed Central

Kepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G.; Cirone, Mara; Colombo, Maria I.; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G.; Faggioni, Alberto; Formenti, Silvia C.; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S.; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A.; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W.; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong-Min; Huang, Xing; Illidge, Tim M.; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V.; Loi, Sherene; Lowenstein, Pedro R.; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A.; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L.; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean-Ehrland; Riganti, Chiara; Rovere-Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J.; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H.; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T.; Yang, Haining; Zong, Wei-Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2014-01-01

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine. PMID:25941621

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.

PubMed

Betsuyaku, Shigeyuki; Katou, Shinpei; Takebayashi, Yumiko; Sakakibara, Hitoshi; Nomura, Nobuhiko; Fukuda, Hiroo

2018-01-01

The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Deletion of angiotensin II type 1 receptor gene or scavenge of superoxide prevents chronic alcohol-induced aortic damage and remodelling.

PubMed

Bai, Yang; Tan, Yi; Wang, Bo; Miao, Xiao; Chen, Qiang; Zheng, Yang; Cai, Lu

2012-10-01

To investigate whether chronic alcohol consumption induces vascular injury via angiotensin II (Ang II) type 1 (AT1) receptor-dependent superoxide generation, male transgenic mice with knockout of AT1 gene (AT1-KO) and age-matched wild-type (WT) C57BL/6 mice were pair-fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 2 months. Ethanol content (%, W/V) in the diet was 4.8 (34% of total calories) at initiation, and gradually increased up to 5.4 (38% of total calories). For some WT mice with and without alcohol treatment, superoxide dismutase mimetic (MnTMPyP) was given simultaneously by intraperitoneal injection at 5 mg/kg body weight daily for 2 months. At the end of studies, aortas were harvested for histopathological and immunohistochemical examination. Significant increases in the wall thickness and structural disarrangement of aorta were found in alcohol group, along with significant increases in aortic oxidative and/or nitrosative damage, expressions of NADPH oxidases (NOXs), inflammatory response, cell death and proliferation, and remodelling (fibrosis). However, these pathological changes were completely attenuated in alcohol-treated AT1-KO mice or in alcohol-treated WT mice that were also simultaneously treated with MnTMPyP for 2 months. These results suggest that chronic alcohol consumption may activate NOX via Ang II/AT1 receptor, to generate superoxide and associated peroxynitrite that in turn causes aortic nitrosative damage, inflammation, cell death and proliferation, and remodelling. Therefore, blocking Ang II/AT1 system or scavenging superoxide may become a potential preventive and/therapeutic approach to alcoholic vascular damage. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Evidence For Multiple Cell Death Pathways during Development of Experimental Cytomegalovirus Retinitis in Mice with Retrovirus-Induced Immunosuppression: Apoptosis, Necroptosis, and Pyroptosis

PubMed Central

Chien, Hsin

2012-01-01

AIDS-related human cytomegalovirus (HCMV) retinitis remains a major ophthalmologic problem worldwide. Although this sight-threatening disease is well characterized clinically, many pathogenic issues remain unresolved, among them a basic understanding of the relative roles of cell death pathways during development of retinal tissue destruction. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS), we initially investigated MCMV-infected eyes for evidence of apoptosis-associated molecules in mice with MAIDS of 4 weeks' (MAIDS-4) and 10 weeks' (MAIDS-10) duration, which were resistant and susceptible to retinal disease, respectively, but which harbored equivalent amounts of infectious MCMV. Whereas MCMV-infected eyes of MAIDS-4 mice showed little evidence of apoptosis-associated molecules, MCMV-infected eyes of MAIDS-10 mice showed significant amounts of tumor necrosis factor alpha (TNF-α), TNF receptors 1 and 2, active caspase 8, active caspase 3, TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-R(DR5), Fas, and Fas ligand mRNAs and/or proteins, all detected at peak amounts prior to development of most severe retinal disease. Immunohistochemical staining showed macrophages, granulocytes (neutrophils), Müller cells, and microglial cells as TNF-α sources. Remarkably, quantification of apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay suggested that apoptosis contributed minimally to retinal disease in MCMV-infected eyes of MAIDS-10 mice. Subsequent studies demonstrated that MCMV-infected eyes of MAIDS-10 mice, but not MAIDS-4 mice, showed evidence of significant increases in molecules associated with two additional cell death pathways, necroptosis (receptor-interacting protein 1 [RIP1] and RIP3 mRNAs) and pyroptosis (caspase 1, interleukin 1β [IL-1β], and IL-18 mRNAs). We conclude that apoptosis, necroptosis, and pyroptosis participate simultaneously during MAIDS-related MCMV retinitis, and all may play a role during AIDS-related HCMV retinitis. PMID:22837196

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.

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.

Induction of apoptosis in endothelial cells treated with cholesterol oxides.

PubMed Central

Lizard, G.; Deckert, V.; Dubrez, L.; Moisant, M.; Gambert, P.; Lagrost, L.

1996-01-01

Cholesterol oxides have a wide range of cytotoxic effects on vascular cells. Therefore, 7-ketocholesterol, 7 beta-hydroxycholesterol, 19-hydroxycholesterol, cholesterol 5 alpha, 6 alpha-epoxide, and 25-hydroxycholesterol, identified in various foodstuffs and human tissues, were chosen to compare and characterize the mode of cell death they induce, apoptosis or necrosis, on bovine aortic endothelial cells. The toxic potency differed from one compound to another, and 7 beta-hydroxycholesterol and 7-ketocholesterol exhibited the most potent effects. Cytotoxicity was accompanied by a decreased number of adherent cells, an increased number of non-adherent cells, and an enhanced permeability to propidium iodide. By electron and fluorescence microscopy performed after staining with Hoechst 33342, apoptotic cells with fragmented and condensed nuclei were identified mainly among non-adherent cells. By flow cytometry, cells with a lower DNA content than cells in the G0/G1 phase were apparent, giving a characteristic sub-G1 peak. Quantification of apoptosis evaluated either by the proportion of apoptotic cells identified by fluorescence microscopy after staining with Hoechst 33342 or by the percentage of cells present in the sub-G1 peak indicated that the ability of cholesterol oxides in inducing apoptosis was in the following order: 7 beta-hydroxycholesterol > 7-ketocholesterol > 19-hydroxycholesterol > cholesterol 5 alpha, 6 alpha-epoxide > 25-hydroxycholesterol. By using electrophoresis on agarose gel, typical internucleosomal DNA fragmentations were detected; they were no longer observed when bovine aortic endothelial cells were simultaneously incubated with 0.5 mmol/L zinc chloride, known to inhibit Ca2+/Mg2+-dependent endonucleases. None of the cholesterol-oxide-induced apoptotic features described above were noted with cholesterol. It is concluded that cholesterol oxides constitute a new class of cholesterol derivatives that can induce cell death by apoptosis in cultured endothelial cells. Images Figure 3 Figure 4 Figure 7 PMID:8623930

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.

The combined effect of thermal and chemotherapy on HeLa cells using magnetically actuated smart textured fibrous system.

PubMed

Tiwari, Pranav; Agarwal, Sakshi; Srivastava, Sachchidanand; Jain, Shilpee

2018-01-01

Thermal therapy combined with chemotherapy is one of the advanced and efficient methods to eradicate cancer. In this work, we fabricated magnetically actuated smart textured (MAST) fibrous systems and studied their candidacy for cancer treatment. The polycaprolactone-Fe 3 O 4 based MAST fibers were fabricated using electrospinning technique. These MAST fibrous systems contained carbogenic quantum dots as a tracking agent and doxorubicin hydrochloride anticancer drug. Additionally, as fabricated MAST fibrous systems were able to deliver anticancer drug and heat energy simultaneously to kill HeLa cells in a 10 min period in vitro. After treatment, the metabolic activity and morphology of HeLa cells were analyzed. In addition, the mechanism of cell death was studied using flow cytometry. Interestingly, the navigation of these systems in the fluid can be controlled with the application of gradient magnetic field. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 40-51, 2018. © 2016 Wiley Periodicals, Inc.

Mammary Gland Involution Provides a Unique Model to Study the TGF-β Cancer Paradox

PubMed Central

Guo, Qiuchen; Betts, Courtney; Pennock, Nathan; Mitchell, Elizabeth; Schedin, Pepper

2017-01-01

Transforming Growth Factor-β (TGF-β) signaling in cancer has been termed the “TGF-β paradox”, acting as both a tumor suppresser and promoter. The complexity of TGF-β signaling within the tumor is context dependent, and greatly impacted by cellular crosstalk between TGF-β responsive cells in the microenvironment including adjacent epithelial, endothelial, mesenchymal, and hematopoietic cells. Here we utilize normal, weaning-induced mammary gland involution as a tissue microenvironment model to study the complexity of TGF-β function. This article reviews facets of mammary gland involution that are TGF-β regulated, namely mammary epithelial cell death, immune activation, and extracellular matrix remodeling. We outline how distinct cellular responses and crosstalk between cell types during physiologically normal mammary gland involution contribute to simultaneous tumor suppressive and promotional microenvironments. We also highlight alternatives to direct TGF-β blocking anti-cancer therapies with an emphasis on eliciting concerted microenvironmental-mediated tumor suppression. PMID:28098775

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.

Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells

PubMed Central

Janssen, Aniek; Kops, Geert J. P. L.; Medema, René H.

2009-01-01

The mitotic checkpoint has evolved to prevent chromosome mis-segregations by delaying mitosis when unattached chromosomes are present. Inducing severe chromosome segregation errors by ablating the mitotic checkpoint causes cell death. Here we have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in four tumor cell lines caused mild chromosome mis-segregations, but no lethality. These cells were, however, remarkably more sensitive to low doses of taxol, which enhanced the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing levels of Mps1 or BubR1, proteins having dual roles in checkpoint activation and chromosome alignment, but not by reducing Mad2, functioning solely in the mitotic checkpoint. Moreover, we find that untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sublethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells by enhancing chromosome mis-segregations. PMID:19855003

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.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

PubMed

Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

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.

Interferon-gamma induces apoptosis and augments the expression of Fas and Fas ligand by microglia in vitro.

PubMed

Badie, B; Schartner, J; Vorpahl, J; Preston, K

2000-04-01

Activation of microglia by interferon-gamma (IFN-gamma) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-gamma has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-gamma-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-gamma (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-gamma concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-gamma also potentiated the expression of Fas and FasL in a similar dose-response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-gamma may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-gamma modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes. Copyright 2000 Academic Press.

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

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

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy.

PubMed

Wang, Junjian; Wang, Haibin; Wang, Ling-Yu; Cai, Demin; Duan, Zhijian; Zhang, Yanhong; Chen, Peng; Zou, June X; Xu, Jianzhen; Chen, Xinbin; Kung, Hsing-Jien; Chen, Hong-Wu

2016-11-01

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy

PubMed Central

Wang, Junjian; Wang, Haibin; Wang, Ling-Yu; Cai, Demin; Duan, Zhijian; Zhang, Yanhong; Chen, Peng; Zou, June X; Xu, Jianzhen; Chen, Xinbin; Kung, Hsing-Jien; Chen, Hong-Wu

2016-01-01

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics. PMID:27612013

Local mechanical stimulation induces components of the pathogen defense response in parsley

PubMed Central

Gus-Mayer, Sabine; Naton, Beatrix; Hahlbrock, Klaus; Schmelzer, Elmon

1998-01-01

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death. PMID:9653198

Ghrelin Partially Protects Against Cisplatin-Induced Male Murine Gonadal Toxicity in a GHSR-1a-Dependent Manner1

PubMed Central

Whirledge, Shannon D.; Garcia, Jose M.; Smith, Roy G.; Lamb, Dolores J.

2015-01-01

ABSTRACT The chemotherapeutic drug cisplatin causes a number of dose-dependent side effects, including cachexia and testicular damage. Patients receiving a high cumulative dose of cisplatin may develop permanent azoospermia and subsequent infertility. Thus, the development of chemotherapeutic regimens with the optimal postsurvival quality of life (fertility) is of high importance. This study tested the hypothesis that ghrelin administration can prevent or minimize cisplatin-induced testicular damage and cachexia. Ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR-1a), are expressed and function in the testis. Targeted deletion of ghrelin, or its receptor, significantly increases the rate of cell death in the testis, suggesting a protective role. Intraperitoneal administration of vehicle, ghrelin, or cisplatin alone or in combination with ghrelin, in cycles of 9 or 18 days, to adult male C57Bl/6 mice was performed. Body weight was measured daily and testicular and epididymal weight, sperm density and motility, testicular histology, and testicular cell death were analyzed at the time of euthanization. Ghrelin coadministration decreased the severity of cisplatin-induced cachexia and gonadal toxicity. Body, testicular, and epididymal weights significantly increased as testicular cell death decreased with ghrelin coadministration. The widespread damage to the seminiferous epithelium induced by cisplatin administration was less severe in mice simultaneously treated with ghrelin. Furthermore, ghrelin diminished the deleterious effects of cisplatin on testis and body weight homeostasis in wild-type but not Ghsr−/− mice, showing that ghrelin's actions are mediated via GHSR. Ghrelin or more stable GHSR agonists potentially offer a novel therapeutic approach to minimize the testicular damage that occurs after gonadotoxin exposure. PMID:25631345

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.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

PubMed

Oh, Yumin; Park, Ogyi; Swierczewska, Magdalena; Hamilton, James P; Park, Jong-Sung; Kim, Tae Hyung; Lim, Sung-Mook; Eom, Hana; Jo, Dong Gyu; Lee, Choong-Eun; Kechrid, Raouf; Mastorakos, Panagiotis; Zhang, Clark; Hahn, Sei Kwang; Jeon, Ok-Cheol; Byun, Youngro; Kim, Kwangmeyung; Hanes, Justin; Lee, Kang Choon; Pomper, Martin G; Gao, Bin; Lee, Seulki

2016-07-01

Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223). © 2015 by the American Association for the Study of Liver Diseases.

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.

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.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

PubMed

Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

2013-01-01

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

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.

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

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.

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.

PR01 Molecular Pathogenesis of Rickettsioses and Development of Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2006-02-01

likely reflecting similar cell death rates in all monolayers at late time points. By the end of the experiment at 120 hours, all monolayers showed a...50-55% increase in permeability when compared to the controls. 2. Cell death rates in rickettsiae-infected SV-HCEC monolayers In order to...necrotic cell death. Quantification of cell death was performed by determining the percent of total cells staining positive for PI. Cell death rates did

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment

PubMed Central

Kojima, Kensuke; Shikami, Masato; Benito, Julina; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Ciurea, Stefan O.; Miida, Takashi; Andreeff, Michael; Konopleva, Marina

2013-01-01

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737–induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. PMID:23955073

[Antiviral activity of phosprenyl and gamapren against infection caused by influenza a (H5N1) virus in cell culture].

PubMed

Sanin, A V; Deryabin, P G; Narovlyansky, A N; Pronin, A V; Kozhevnikova, T N

2017-01-01

The antiviral activity of Phosprenyl and Gamapren in vitro against highly pathogenic strain of avian influenza H5N1 virus was studied. Inoculation of the virus to the susceptible cell culture led to development of the cytopathogenic effect. Preliminary introduction of Phosprenyl and Gamapren an hour prior to infecting the cells with virus 10.0 TCID50 dose completely inhibited the cytopathogenic activity of the virus. At higher doses of virus (100.0 TCID50) significant inhibition of the infectious activity of the virus was observed: 70% of infected cells survived under the action of Phosprenyl, and 90% under the action of Gamapren. With the introduction of the preparations simultaneously with the infection of cells with virus at a dose of 10.0 TCID50 virtually 100% of infected cells survived, while in control cultures death of 100% of the cells occurred. After infection with the virus at a dose of 100.0 TCID50 Phosprenyl and Gamapren caused 50% protection of the cells. The antiviral effect of the drugs Phosprenyl and Gamapren may be associated not only with their virulicidal, but with anti-viral activity as well.

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.

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

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.

Cytoskeletal and morphologic impact of cellular oxidant injury.

PubMed Central

Hinshaw, D. B.; Sklar, L. A.; Bohl, B.; Schraufstatter, I. U.; Hyslop, P. A.; Rossi, M. W.; Spragg, R. G.; Cochrane, C. G.

1986-01-01

The relationship between changes in cell morphology and the cytoskeleton in oxidant injury was examined in the P388D1 cell line. Flow cytometry of cells stained with NBD-phallacidin, a fluorescent probe specific for filamentous (F) actin, revealed a substantial increase in F actin content in H2O2-injured cells over 3-4 hours. Doses of H2O2 as low as 500 microM produced sustained increases in F actin content. Experiments where catalase was used to interrupt H2O2 exposure over a long time course revealed 15-30 minutes to be the critical period of exposure to 5 mM H2O2 necessary for a sustained increase in F actin as well as large increases in membrane blebbing and later cell death. The increase in F actin with H2O2 injury was confirmed with the use of electrophoresis in acrylamide gels of 1% Triton X-100 cytoskeletal extracts from P388D1 cells. Scanning electron microscopy revealed major loss of surface convolutions in addition to the formation of blebs. Fluorescence microscopy of adherent cells using rhodamine phalloidin showed considerable cell rounding and rearrangement of cellular F actin by 30 minutes of exposure to H2O2. Transmission electron microscopy revealed side to side aggregation of F actin bundles (microfilaments) developing during this time. Considerable swelling of mitochondria and other subcellular organelles was seen after 2 hours of injury. The apparent area of attachment to the substrate was markedly diminished in injured cells. H2O2 injury produced a marked increase in F actin with an associated rearrangement of the microfilaments and simultaneous changes in the plasma membrane prior to cell death in the P388D1 cell line. Images Figure 5 Figure 6 Figure 7 Figure 8 PMID:3717299

BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells.

PubMed

Das, Chandan Kanta; Linder, Benedikt; Bonn, Florian; Rothweiler, Florian; Dikic, Ivan; Michaelis, Martin; Cinatl, Jindrich; Mandal, Mahitosh; Kögel, Donat

2018-03-01

Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549 r DOX 20 ) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468 r 5-FU 2000 ) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549 r DOX 20 and MDA-MB-468 r 5-FU 2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Partial T-cell depletion improves the composite endpoint graft-versus-host disease-free, relapse-free survival after allogeneic hematopoietic stem cell transplantation.

PubMed

Simonetta, Federico; Masouridi-Levrat, Stavroula; Beauverd, Yan; Tsopra, Olga; Tirefort, Yordanka; Koutsi, Aikaterini; Stephan, Caroline; Polchlopek-Blasiak, Karolina; Pradier, Amandine; Dantin, Carole; Ansari, Marc; Roosnek, Eddy; Chalandon, Yves

2018-03-01

Graft-versus-host disease (GvHD)-free, relapse-free survival (GRFS) is a recently reported composite endpoint that allows to simultaneously estimate risk of death, relapse and GvHD after allogeneic hematopoietic stem cell transplantation (HSCT). In this retrospective study comprising 333 patients transplanted for hematologic malignancies, we compared GRFS in patients receiving partial T-cell-depleted (pTCD) grafts with patients receiving T-cell-replete grafts (No-TCD). pTCD was associated with a significantly improved GRFS. The beneficial effect of pTCD on GRFS remained highly significant in multivariable analysis taking into account clinical factors differing between patient groups. We observed no effect of pTCD on overall survival, progression-free survival, and relapse cumulative incidence, while non-relapse mortality cumulative incidence was significantly lower in patients receiving pTCD. The results of our retrospective analysis suggest that pTCD could improve GRFS in allogeneic HSCT recipients without significantly affecting OS and PFS, thus improving patients' quality of life without impairing the curative potential of allogeneic HSCT.

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.

Acute collective gas poisoning at work in a manure storage tank.

PubMed

Żaba, Czesław; Marcinkowski, Jerzy T; Wojtyła, Andrzej; Tężyk, Artur; Tobolski, Jarosław; Zaba, Zbigniew

2011-01-01

Cases of deaths in manure or septic tanks are rare in legal-medical practice, more frequently as unfortunate occupational accidents. Poisoning with toxic gases, especially with hydrogen sulfide, is reported as the cause of death, while the exhaustion of oxygen in the air is omitted with the simultaneous excess of carbon dioxide. In such cases, determination of the direct cause of death constitutes a big problem because post-mortem examination does not reveal the specific changes. A case of acute collective poisoning by gases in a manure storage tank is presented of 5 agricultural workers, 2 of whom died. While explaining the cause of poisoning and deaths, toxicological blood tests were performed in the victims of the accident, as well as gases inside the manure storage tank. The post-mortem examinations and toxicological blood tests performed did not allow determination of the direct cause of death. Toxicological tests of gases from inside the manure tank showed a very low concentration of oxygen, with a simultaneous very high concentration of carbon dioxide, and a considerable level of hydrogen sulfide. The cause of fainting of three and deaths of two workers was not the poisoning with hydrogen sulfide, but oxygen deficiency in the air of the tank.

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.

Suicidal death of erythrocytes in cancer and its chemotherapy: A potential target in the treatment of tumor-associated anemia.

PubMed

Lang, Elisabeth; Bissinger, Rosi; Qadri, Syed M; Lang, Florian

2017-10-15

In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis characterized by cell shrinkage and cell membrane scrambling. Eryptotic erythrocytes are rapidly cleared from circulating blood and may adhere to the vascular wall. Stimulation of eryptosis thus impairs microcirculation and leads to anemia as soon as the loss of erythrocytes cannot be fully compensated by enhanced erythropoiesis. Signaling stimulating eryptosis includes increase of cytosolic Ca 2+ -activity, ceramide, caspases, calpain, p38-kinase, protein-kinase C, Janus-activated kinase 3, casein-kinase 1α, and cyclin-dependent kinase 4. Eryptosis is inhibited by AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein-kinase, mitogen- and stress-activated kinase, and sorafenib- and sunitinib-sensitive tyrosine-kinases. Eryptosis is triggered by complement, hyperosmotic shock, energy-depletion, oxidative stress, multiple xenobiotics including diverse cytostatic drugs, diabetes, hepatic failure, iron-deficiency, chronic kidney disease, hemolytic-uremic-syndrome, fever, systemic lupus erythematosus, infections, sepsis, sickle cell anemia, thalassemia, glucose-6-phosphate-dehydrogenase deficiency, and Wilson´s disease. Compelling evidence points to a decisive role of eryptosis in anemia of malignancy. As shown for lung cancer, eryptosis inducing plasma components accumulate in cancer patients and trigger oxidative stress and ceramide. The tumor-induced eryptosis leads to anemia despite increased erythropoiesis. The stimulation of eryptosis in malignancy is compounded by cytostatic treatment, as a large number of cytostatic agents trigger eryptosis. Inhibiting eryptosis may be a useful strategy in reducing tumor-induced anemia and impaired microcirculation. Inhibitors of eryptosis may, however, be harmful, if they similarly interfere with death of tumor cells. Clearly, additional experimental effort is required to achieve killing of tumor cells with simultaneous avoidance of stimulated eryptosis. © 2017 UICC.

"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.

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

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.

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.

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.

[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.

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.

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis.

PubMed

Brünker, Peter; Wartha, Katharina; Friess, Thomas; Grau-Richards, Sandra; Waldhauer, Inja; Koller, Claudia Ferrara; Weiser, Barbara; Majety, Meher; Runza, Valeria; Niu, Huifeng; Packman, Kathryn; Feng, Ningping; Daouti, Sherif; Hosse, Ralf J; Mössner, Ekkehard; Weber, Thomas G; Herting, Frank; Scheuer, Werner; Sade, Hadassah; Shao, Cuiying; Liu, Bin; Wang, Peng; Xu, Gary; Vega-Harring, Suzana; Klein, Christian; Bosslet, Klaus; Umaña, Pablo

2016-05-01

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR. ©2016 American Association for Cancer Research.

Chemosensitizing effects of carbon-based nanomaterials in cancer cells: enhanced apoptosis and inhibition of proliferation as underlying mechanisms

NASA Astrophysics Data System (ADS)

Erdmann, Kati; Ringel, Jessica; Hampel, Silke; Rieger, Christiane; Huebner, Doreen; Wirth, Manfred P.; Fuessel, Susanne

2014-10-01

Recent studies have shown that carbon nanomaterials such as carbon nanofibres (CNFs) and multi-walled carbon nanotubes (CNTs) can exert antitumor activities themselves and sensitize cancer cells to conventional chemotherapeutics such as carboplatin and cisplatin. In the present study, the chemosensitizing effect of CNFs and CNTs on cancer cells of urological origin was investigated regarding the underlying mechanisms. Prostate cancer (DU-145, PC-3) and bladder cancer (EJ28) cells were treated with carbon nanomaterials (CNFs, CNTs) and chemotherapeutics (carboplatin, cisplatin) alone as well as in combination for 24 h. Forty-eight (EJ28) or 72 h (DU-145, PC-3) after the end of treatment the effects on cellular proliferation, clonogenic survival, cell death rate and cell cycle distribution were evaluated. Depending on the cell line, simultaneous administration of chemotherapeutics and carbon nanomaterials produced an additional inhibition of cellular proliferation and clonogenic survival of up to 77% and 98%, respectively, compared to the inhibitory effects of the chemotherapeutics alone. These strongly enhanced antiproliferative effects were accompanied by an elevated cell death rate, which was predominantly mediated via apoptosis and not by necrosis. The antitumor effects of combinations with CNTs were less pronounced than those with CNFs. The enhanced effects of the combinatory treatments on cellular function were mostly of additive to partly synergistic nature. Furthermore, cell cycle analysis demonstrated an arrest at the G2/M phase mediated by a monotreatment with chemotherapeutics. Following combinatory treatments, mostly less than or nearly additive increases of cell fractions in the G2/M phase could be observed. In conclusion, the pronounced chemosensitizing effects of CNFs and CNTs were mediated by an enhanced apoptosis and inhibition of proliferation. The combination of carbon-based nanomaterials and conventional chemotherapeutics represents a novel approach in cancer therapy to bypass chemoresistance by minimizing the chemotherapeutic dosing.

Early induction of c-Myc is associated with neuronal cell death.

PubMed

Lee, Hyun-Pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2011-11-14

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Measurement and Characterization of Apoptosis by Flow Cytometry.

PubMed

Telford, William; Tamul, Karen; Bradford, Jolene

2016-07-01

Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Mitochondria are targets for the antituberculosis drug rifampicin in cultured epithelial cells.

PubMed

Erokhina, M V; Kurynina, A V; Onishchenko, G E

2013-10-01

Rifampicin is a widely used drug for antituberculosis therapy. Its target is the bacterial RNA polymerase. After entry into the human or mammalian organism, rifampicin is accumulated in cells of epithelial origin (kidneys, liver, lungs) where it induces apoptosis, necrosis, and fibrosis. The purpose of this study was to determine the intracellular mechanisms leading to rifampicin-induced pathological changes and cell death. We analyzed the survival and state of the chondriome of cultured epithelial cells of the SPEV line under the influence of rifampicin. Our data show that the drug induces pronounced pathological changes in the network and ultrastructure of mitochondria, and their dysfunction results in excessive production of reactive oxygen species and release of cytochrome c. These data suggest the initiation of the mitochondrial pathway of apoptosis. Simultaneously, we observed inhibition of cell proliferation and changes in morphology of the epithelial cells toward fibroblast-like appearance, which could indicate induction of epithelial-mesenchymal transition. Thus, mitochondria are the main potential target for rifampicin in cells of epithelial origin. We suggest that similar mechanisms of pathological changes can be induced in vivo in organs and tissues accumulating rifampicin during chemotherapy of bacterial infectious diseases.

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.

Identification of the Calmodulin-Binding Domains of Fas Death Receptor

PubMed Central

Chang, Bliss J.; Samal, Alexandra B.; Vlach, Jiri; Fernandez, Timothy F.; Brooke, Dewey; Prevelige, Peter E.; Saad, Jamil S.

2016-01-01

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas–mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209–239 (Fas-Pep1) and 251–288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD–CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway. PMID:26735300

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

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.

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage.

PubMed

Yang, Yu; Bolnick, Alan; Shamir, Alexandra; Abdulhasan, Mohammed; Li, Quanwen; Parker, G C; Puscheck, Elizabeth E; Rappolee, D A

2017-08-01

Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

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

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).

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.

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

Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

PubMed

Chio, Chung-Ching; Wei, Li; Chen, Tyng Guey; Lin, Chien-Min; Shieh, Ja-Ping; Yeh, Poh-Shiow; Chen, Ruei-Ming

2016-06-01

OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

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

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.

Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma.

PubMed

Maser, Tyler; Rich, Maria; Hayes, David; Zhao, Ping; Nagulapally, Abhinav B; Bond, Jeffrey; Saulnier Sholler, Giselle

2017-06-01

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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.

Development of therapeutic Au-methylene blue nanoparticles for targeted photodynamic therapy of cervical cancer cells.

PubMed

Yu, Jiashing; Hsu, Che-Hao; Huang, Chih-Chia; Chang, Po-Yang

2015-01-14

Photodynamic therapy (PDT) involves the cellular uptake of a photosensitizer (PS) combined with oxygen molecules and light at a specific wavelength to be able to trigger cancer cell death via the apoptosis pathway, which is less harmful and has less inflammatory side effect than necrosis. However, the traditional PDT treatment has two main deficiencies: the dark toxicity of the PS and the poor selectivity of the cellular uptake of PS between the target cells and normal tissues. In this work, methylene blue (MB), a known effective PS, combined with Au nanoparticles (NPs) was prepared using an intermolecular interaction between a polystyrene-alt-maleic acid (PSMA) layer on the Au NPs and MB. The Au@polymer/MB NPs produced a high quantum yield of singlet oxygen molecules, over 50% as much as that of free MB, when they were excited by a dark red light source at 660 nm, but without significant dark toxicity. Furthermore, transferrin (Tf) was conjugated on the Au@polymer/MB NPs via an EDC/NHS reaction to enhance the selectivity to HeLa cells compared to 3T3 fibroblasts. With a hand-held single laser treatment (32 mW/cm) for 4 min, the new Au@polymer/MB-Tf NPs showed a 2-fold enhancement of PDT efficiency toward HeLa cells over the use of free MB at 4 times dosage. Cellular staining examinations showed that the HeLa cells reacted with Au@polymer/MB-Tf NPs and the 660 nm light excitation triggered PDT, which caused the cells to undergo apoptosis ("programmed" cell death). We propose that applying this therapeutic Au@polymer/MB-Tf nanoagent is facile and safe for delivery and cancer cell targeting to simultaneously minimize side effects and accomplish a significant enhancement in photodynamic therapeutic efficiency toward next-generation nanomedicine development.

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.

Combining Portable Raman Probes with Nanotubes for Theranostic Applications

PubMed Central

Bhirde, Ashwinkumar A.; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A.; Leapman, Richard D.; Gutkind, J. Silvio; Lee, Seulki; Chen, Xiaoyuan

2011-01-01

Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer. PMID:21769298

Combining portable Raman probes with nanotubes for theranostic applications.

PubMed

Bhirde, Ashwinkumar A; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A; Leapman, Richard D; Gutkind, J Silvio; Lee, Seulki; Chen, Xiaoyuan

2011-01-01

Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer.

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.

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death.

PubMed

Liu, Sijia; Fu, Chunxiang; Gou, Jiqing; Sun, Liang; Huhman, David; Zhang, Yunwei; Wang, Zeng-Yu

2017-01-01

Switchgrass ( Panicum virgatum ) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O -methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT -deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death

PubMed Central

Liu, Sijia; Fu, Chunxiang; Gou, Jiqing; Sun, Liang; Huhman, David; Zhang, Yunwei; Wang, Zeng-Yu

2017-01-01

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance. PMID:28676804

The effect of co-administration of death camas (Zigadenus spp.) and low larkspur (Delphinium spp.) in cattle

USDA-ARS?s Scientific Manuscript database

In many rangeland settings, there is more than one potential poisonous plant. Two poisonous plants that are often found growing simultaneously in the same location are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). The objective of this study was to determine if co-administration...

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

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.

The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

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

Lyu, Qing; Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055; Tou, Fangfang

Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary,more » our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.« less

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

Proposal for a recovery prediction method for patients affected by acute mediastinitis

PubMed Central

2012-01-01

Background An attempt to find a prediction method of death risk in patients affected by acute mediastinitis. There is not such a tool described in available literature for that serious disease. Methods The study comprised 44 consecutive cases of acute mediastinitis. General anamnesis and biochemical data were included. Factor analysis was used to extract the risk characteristic for the patients. The most valuable results were obtained for 8 parameters which were selected for further statistical analysis (all collected during few hours after admission). Three factors reached Eigenvalue >1. Clinical explanations of these combined statistical factors are: Factor1 - proteinic status (serum total protein, albumin, and hemoglobin level), Factor2 - inflammatory status (white blood cells, CRP, procalcitonin), and Factor3 - general risk (age, number of coexisting diseases). Threshold values of prediction factors were estimated by means of statistical analysis (factor analysis, Statgraphics Centurion XVI). Results The final prediction result for the patients is constructed as simultaneous evaluation of all factor scores. High probability of death should be predicted if factor 1 value decreases with simultaneous increase of factors 2 and 3. The diagnostic power of the proposed method was revealed to be high [sensitivity =90%, specificity =64%], for Factor1 [SNC = 87%, SPC = 79%]; for Factor2 [SNC = 87%, SPC = 50%] and for Factor3 [SNC = 73%, SPC = 71%]. Conclusion The proposed prediction method seems a useful emergency signal during acute mediastinitis control in affected patients. PMID:22574625

Development of Novel Tumor-Targeted Theranostic Nanoparticles Activated by Membrane-Type Matrix Metalloproteinases for Combined Cancer Magnetic Resonance Imaging and Therapy

PubMed Central

Ansari, Celina; Tikhomirov, Grigory A.; Hong, Su Hyun; Falconer, Robert A.; Loadman, Paul M.; Gill, Jason H.; Castaneda, Rosalinda; Hazard, Florette K.; Tong, Ling; Lenkov, Olga D.; Felsher, Dean W.; Rao, Jianghong; Daldrup-Link, Heike E.

2013-01-01

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, we describe the design and characterization of novel multifunctional “theranostic” nanoparticles (TNPs) for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs were synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death was observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrated significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induced a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death was observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. Our findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens. PMID:24038954

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.

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.

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.

Quantitative non-invasive intracellular imaging of Plasmodium falciparum infected human erythrocytes

NASA Astrophysics Data System (ADS)

Edward, Kert; Farahi, Faramarz

2014-05-01

Malaria is a virulent pathological condition which results in over a million annual deaths. The parasitic agent Plasmodium falciparum has been extensively studied in connection with this epidemic but much remains unknown about its development inside the red blood cell host. Optical and fluorescence imaging are among the two most common procedures for investigating infected erythrocytes but both require the introduction of exogenous contrast agents. In this letter, we present a procedure for the non-invasive in situ imaging of malaria infected red blood cells. The procedure is based on the utilization of simultaneously acquired quantitative phase and independent topography data to extract intracellular information. Our method allows for the identification of the developmental stages of the parasite and facilitates in situ analysis of the morphological changes associated with the progression of this disease. This information may assist in the development of efficacious treatment therapies for this condition.

Early detection of ventricular tachycardia with sending messages to cell phone

NASA Astrophysics Data System (ADS)

Ramirez, L. J.; Lozano, F. A.; Rondon, C. R.

2011-09-01

Sustained ventricular tachycardia (VTs) can be asymptomatic for some people, but for other is deadly because it is a major cause of sudden cardiac death [1]. Some patients may present this arrhythmia, and even so, they decide to drive car increasing the likelihood of VTs, putting at risk not only his life but that of the other drivers. We developed a system for early detection of VTs, consisting of EKG sensors, a card of processing and a cell phone, which detects this arrhythmia, gives an alarm signal to the driver, and it simultaneously sending to text messages a specialist doctor and a relative or friend, all in real time. This design was conditioned to the car, is light and comfortable, that allowed that work of car's driver without discomfort. This system will save lives, since in case of emergency sends a help message, no matter where you are in the driver.

Molecular demultiplexer as a terminator automaton.

PubMed

Turan, Ilke S; Gunaydin, Gurcan; Ayan, Seylan; Akkaya, Engin U

2018-02-23

Molecular logic gates are expected to play an important role on the way to information processing therapeutic agents, especially considering the wide variety of physical and chemical responses that they can elicit in response to the inputs applied. Here, we show that a 1:2 demultiplexer based on a Zn 2+ -terpyridine-Bodipy conjugate with a quenched fluorescent emission, is efficient in photosensitized singlet oxygen generation as inferred from trap compound experiments and cell culture data. However, once the singlet oxygen generated by photosensitization triggers apoptotic response, the Zn 2+ complex then interacts with the exposed phosphatidylserine lipids in the external leaflet of the membrane bilayer, autonomously switching off singlet oxygen generation, and simultaneously switching on a bright emission response. This is the confirmatory signal of the cancer cell death by the action of molecular automaton and the confinement of unintended damage by excessive singlet oxygen production.

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

Do antioxidants inhibit oxidative-stress-induced autophagy of tenofibroblasts?

PubMed

Kim, Ra-Jeong; Hah, Young-Sool; Sung, Chang-Meen; Kang, Jae-Ran; Park, Hyung Bin

2014-07-01

Recent research on tendinopathy has focused on its relationship to programmed cell death. Increased autophagy has been observed in ruptured rotator cuff tendon tissues, suggesting a causal relationship. We investigated whether autophagy occurs in human rotator cuff tenofibroblast death induced by oxidative stress and whether antioxidants protect against autophagic cell death. We used H2 O2 (0.75 mM) as oxidative stressor, cyanidin (100 µg/ml) as antioxidant, zVAD (20 µM) as apoptosis inhibitor, and 3-MA (10 mM) as autophagy inhibitor. We evaluated cell viability and known autophagic markers: LC3-II expression, GFP-LC3 puncta formation, autolysosomes, and Atg5-12 and Beclin 1 expression. H2 O2 exposure increased the rates of cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. After we induced apoptosis arrest using zVAD, H2 O2 exposure still induced cell death, LC3-II expression, and GFP-LC3 puncta formation. H2 O2 exposure also increased Atg5-12 and Beclin 1 expressions, indicating autophagic cell death. However, cyanidin treatment reduced H2 O2 -induced cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. Cyanidin and 3-MA similarly reduced the cell-death rate, and Atg5-12 and Beclin 1 expression. This study demonstrated that H2 O2 , an oxidative stressor, induces autophagic cell death in rotator cuff tenofibroblasts, and that cyanidin, a natural antioxidant, inhibits autophagic cell death. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Cancer terminator viruses (CTV): A better solution for viral-based therapy of cancer.

PubMed

Emdad, Luni; Das, Swadesh K; Wang, Xiang-Yang; Sarkar, Devanand; Fisher, Paul B

2018-08-01

In principle, viral gene therapy holds significant potential for the therapy of solid cancers. However, this promise has not been fully realized and systemic administration of viruses has not proven as successful as envisioned in the clinical arena. Our research is focused on developing the next generation of efficacious viruses to specifically treat both primary cancers and a major cause of cancer lethality, metastatic tumors (that have spread from a primary site of origin to other areas in the body and are responsible for an estimated 90% of cancer deaths). We have generated a chimeric tropism-modified type 5 and 3 adenovirus that selectively replicates in cancer cells and simultaneously produces a secreted anti-cancer toxic cytokine, melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24), referred to as a Cancer Terminator Virus (CTV) (Ad.5/3-CTV). In preclinical animal models, injection into a primary tumor causes selective cell death and therapeutic activity is also observed in non-injected distant tumors, that is, "bystander anti-tumor activity." To enhance the impact and therapeutic utility of the CTV, we have pioneered an elegant approach in which viruses are encapsulated in microbubbles allowing "stealth delivery" to tumor cells that when treated with focused ultrasound causes viral release killing tumor cells through viral replication, and producing and secreting MDA-7/IL-24, which stimulates the immune system to attack distant cancers, inhibits tumor angiogenesis and directly promotes apoptosis in distant cancer cells. This strategy is called UTMD (ultrasound-targeted microbubble-destruction). This novel CTV and UTMD approach hold significant promise for the effective therapy of primary and disseminated tumors. © 2017 Wiley Periodicals, Inc.

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Folate and Heptamethine Cyanine Modified Chitosan-Based Nanotheranostics for Tumor Targeted Near-Infrared Fluorescence Imaging and Photodynamic Therapy.

PubMed

Zhang, Yingying; Lv, Tingting; Zhang, Huijuan; Xie, Xiaodong; Li, Ziying; Chen, Haijun; Gao, Yu

2017-07-10

Folate (FA) and heptamethine cyanine (Cy7)-modified chitosan (CF7) was synthesized by click chemistry and its self-assembled nanoparticles (CF7Ns) were developed for tumor-specific imaging and photodynamic therapy. The characterization spectrum confirmed CF7 had a good FA and Cy7 conjugation efficacy. The diameter of CF7Ns measured by DLS was about 291.6 nm, and the morphology observed with AFM showed filamentous clusters of particles. The results of targeting ability of CF7Ns demonstrated enhanced targeting behaviors of CF7Ns compared with non-FA-modified nanoparticles C7Ns in FA receptor-positive HeLa cells. The cytotoxicity and cell apoptosis assay showed that CF7Ns under near-infrared light irradiation led to more apoptotic cell death in HeLa cells to improve the therapeutic efficacy. The mechanisms of the photodynamic effects of CF7Ns were demonstrated through measurement of intracellular reactive oxygen species and the apoptosis-related cytokines. These results suggested that CF7Ns are promising tumor targeting carriers for simultaneous fluorescence imaging and photodynamic therapy.

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

PubMed Central

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

2011-01-01

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

Multi-drug loaded micelles delivering chemotherapy and targeted therapies directed against HSP90 and the PI3K/AKT/mTOR pathway in prostate cancer.

PubMed

Le, Bao; Powers, Ginny L; Tam, Yu Tong; Schumacher, Nicholas; Malinowski, Rita L; Steinke, Laura; Kwon, Glen; Marker, Paul C

2017-01-01

Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy.

'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.

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

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

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

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

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

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

Selective resistance of CD44hi T cells to p53-dependent cell death results in persistence of immunologic memory after total body irradiation.

PubMed

Yao, Zhenyu; Jones, Jennifer; Kohrt, Holbrook; Strober, Samuel

2011-10-15

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4(+)CD44(hi) NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4(+) T cells were markedly more resistant than CD8(+) T cells, and CD44(hi) T cells, including NKT cells and memory T cells, were markedly more resistant than CD44(lo) (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53(-/-) mice, there was progressive T cell death in p53(-/-) mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44(hi) T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.

Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity

PubMed Central

Franco, Rodrigo

2013-01-01

Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP+-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP+-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP+-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP+, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP+ but not during rotenone or 6-OHDA toxicity. PMID:23997112

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

PubMed

Flint, D J; Boutinaud, M; Tonner, E; Wilde, C J; Hurley, W; Accorsi, P A; Kolb, A F; Whitelaw, C B A; Beattie, J; Allan, G J

2005-08-01

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.

Scorched earth strategy

PubMed Central

Wrzaczek, Michael; Brosché, Mikael

2009-01-01

Programmed cell death is a common feature of developmental processes and responses to environmental cues in many multicellular organisms. Examples of programmed cell death in plants are leaf abscission in autumn and the hypersensitive response during pathogen attack. Reactive oxygen species (ROS) have been implicated in the regulation of various types of cell death.1,2 However, the precise mechanics of the involvement of ROS in the processes leading to initiation of cell death and subsequent containment are currently unknown. We recently showed the involvement of an Arabidopsis protein GRIM REAPER in the regulation of ROS-induced cell death under stress conditions.3 Our results indicated that the presence of a truncated protein primes plants for cell death in the presence of ROS leading to ozone sensitivity and increased resistance to hemibiotrophic pathogens. PMID:19820355

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

PubMed

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

2008-01-01

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

The GYF domain protein PSIG1 dampens the induction of cell death during plant-pathogen interactions

PubMed Central

Matsui, Hidenori; Nomura, Yuko; Egusa, Mayumi; Hamada, Takahiro; Hyon, Gang-Su; Kaminaka, Hironori; Ueda, Takashi

2017-01-01

The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system. PMID:29073135

Necroptosis: an emerging type of cell death in liver diseases.

PubMed

Saeed, Waqar Khalid; Jun, Dae Won

2014-09-21

Cell death has been extensively evaluated for decades and it is well recognized that pharmacological interventions directed to inhibit cell death can prevent significant cell loss and can thus improve an organ's physiological function. For long, only apoptosis was considered as a sole form of programmed cell death. Recently necroptosis, a RIP1/RIP3-dependent programmed cell death, has been identified as an apoptotic backup cell death mechanism with necrotic morphology. The evidences of necroptosis and protective effects achieved by blocking necroptosis have been extensively reported in recent past. However, only a few studies reported the evidence of necroptosis and protective effects achieved by inhibiting necroptosis in liver related disease conditions. Although the number of necroptosis initiators is increasing; however, interestingly, it is still unclear that what actually triggers necroptosis in different liver diseases or if there is always a different necroptosis initiator in each specific disease condition followed by specific downstream signaling molecules. Understanding the precise mechanism of necroptosis as well as counteracting other cell death pathways in liver diseases could provide a useful insight towards achieving extensive therapeutic significance. By targeting necroptosis and/or other parallel death pathways, a significant cell loss and thus a decrement in an organ's physiological function can be prevented.

Evaluation of Dying Vocal Fold Epithelial Cells by Ultrastructural Features and TUNEL Method.

PubMed

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 2 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, the 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 necrosis 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. © 2016 S. Karger AG, Basel.

Ethylene Biosynthesis and Signaling Is Required for Rice Immune Response and Basal Resistance Against Magnaporthe oryzae Infection.

PubMed

Helliwell, Emily E; Wang, Qin; Yang, Yinong

2016-11-01

Recent studies have suggested that ethylene enhances host resistance to fungal pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Among the six 1-aminocyclopropane-1-carboxylic acid synthase genes in rice, OsACS1 and OsACS2 are induced within 24 h of inoculation by M. oryzae. This induction occurs simultaneously with an increase in ethylene production that is noticeable 12 h postinoculation. The purpose of this study was to examine the dynamics of ethylene production and signaling in wild type and RNA interference-mediated suppression lines deficient in ethylene production (acs2) or signaling (eil1) after challenge with M. oryzae as well as fungal cell-wall elicitors. Ethylene-insensitive mutant lines show an attenuated basal defense response including lower basal expression of the genes encoding a chitin-binding receptor, pathogenesis-related (PR) proteins, and the enzymes involved in the synthesis of diterprenoid phytoalexins, a reduction on early hypersensitive response (HR)-like cell death, and reduced incidence of callose deposition. Ethylene-deficient mutants showed an intermediate phenotype, with a significant reduction in expression of defense-related genes and callose deposition, but only a slight reduction in HR-like cell death. As a result, all ethylene-insensitive mutants show increased susceptibility to M. oryzae, whereas the ethylene-deficient lines show a slight but less significant increase in disease severity. These results show that ethylene signaling and, to some extent, ethylene production are required for rice basal resistance against the blast fungus Magnaporthe oryzae.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

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

Gonzalez-Rubio, Sandra; Linares, Clara I.; Bello, Rosario I.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca{sup 2+} on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca{sup 2+} pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca{sup 2+} entrance was induced by A23187 in HepG2. Cell death, Ca{sup 2+} mobilization, NOS-1, -2 and -3 expression, AP-1 activation,more » and NO production were evaluated. GCDCA reduced intracellular Ca{sup 2+} concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca{sup 2+} entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca{sup 2+} entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca{sup 2+} concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca{sup 2+}-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.« less

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

PubMed Central

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

2015-01-01

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

Dying dangerously: Necrotic cell death and chronic inflammation promote tumor growth.

PubMed

Lotze, Michael T; Demarco, Richard A

2004-12-01

Extract: We all shudder about untimely deaths or those that we were not prepared for. As such we perceive such "unscheduled" deaths as dangerous. Similarly, apoptotic death (literally falling leaves) or the programmed cell death of cells in multicellular organisms ranging from slime mold and simple worms through to mammals, has a level of tidiness and well-orchestrated activities with literally hundreds if not thousands of gene products employed with either the primary or secondary purpose of coordinating the orderly death of cells throughout life. During inflammation of any sort, driven by tissue damage or injury or infection by pathogens (virus, bacteria, and parasites), apoptotic death similarly serves to quickly rid the host of damaged cells, promote removal and digestion of the infected cell, and prepare the way for tissue remodeling and repair. When this goes awry, for example during periods of chronic inflammation, tissues are subjected to the contrasting needs of driving apoptotic death whilst maintaining the barrier function of the epithelia (such as skin cells) as well as the selective permeability of mucosal sites (i.e., areas where mucus is secreted to protect the cells from their surroundings, such as gut cells protecting themselves from the gastric acids). Prudently, they need to limit and husband local resources sufficiently for the maintenance of tissue integrity and renewal. It is our provocative and novel contention that cancer in adults (and not children) most often arises in a setting of chronic inflammation and disordered cell death rather than one associated primarily with disordered cell growth as it is popularly imagined by scientists, clinicians, and the general public.

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

PubMed

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

2018-06-01

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

Further considerations on in vitro skeletal muscle cell death

PubMed Central

Battistelli, Michela; Salucci, Sara; Burattini, Sabrina; Falcieri, Elisabetta

2013-01-01

Summary The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death induction. PMID:24596689

DPL-1 DP, LIN-35 Rb and EFL-1 E2F act with the MCD-1 zinc-finger protein to promote programmed cell death in Caenorhabditis elegans.

PubMed

Reddien, Peter W; Andersen, Erik C; Huang, Michael C; Horvitz, H Robert

2007-04-01

The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.

Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

PubMed

Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

2015-07-01

We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Effects of HSP27 downregulation on PDT resistance through PDT-induced autophagy in head and neck cancer cells.

PubMed

Kim, Jisun; Lim, Haesoon; Kim, Sangwoo; Cho, Hyejung; Kim, Yong; Li, Xiaojie; Choi, Hongran; Kim, Okjoon

2016-04-01

We previously reported that photodynamic therapy (PDT) induces cell death in head and neck cancer through both autophagy and apoptosis. Regulation of cell death by autophagy and apoptosis is important to enhance the effects of PDT. Autophagy maintains a balance between cell death and PDT resistance. Downregulation of heat shock protein 27 (HSP27) induces PDT resistance in head and neck cancer cells. Furthermore, HSP70 regulates apoptosis during oxidative stress. However, the role of HSPs in PDT-induced cell death through autophagy and apoptosis is unclear. Therefore, in the present study, we investigated the effects of HSP27 and HSP70 on PDT-induced cell death of oral cancer cells through autophagy and apoptosis. Cancer cells were treated with hematoporphyrin at varying doses, followed by irradiation at 635 nm with an energy density of 5 mW/cm2. We determined the changes in HSP expression by determining the levels of PARP-1 and LC3II in PDT-resistant cells. Furthermore, we assessed cell death signaling after downregulating HSPs by transfecting specific siRNAs. We observed that PDT decreased HSP27 expression but increased HSP70 expression in the head and neck cancer cells. Treatment of cells with LC3II and PARP-1 inhibitors resulted in upregulation of HSP70 and HSP27 expression, respectively. Downregulation of HSP27 and HSP70 induced cell death and PDT resistance through autophagy and apoptosis. Moreover, downregulation of HSP27 in PDT-resistant cells resulted in enhanced survival. These results indicate that the regulation of HSP27 and HSP70 plays a principal role in increasing the effects of PDT by inducing autophagic and apoptotic cell death.

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia.

PubMed

Chen, Yongqiang; Henson, Elizabeth S; Xiao, Wenyan; Huang, Daniel; McMillan-Ward, Eileen M; Israels, Sara J; Gibson, Spencer B

2016-06-02

Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

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

Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp

Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in severalmore » cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.« less

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

PubMed

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

2009-01-01

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

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

PubMed Central

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

2009-01-01

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

Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment.

PubMed

Tichy, Elisia D; Stephan, Zachary A; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J

2013-05-01

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. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

NASA Astrophysics Data System (ADS)

Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

2015-05-01

Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00211g

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.

Programmed cell death of tobacco BY-2 cells induced by still culture conditions is affected by the age of the culture under agitation.

PubMed

Hiraga, Asahi; Kaneta, Tsuyoshi; Sato, Yasushi; Sato, Seiichi

2010-01-25

Evans Blue staining indicated that actively growing tobacco BY-2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential- and stationary-phase cells, respectively. Actively growing cells became TUNEL (transferase-mediated dUTP nick end labelling)-positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical 'DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY-2 cells induced by still conditions is PCD (programmed cell death).

Selective Resistance of CD44hi T Cells to p53 Dependent Cell Death Results in Persistence of Immunologic Memory after Total Body Irradiation1,2,3

PubMed Central

Yao, Zhenyu; Jones, Jennifer; Kohrt, Holbrook; Strober, Samuel

2011-01-01

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation (TLI) markedly changes the balance of residual T cell subsets to favor CD4+CD44hi natural killer T (NKT) cells due to differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results show that CD4+ T cells were markedly more resistant than CD8+ T cells, and CD44hi T cells including NKT cells and memory T cells were markedly more resistant than CD44lo (naïve) T cells. The memory T cells immunized to alloantigens persisted even after myeloabloative (1,000cGy) TBI, and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1,000cGy was prevented in p53−/− mice, there was progressive T cell death in p53−/− mice at higher doses. Whereas, p53 dependent T cell death changed the balance of subsets, the p53 independent T cell death did not. In conclusion, resistance of CD44hi T cells to p53 dependent cell death results in the persistence of immunological memory after TBI, and can explain the immune mediated rejection of marrow transplants in sensitized recipients. PMID:21930972

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

PubMed

Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

2015-11-01

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

PubMed Central

Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

2015-01-01

Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

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

PubMed

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

2008-12-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-09-01

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

Using natural products to promote caspase-8-dependent cancer cell death.

PubMed

Tewary, Poonam; Gunatilaka, A A Leslie; Sayers, Thomas J

2017-02-01

The selective killing of cancer cells without toxicity to normal nontransformed cells is an idealized goal of cancer therapy. Thus, there has been much interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that appears to selectively kill cancer cells. TRAIL has been reported to trigger apoptosis and under some circumstances, an alternate death signaling pathway termed necroptosis. The relative importance of necroptosis for cell death induction in vivo is under intensive investigation. Nonetheless, many cancer cells (particularly those freshly isolated from cancer patients) are highly resistant to TRAIL-mediated cell death. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. There are numerous reports in which combination of TRAIL with standard antineoplastic drugs has resulted in enhanced cancer cell death in vitro. However, many of these chemotherapeutic drugs are nonspecific and associated with adverse effects, which raise serious concerns for cancer therapy in patients. By contrast, natural products have been shown to be safer and efficacious alternatives. Recently, a number of studies have suggested that certain natural products when combined with TRAIL can enhance cancer cell death. In this review, we highlight molecular pathways that might be targeted by various natural products to promote cell death, and focus on our recent work with withanolides as TRAIL sensitizers. Finally, we will suggest synergistic approaches for combining active withanolides with various forms of immunotherapy to promote cancer cell death and an effective antitumor immune response.

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

PubMed Central

LaBelle, James L.; Katz, Samuel G.; Bird, Gregory H.; Gavathiotis, Evripidis; Stewart, Michelle L.; Lawrence, Chelsea; Fisher, Jill K.; Godes, Marina; Pitter, Kenneth; Kung, Andrew L.; Walensky, Loren D.

2012-01-01

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death. PMID:22622039

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

PubMed

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

2013-05-03

Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no.

Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons

PubMed Central

Wehner, Amanda B.; Abdesselem, Houari; Dickendesher, Travis L.; Imai, Fumiyasu; Yoshida, Yutaka; Giger, Roman J.; Pierchala, Brian A.

2016-01-01

ABSTRACT During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as ‘competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro. The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death. PMID:27143756

‘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

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

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

Wu, Ping; Zhu, Xueping; Jin, Wei

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

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

PubMed

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

2016-09-01

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

Transglutaminase induction by various cell death and apoptosis pathways.

PubMed

Fesus, L; Madi, A; Balajthy, Z; Nemes, Z; Szondy, Z

1996-10-31

Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

PubMed Central

Lang, Fangfang; Qin, Zhaoyang; Li, Fang; Zhang, Huilin; Fang, Zhenghui; Hao, Enkui

2015-01-01

Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells. PMID:26067645

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Mechanical Stress Promotes Cisplatin-Induced Hepatocellular Carcinoma Cell Death

PubMed Central

Riad, Sandra; Bougherara, Habiba

2015-01-01

Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death). PMID:25685789

Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death.

PubMed

Evans, M; Murofushi, T; Tsuda, H; Mikami, Y; Zhao, N; Ochiai, K; Kurita-Ochiai, T; Yamamoto, M; Otsuka, K; Suzuki, N

2017-06-01

Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram-negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms. The Ca9-22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double-stranded DNA. AMP-activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule-associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription-polymerase chain reaction. Butyrate-induced cell death occurred in a dose-dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate-induced cell death under conditions of starvation, AMPK activation resulted in butyrate-induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate-induced autophagy-dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly induced LC3B production and the induction was attenuated by AMPK inhibition. LC3B knockdown, in turn, significantly decreased butyrate-induced cell death. Therefore, AMPK-dependent LC3B induction apparently plays an important role in butyrate-induced cell death. There was a lack of correspondence between the levels of AMPK activation and LC3B induction; this may reflect the histone deacetylase-inhibitory capacity of butyrate on histone proteins. Taken together, starvation and butyrate exposure promote autophagy via AMPK signaling, while the histone deacetylase-inhibitory effects of butyrate alter chromatin to transcriptionally active state, resulting in strong LC3B induction and subsequent cell death. These findings may help improve the understanding of the cellular processes underlying periodontal disease initiation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

PubMed

Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

2014-09-06

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

PubMed

Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

2013-03-06

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

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.

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

PubMed Central

Inoue, H; Tani, K

2014-01-01

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

Contribution of TMEM16F to pyroptotic cell death.

PubMed

Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Schreiber, Rainer; Kunzelmann, Karl

2018-02-20

Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X 7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca 2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X 7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca 2+ , we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca 2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids.

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

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

Ji, Chao; Yang, Bo; Yang, Zhi

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

Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

PubMed

Ehrhard, Simone; Wernli, Marion; Dürmüller, Ursula; Battegay, Manuel; Gudat, Fred; Erb, Peter

2009-10-01

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

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

Kito, Hiroaki; Yamazaki, Daiju; Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto

Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cellmore » turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.« less

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

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

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

2008-07-15

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

Induction of Inhibitory Receptors on T Cells During Plasmodium vivax Malaria Impairs Cytokine Production

PubMed Central

Costa, Pedro A. C.; Leoratti, Fabiana M. S.; Figueiredo, Maria M.; Tada, Mauro S.; Pereira, Dhelio B.; Junqueira, Caroline; Soares, Irene S.; Barber, Daniel L.; Gazzinelli, Ricardo T.; Antonelli, Lis R. V.

2015-01-01

The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4+ and CD8+ T cells. Higher frequencies of CD4+ express more than 1 regulatory molecule compared to CD8+ T cells. Moreover, lower proportions of CD4+ T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin–3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function. PMID:26019284

Precise stacking of decellularized extracellular matrix based 3D cell-laden constructs by a 3D cell printing system equipped with heating modules.

PubMed

Ahn, Geunseon; Min, Kyung-Hyun; Kim, Changhwan; Lee, Jeong-Seok; Kang, Donggu; Won, Joo-Yun; Cho, Dong-Woo; Kim, Jun-Young; Jin, Songwan; Yun, Won-Soo; Shim, Jin-Hyung

2017-08-17

Three-dimensional (3D) cell printing systems allow the controlled and precise deposition of multiple cells in 3D constructs. Hydrogel materials have been used extensively as printable bioinks owing to their ability to safely encapsulate living cells. However, hydrogel-based bioinks have drawbacks for cell printing, e.g. inappropriate crosslinking and liquid-like rheological properties, which hinder precise 3D shaping. Therefore, in this study, we investigated the influence of various factors (e.g. bioink concentration, viscosity, and extent of crosslinking) on cell printing and established a new 3D cell printing system equipped with heating modules for the precise stacking of decellularized extracellular matrix (dECM)-based 3D cell-laden constructs. Because the pH-adjusted bioink isolated from native tissue is safely gelled at 37 °C, our heating system facilitated the precise stacking of dECM bioinks by enabling simultaneous gelation during printing. We observed greater printability compared with that of a non-heating system. These results were confirmed by mechanical testing and 3D construct stacking analyses. We also confirmed that our heating system did not elicit negative effects, such as cell death, in the printed cells. Conclusively, these results hold promise for the application of 3D bioprinting to tissue engineering and drug development.

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

PubMed

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

2009-10-01

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

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

PubMed

Muntané, Jordi

2011-10-17

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

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

Regulated necrosis and its implications in toxicology.

PubMed

Aki, Toshihiko; Funakoshi, Takeshi; Uemura, Koichi

2015-07-03

Recent research developments have revealed that caspase-dependent apoptosis is not the sole form of regulated cell death. Caspase-independent, but genetically regulated, forms of cell death include pyroptosis, necroptosis, parthanatos, and the recently discovered ferroptosis and autosis. Importantly, regulated necrosis can be modulated by small molecule inhibitors/activators, confirming the cell autonomous mechanism of these forms of cell death. The success of small molecule-mediated manipulation of regulated necrosis has produced great changes in the field of cell death research, and has also brought about significant changes in the fields of pharmacology as well as toxicology. In this review, we intend to summarize the modes of regulated cell death other than apoptosis, and discuss their implications in toxicology. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Live to die another way: modes of programmed cell death and the signals emanating from dying cells

PubMed Central

Fuchs, Yaron; Steller, Hermann

2015-01-01

Preface All life ends in death, but perhaps one of life’s grander ironies is that it also depends on death. Cell-intrinsic suicide pathways, termed programmed cell death (PCD), are crucial for animal development, tissue homeostasis and pathogenesis. Originally, PCD was virtually synonymous with apoptosis, but recently, alternative PCD mechanisms have been reported. Here, we provide an overview of several distinct PCD mechanisms, namely apoptosis, autophagy and necroptosis. In addition, we discuss the complex signals emanating from dying cells, which can either fuel regeneration or instruct additional killing. Further advances in understanding the physiological role of multiple cell death mechanisms and associated signals will be important to selectively manipulate PCD for therapeutic purposes. PMID:25991373

Cytoplasmic vacuolization in cell death and survival

PubMed Central

Komissarov, Alexey A.; Rafieva, Lola M.; Kostrov, Sergey V.

2016-01-01

Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival. PMID:27331412

Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

BID links ferroptosis to mitochondrial cell death pathways.

PubMed

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

2017-08-01

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Mechanisms underlying 3-bromopyruvate-induced cell death in colon cancer.

PubMed

Sun, Yiming; Liu, Zhe; Zou, Xue; Lan, Yadong; Sun, Xiaojin; Wang, Xiu; Zhao, Surong; Jiang, Chenchen; Liu, Hao

2015-08-01

3-Bromopyruvate (3BP) is an energy-depleting drug that inhibits Hexokinase II activity by alkylation during glycolysis, thereby suppressing the production of ATP and inducing cell death. As such, 3BP can potentially serve as an anti-tumorigenic agent. Our previous research showed that 3BP can induce apoptosis via AKT /protein Kinase B signaling in breast cancer cells. Here we found that 3BP can also induce colon cancer cell death by necroptosis and apoptosis at the same time and concentration in the SW480 and HT29 cell lines; in the latter, autophagy was also found to be a mechanism of cell death. In HT29 cells, combined treatment with 3BP and the autophagy inhibitor 3-methyladenine (3-MA) exacerbated cell death, while viability in 3BP-treated cells was enhanced by concomitant treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and the necroptosis inhibitor necrostatin (Nec)-1. Moreover, 3BP inhibited tumor growth in a SW480 xenograft mouse model. These results indicate that 3BP can suppress tumor growth and induce cell death by multiple mechanisms at the same time and concentration in different types of colon cancer cell by depleting cellular energy stores.

Detecting cell death with optical coherence tomography and envelope statistics

NASA Astrophysics Data System (ADS)

Farhat, Golnaz; Yang, Victor X. D.; Czarnota, Gregory J.; Kolios, Michael C.

2011-02-01

Currently no standard clinical or preclinical noninvasive method exists to monitor cell death based on morphological changes at the cellular level. In our past work we have demonstrated that quantitative high frequency ultrasound imaging can detect cell death in vitro and in vivo. In this study we apply quantitative methods previously used with high frequency ultrasound to optical coherence tomography (OCT) to detect cell death. The ultimate goal of this work is to use these methods for optically-based clinical and preclinical cancer treatment monitoring. Optical coherence tomography data were acquired from acute myeloid leukemia cells undergoing three modes of cell death. Significant increases in integrated backscatter were observed for cells undergoing apoptosis and mitotic arrest, while necrotic cells induced a decrease. These changes appear to be linked to structural changes observed in histology obtained from the cell samples. Signal envelope statistics were analyzed from fittings of the generalized gamma distribution to histograms of envelope intensities. The parameters from this distribution demonstrated sensitivities to morphological changes in the cell samples. These results indicate that OCT integrated backscatter and first order envelope statistics can be used to detect and potentially differentiate between modes of cell death in vitro.

Apoptosis evaluation in epithelial cells exposed to different chemicals: relevance of floating cells.

PubMed

Turco, L; De Angelis, I; Stammati, A; Zucco, F

2000-01-01

The recent increase in understanding of cell death has promoted new approaches in toxicological studies, mainly those dealing with in vitro systems where the evaluation of cell death has been the most widely adopted end-point in measuring the effects of chemical toxicants. The aim of this study was to investigate the possibility of improving the traditional cytotoxicity test protocols in order to produce more specific information on the type of cell death induced by exposure to toxicants. In particular, we characterized the mode of cell death in an established epithelial cell line, HEp-2 cells, which is frequently used in cytotoxicity testing owing to its easy handling and standardization of culture conditions. Reference chemicals for apoptosis and necrosis were selected as controls, together with other molecules that have been shown, in preliminary studies, to induce various morphological and structural modifications in relation to cell death. The results obtained show that: (a) the floating fraction of treated cells gives the clearest picture of the necrotic/apoptotic distribution; (b) morphological analysis is crucial for characterization of apoptosis; (c) more than one cytotoxic end-point is necessary to clearly identify the type of cell death.

Autophagy promotes caspase-dependent cell death during Drosophila development.

PubMed

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

2009-04-01

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

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

PubMed

Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

2018-05-23

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

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

Research highlights: {yields} Ischemia induces high level of iPLA{sub 2} resulting in caspase-independent myocyte death. {yields} Urocortin causes iPLA{sub 2} down-regulation leading to avoidance of non-apoptotic death. {yields} The survival-promoting effect of urocortin is abrogated by CRH receptor antagonist. -- Abstract: Caspase-independent, non-apoptotic cell death in ischemic heart disease is considered to be one of the important therapeutic targets, however, the detailed mechanisms of this cell death process are not clear. In this study, we investigated the mechanisms of non-apoptotic cell death in cultured neonatal rat cardiomyocytes during ischemia, and the cardioprotection by preventing the mechanisms. We found that ischemiamore » caused elevation of the phospholipase A{sub 2} (iPLA{sub 2}) expression in the myocytes, leading to distinctive non-apoptotic nuclear shrinkage, and cell death. Moreover, we investigated whether the potent cardioprotective corticotropin-releasing hormone (CRH), urocortin, which had been less focused on non-apoptotic cell death, inhibits the ischemic myocyte death. Ischemia-augmented nuclear shrinkage of the myocytes was suppressed by the pretreatment of {approx}10 nM urocortin before the cells were exposed to ischemia. Urocortin could significantly suppress the expression and activity of iPLA{sub 2}, resulting in preventing the ischemia-induced cell death. The survival-promoting effect of urocortin was abrogated by the CRH receptor antagonist astressin. These findings provide the first evidence linking the targets of the urocortin-mediated cardioprotection to the suppression of the caspase-independent, non-apoptotic death in cardiac myocytes exposed to ischemia.« less

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

PubMed

Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

2017-03-01

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

Steen, Hakan; Lindholm, Dan; Minerva Institute for Medical Research, Biomedicum Helsinki, Helsinki

2008-02-08

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

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.

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

PubMed

Sato, Akira

2017-01-01

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

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

PubMed Central

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

2008-01-01

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

Sickle cell trait and sudden death--bringing it home.

PubMed Central

Mitchell, Bruce L.

2007-01-01

Sickle cell trait continues to be the leading cause of sudden death for young African Americans in military basic training and civilian organized sports. The syndrome may have caused the death of up to 10 college football players since 1974 and, as recently as 2000, was suspected as the cause of death of three U.S. Army recruits. The penal military-style boot camps in the United States and the recent death of two teenagers with sickle cell trait merits renewed vigor in the education of athletic instructors, the military and the public about conditions associated with sudden death in individuals with sickle cell trait. Images Figure 1 Figure 2 PMID:17393956

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

PubMed

Muñoz-Pinedo, Cristina

2012-01-01

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

Utility in Treating Kidney Failure in End-Stage Liver Disease With Simultaneous Liver-Kidney Transplantation.

PubMed

Cheng, Xingxing S; Stedman, Margaret R; Chertow, Glenn M; Kim, W Ray; Tan, Jane C

2017-05-01

Simultaneous liver-kidney (SLK) transplantation plays an important role in treating kidney failure in patients with end-stage liver disease. It used 5% of deceased donor kidney transplanted in 2015. We evaluated the utility, defined as posttransplant kidney allograft lifespan, of this practice. Using data from the Scientific Registry of Transplant Recipients, we compared outcomes for all SLK transplants between January 1, 1995, and December 3, 2014, to their donor-matched kidney used in kidney-alone (Ki) or simultaneous pancreas kidney (SPK) transplants. Primary outcome was kidney allograft lifespan, defined as the time free from death or allograft failure. Secondary outcomes included death and death-censored allograft failure. We adjusted all analyses for donor, transplant, and recipient factors. The adjusted 10-year mean kidney allograft lifespan was higher in Ki/SPK compared with SLK transplants by 0.99 years in the Model for End-stage Liver Disease era and 1.71 years in the pre-Model for End-stage Liver Disease era. Death was higher in SLK recipients relative to Ki/SPK recipients: 10-year cumulative incidences 0.36 (95% confident interval 0.33-0.38) versus 0.19 (95% confident interval 0.17-0.21). SLK transplantation exemplifies the trade-off between the principles of utility and medical urgency. With each SLK transplantation, about 1 year of allograft lifespan is traded so that sicker patients, that is, SLK transplant recipients, are afforded access to the organ. These data provide a basis against which benefits derived from urgency-based allocation can be measured.

Utility in Treating Kidney Failure in End-Stage Liver Disease With Simultaneous Liver-Kidney Transplantation

PubMed Central

Cheng, Xingxing S.; Stedman, Margaret R.; Chertow, Glenn M.; Kim, W. Ray; Tan, Jane C.

2017-01-01

Background Simultaneous liver-kidney (SLK) transplantation plays an important role in treating kidney failure in patients with end-stage liver disease. It used 5% of deceased donor kidney transplanted in 2015. We evaluated the utility, defined as posttransplant kidney allograft lifespan, of this practice. Methods Using data from the Scientific Registry of Transplant Recipients, we compared outcomes for all SLK transplants between January 1, 1995, and December 3, 2014, to their donor-matched kidney used in kidney-alone (Ki) or simultaneous pancreas kidney (SPK) transplants. Primary outcome was kidney allograft lifespan, defined as the time free from death or allograft failure. Secondary outcomes included death and death-censored allograft failure. We adjusted all analyses for donor, transplant, and recipient factors. Results The adjusted 10-year mean kidney allograft lifespan was higher in Ki/SPK compared with SLK transplants by 0.99 years in the Model for End-stage Liver Disease era and 1.71 years in the pre-Model for End-stage Liver Disease era. Death was higher in SLK recipients relative to Ki/SPK recipients: 10-year cumulative incidences 0.36 (95% confident interval 0.33-0.38) versus 0.19 (95% confident interval 0.17-0.21). Conclusions SLK transplantation exemplifies the trade-off between the principles of utility and medical urgency. With each SLK transplantation, about 1 year of allograft lifespan is traded so that sicker patients, that is, SLK transplant recipients, are afforded access to the organ. These data provide a basis against which benefits derived from urgency-based allocation can be measured. PMID:28437790

NAD+ depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death?

PubMed

Siegel, C; McCullough, L D

2011-09-01

Multiple cell death pathways are activated in cerebral ischaemia. Much of the initial injury, especially in the core of the infarct where cerebral blood flow is severely reduced, is necrotic and secondary to severe energy failure. However, there is considerable evidence that delayed cell death continues for several days, primarily in the penumbral region. As reperfusion therapies grow in number and effectiveness, restoration of blood flow early after injury may lead to a shift towards apoptosis. It is important to elucidate what are the key mediators of apoptotic cell death after stroke, as inhibition of apoptosis may have therapeutic implications. There are two well described pathways that lead to apoptotic cell death; the caspase pathway and the more recently described caspase-independent pathway triggered by poly-ADP-ribose polymers (PARP) activation. Caspase-induced cell death is initiated by release of mitochondrial cytochrome c, formation of the cytosolic apoptosome, and activation of endonucleases leading to a multitude of small randomly cleaved DNA fragments. In contrast caspase-independent cell death is secondary to activation of apoptosis inducing factor (AIF). Mitochondrial AIF translocates to the nucleus, where it induces peripheral chromatin condensation, as well as characteristic high-molecular-weight (50 kbp) DNA fragmentation. Although caspase-independent cell death has been recognized for some time and is known to contribute to ischaemic injury, the upstream triggering events leading to activation of this pathway remain unclear. The two major theories are that ischaemia leads to nicotinamide adenine dinucleotide (NAD+) depletion and subsequent energy failure, or alternatively that cell death is directly triggered by a pro-apoptotic factor produced by activation of the DNA repair enzyme PARP. PARP activation is robust in the ischaemic brain producing variable lengths of poly-ADP-ribose (PAR) polymers as byproducts of PARP activation. PAR polymers may be directly toxic by triggering mitochondrial AIF release independently of NAD+ depletion. Recently, sex differences have been discovered that illustrate the importance of understanding these molecular pathways, especially as new therapeutics targeting apoptotic cell death are developed. Cell death in females proceeds primarily via caspase activation whereas caspase-independent mechanisms triggered by the activation of PARP predominate in the male brain. This review summarizes the current literature in an attempt to clarify the roles of NAD+ and PAR polymers in caspase-independent cell death, and discuss sex specific cell death to provide an example of the possible importance of these downstream mediators. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

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

PubMed

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

2018-01-01

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

Human colon cancer HT-29 cell death responses to doxorubicin and Morus Alba leaves flavonoid extract.

PubMed

Fallah, S; Karimi, A; Panahi, G; Gerayesh Nejad, S; Fadaei, R; Seifi, M

2016-03-31

The mechanistic basis for the biological properties of Morus alba flavonoid extract (MFE) and chemotherapy drug of doxorubicin on human colon cancer HT-29 cell line death are unknown. The effect of doxorubicin and flavonoid extract on colon cancer HT-29 cell line death and identification of APC gene expression and PARP concentration of HT-29 cell line were investigated. The results showed that flavonoid extract and doxorubicin induce a dose dependent cell death in HT-29 cell line. MFE and doxorubicin exert a cytotoxic effect on human colon cancer HT-29 cell line by probably promoting or induction of apoptosis.

Cylindromatosis mediates neuronal cell death in vitro and in vivo.

PubMed

Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

2018-01-19

The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

Fight or Flight - Regulation of Emergency Hematopoiesis by Pyroptosis and Necroptosis

PubMed Central

Croker, Ben A.; Silke, John; Gerlic, Motti

2015-01-01

Purpose of review A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis, a caspase-1-dependent cell death, and necroptosis, a RIPK3/MLKL-dependent, caspase-independent cell death, on emergency hematopoiesis. Recent findings Activation of non-apoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns (DAMPs) such as IL-1α, IL-1β, IL-18, IL-33, HMGB1 and mtDNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia and immunosuppression. Although originally described as discrete entities, there now appears to be more intimate connections between the non-apoptotic and death receptor signaling pathways. Summary The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis. PMID:26049749

Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

PubMed

Viringipurampeer, Ishaq A; Gregory-Evans, Cheryl Y; Metcalfe, Andrew L; Bashar, Emran; Moritz, Orson L; Gregory-Evans, Kevin

2018-06-18

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Using stochastic cell division and death to probe minimal units of cellular replication

NASA Astrophysics Data System (ADS)

Chib, Savita; Das, Suman; Venkatesan, Soumya; Sai Narain Seshasayee, Aswin; Thattai, Mukund

2018-03-01

The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in Escherichia coli populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term ‘widgets’ reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget’s molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

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

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.

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

PubMed Central

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-01-01

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2−), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2− production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2− is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2−. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment. PMID:26655501

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.

PubMed

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-04-12

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.

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.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

PubMed Central

Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

2013-01-01

Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

PubMed

Erental, Ariel; Sharon, Idith; Engelberg-Kulka, Hanna

2012-01-01

In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

PubMed

Wu, Yuqiu; Kazumura, Kimiko; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

2015-10-01

Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria

PubMed Central

Bonhoeffer, Sebastian

2018-01-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm. PMID:29750784

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

PubMed

Frenoy, Antoine; Bonhoeffer, Sebastian

2018-05-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm.

Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death.

PubMed

Zamulaeva, I A; Lekakh, I V; Kiseleva, V I; Gabai, V L; Saenko, A S; Shevchenko, A S; Poverenny, A M

1997-08-18

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

ERK1/2 and the Bcl-2 Family Proteins Mcl-1, tBid, and Bim Are Involved in Inhibition of Apoptosis During Persistent Chlamydia psittaci Infection.

PubMed

Li, Li; Wang, Chuan; Wen, Yating; Hu, Yuming; Xie, Yafeng; Xu, Man; Liang, Mingxing; Liu, Wei; Liu, Liangzhuan; Wu, Yimou

2018-04-18

Chlamydia psittaci is an obligate intracellular pathogen that can cause zoonosis. Persistent C. psittaci infection can inhibit apoptosis in host cells, thus extending their survival and enabling them to complete their growth cycle. In this study, the antiapoptotic effects of persistent C. psittaci infection, induced by treatment with IFN-γ, were found to be associated with both the death receptor and the mitochondrial pathways of apoptosis. These effects were mediated by Bcl-2 family members, as evidenced by the decreased expression of proapoptotic proteins, such as tBid and Bim. Simultaneously, the antiapoptotic protein Mcl-1 was upregulated by persistent C. psittaci infection. Increased phosphorylation of ERK1/2 was observed; however, the expression of Bad, unlike that of other proapoptotic proteins, did not seem to be involved in this process. In summary, persistent chlamydial infection exerts antiapoptotic effects through both the death receptor and the mitochondrial pathways, in a process that is regulated by the ERK1/2 and apoptotic proteins of the Bcl-2 family.

Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system

PubMed Central

Moya, Bartolomé; Munar-Bestard, Marta; Zamorano, Laura; Cabot, Gabriel; Blázquez, Jesús; Ayala, Juan A.; Oliver, Antonio

2017-01-01

Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β–lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons. PMID:28742861

Consequences of Neurite Transection In Vitro

PubMed Central

Cengiz, Nurettin; Erdoğan, Ender; Him, Aydın; Oğuz, Elif Kaval

2012-01-01

Abstract In order to quantify degenerative and regenerative changes and analyze the contribution of multiple factors to the outcome after neurite transection, we cultured adult mouse dorsal root ganglion neurons, and with a precise laser beam, we transected the nerve fibers they extended. Cell preparations were continuously visualized for 24 h with time-lapse microscopy. More distal cuts caused a more elongated field of degeneration, while thicker neurites degenerated faster than thinner ones. Transected neurites degenerated more if the uncut neurites of the same neuron simultaneously degenerated. If any of these uncut processes regenerated, the transected neurites underwent less degeneration. Regeneration of neurites was limited to distal cuts. Unipolar neurons had shorter regeneration than multipolar ones. Branching slowed the regenerative process, while simultaneous degeneration of uncut neurites increased it. Proximal lesions, small neuronal size, and extensive and rapid neurite degeneration were predictive of death of an injured neuron, which typically displayed necrotic rather than apoptotic form. In conclusion, this in vitro model proved useful in unmasking many new aspects and correlates of mechanically-induced neurite injury. PMID:20121423

Oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signaling loop to induce cell death in cancer cells.

PubMed

Wang, Qian; Huang, Lihong; Yue, Jianbo

2017-06-01

High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2), a Ca 2+ -permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of TRPM2-mediated Ca 2+ influx on oxidative stress-induced cell death in cancer cells. We found that oxidative stress activated the TRPM2-Ca 2+ -CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondrial membrane potential. In summary, our data demonstrated that oxidative stress activates the TRPM2-Ca 2+ -CaMKII-ROS signal loop to inhibit autophagy and induce cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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

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

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

Discovery of a BTK/MNK Dual Inhibitor for Lymphoma and Leukemia

PubMed Central

Wu, Hong; Hu, Chen; Wang, Aoli; Weisberg, Ellen L.; Chen, Yongfei; Yun, Cai-Hong; Wang, Wenchao; Liu, Yan; Liu, Xiaochuan; Tian, Bei; Wang, Jinhua; Zhao, Zheng; Liang, Yanke; Li, Binhua; Wang, Li; Wang, Beilei; Chen, Cheng; Buhrlage, Sara J.; Nonami, Atsushi; Li, Yuyang; Fernandes, Stacey M.; Adamia, Sophia; Stone, Richard M.; Galinsky, Ilene A.; Wang, Xianhuo; Yang, Guang; Griffin, James D.; Brown, Jennifer R.; Eck, Michael J.; Liu, Jing; Gray, Nathanael S.; Liu, Qingsong

2016-01-01

BTK kinase is a member of the TEC kinase family and is a key regulator of the B-cell Receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as MCL, CLL and AML. MNK kinase is one of the key downstream regulators in the RAF-MEK-ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has shown moderate efficacy for AML cell lines proliferation. Through a structure-based drug design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and non-covalent binding to MNK. Compared to the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 displays a stronger anti-proliferative effect against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0–G1 stage and can induce strong apoptotic cell death. These results demonstrated that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances. PMID:26165234

Cell death and cell lysis are separable events during pyroptosis

PubMed Central

DiPeso, Lucian; Ji, Daisy X; Vance, Russell E; Price, Jordan V

2017-01-01

Although much insight has been gained into the mechanisms by which activation of the inflammasome can trigger pyroptosis in mammalian cells, the precise kinetics of the end stages of pyroptosis have not been well characterized. Using time-lapse fluorescent imaging to analyze the kinetics of pyroptosis in individual murine macrophages, we observed distinct stages of cell death and cell lysis. Our data demonstrate that cell membrane permeability resulting from gasdermin D pore formation is coincident with the cessation of cell movement, loss of mitochondrial activity, and cell swelling, events that can be uncoupled from cell lysis. We propose a model of pyroptosis in which cell death can occur independently of cell lysis. The uncoupling of cell death from cell lysis may allow for better control of cytosolic contents upon activation of the inflammasome. PMID:29147575

Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells

PubMed Central

Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

2017-01-01

Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity. PMID:28139717

Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells.

PubMed

Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

2017-01-31

Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity.

The simultaneous death of seven people due to the detonation of an antipersonnel landmine at the land borders of the European Union during peacetime.

PubMed

Pavlidis, Pavlos; Karakasi, Valeria

2015-01-01

This incident concerns the simultaneous death of seven people as a result of the accidental triggering of an antipersonnel landmine during peacetime. The victims were illegal migrants who attempted to cross the Greek-Turkish border zone and accidentally entered a demarcated minefield. This incident is presented because of its rarity and highlights the devastating consequences of the residual mines on the European Union eastern frontiers in peacetime. It also showcases the difficulties and risks that arise during the identification process in illegal migration issues. The victims' positions at the moment of explosion are indicated by the detailed forensic examination and comparison of the injuries' anatomical dispersion and their severity.

Expressions of Mast Cell Tryptase and Brain Natriuretic Peptide in Myocardium of Sudden Death due to Hypersensitivity and Coronary Atherosclerotic Heart Disease.

PubMed

Shi, J R; Tian, C J; Zeng, Q; Guo, X J; Lu, J; Gao, C R

2016-06-01

To explore the value of mast cell tryptase and brain natriuretic peptide（BNP） in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart disease. Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010-2015. All samples were divided into three groups： death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting. Immunofluorescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison （ P <0.05）; The expression level of BNP in sudden death of coronary atherosclerotic heart disease group were significantly higher than the sudden death of hypersensitivity group and death of craniocerebral injury group （ P <0.05）. The difference of the expression level of BNP between the sudden death of hypersensitivity group and the death of craniocerebral injury group had no statistical significance （ P >0.05）. The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease. Copyright© by the Editorial Department of Journal of Forensic Medicine

Stem cell death and survival in heart regeneration and repair.

PubMed

Abdelwahid, Eltyeb; Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

2016-03-01

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function.

Stem cell death and survival in heart regeneration and repair

PubMed Central

Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

2016-01-01

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function. PMID:26687129

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

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

PubMed

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

2017-11-17

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

THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY

EPA Science Inventory

Apoptosis, a form of programmed cell death, occurs in the nervous system throughout development, but with a preponderance of cell death occurring during the prenatal and perinatal periods. Aberrant periods of increased or decreased cell death, induced by toxicants in air, water,...

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death*

PubMed Central

Itakura, Masanori; Kubo, Takeya; Kaneshige, Akihiro; Harada, Naoki; Izawa, Takeshi; Azuma, Yasu-Taka; Kuwamura, Mitsuru; Yamaji, Ryouichi; Takeuchi, Tadayoshi

2017-01-01

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death. PMID:28167533

The Sigma Receptor Ligand (+)-Pentazocine Prevents Apoptotic Retinal Ganglion Cell Death induced in vitro by Homocysteine and Glutamate

PubMed Central

Martin, Pamela Moore; Ola, Mohammad S.; Agarwal, Neeraj; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Recent studies demonstrated that the excitotoxic amino acid homocysteine induces apoptotic death of retinal ganglion cells in vivo. In the present study, an in vitro rat retinal ganglion cell (RGC-5) culture system was used to analyze the toxicity of acute exposure to high levels of homocysteine, the mechanism of homocysteine-induced toxicity and the usefulness of σR1 ligands as neuroprotectants. When cultured RGC-5 cells were subjected to treatment with 1 mM D, L- homocysteine, a significant increase in cell death was detected by TUNEL analysis and analysis of activated caspase. When cells were treated with homocysteine- or glutamate in the presence of MK-801, an antagonist of the NMDA receptor, the cell death was inhibited significantly. In contrast, NBQX, an antagonist of the AMPA/Kainate receptor, and nifedipine, a calcium channel blocker, did not prevent the homocysteine- or glutamate-induced cell death. Semi-quantitative RT-PCR and immunocytochemical analysis demonstrated that RGC-5 cells exposed to homocysteine or glutamate express type 1 sigma receptor at levels similar to control cells. Treatment of RGC-5 cells with 3 µM or 10 µM concentrations of the σR1-specific ligand (+)-pentazocine inhibited significantly the apoptotic cell death induced by homocysteine or glutamate. The results suggest that homocysteine is toxic to ganglion cells in vitro, that the toxicity is mediated via NMDA receptor activation, and that the σR1-specific ligand (+)-pentazocine can block the RGC-5 cell death induced by homocysteine and glutamate. PMID:15046867

Engineering death receptor ligands for cancer therapy.

PubMed

Wajant, Harald; Gerspach, Jeannette; Pfizenmaier, Klaus

2013-05-28

CD95, TNFR1, TRAILR1 and TRAILR2 belong to a subgroup of TNF receptors which is characterized by a conserved cell death-inducing protein domain that connects these receptors to the apoptotic machinery of the cell. Activation of death receptors in malignant cells attracts increasing attention as a principle to fight cancer. Besides agonistic antibodies the major way to stimulate death receptors is the use of their naturally occurring "death ligands" CD95L, TNF and TRAIL. However, dependent from the concept followed to develop a death ligand-based therapy various limiting aspects have to be taken into consideration on the way to a "bedside" usable drug. Problems arise in particular from the cell associated transmembrane nature of the death ligands, the poor serum half life of the soluble fragments derived from the transmembrane ligands, the ubiquitous expression of the death receptors and the existence of additional non-death receptors of the death ligands. Here, we summarize strategies how these limitations can be overcome by genetic engineering. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

[Progression of the manner of cardiomyocyte death and its potential significance in forensic medicine].

PubMed

Feng, Xiang-ping; Wu, Wei; Chen, Xin-shan

2004-01-01

The manner of cell death is a hotspot of medical researchers. Apoptosis and necrosis were considered as two manners of cell death in the past. But recently a new manner of cell death--oncosis is gradually accepted by the pathologists. Oncosis is different from apoptosis in morphologic, mechanism and the role in cardiovascular diseases. In this paper, the progression of the research about manner of the cardiomyocyte death and its significance in forensic medicine in recent years was reviewed.

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

PubMed Central

Phizacklea, Mark; Gay, Nicholas J.

2017-01-01

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88–NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts. PMID:28373203

6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells.

PubMed

Li, Ting-Yi; Chiang, Been-Huang

2017-09-01

6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6-shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6h, 6-shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death

PubMed Central

Cao, Chike; Liu, Yanling; Lehmann, Michael

2007-01-01

Cell death during Drosophila melanogaster metamorphosis is controlled by the steroid hormone 20-hydroxyecdysone (20E). Elements of the signaling pathway that triggers death are known, but it is not known why some tissues, and not others, die in response to a particular hormone pulse. We found that loss of the tissue-specific transcription factor Fork head (Fkh) is both required and sufficient to specify a death response to 20E in the larval salivary glands. Loss of fkh itself is a steroid-controlled event that is mediated by the 20E-induced BR-C gene, and that renders the key death regulators hid and reaper hormone responsive. These results implicate the D. melanogaster FOXA orthologue Fkh with a novel function as a competence factor for steroid-controlled cell death. They explain how a specific tissue is singled out for death, and why this tissue survives earlier hormone pulses. More generally, they suggest that cell identity factors like Fkh play a pivotal role in the normal control of developmental cell death. PMID:17339378

Comparative analysis of the role of small G proteins in cell migration and cell death: Cytoprotective and promigratory effects of RalA

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

Jeon, Hyejin; Zheng, Long Tai; Lee, Shinrye

2011-08-15

Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show thatmore » small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.« less

Zinc Oxide Nanoparticle Induces Microglial Death by NADPH-Oxidase-Independent Reactive Oxygen Species as well as Energy Depletion.

PubMed

Sharma, Anuj Kumar; Singh, Vikas; Gera, Ruchi; Purohit, Mahaveer Prasad; Ghosh, Debabrata

2017-10-01

Zinc oxide nanoparticle (ZnO-NP) is one of the most widely used engineered nanoparticles. Upon exposure, nanoparticle can eventually reach the brain through various routes, interact with different brain cells, and alter their activity. Microglia is the fastest glial cell to respond to any toxic insult. Nanoparticle exposure can activate microglia and induce neuroinflammation. Simultaneous to activation, microglial death can exacerbate the scenario. Therefore, we focused on studying the effect of ZnO-NP on microglia and finding out the pathway involved in the microglial death. The present study showed that the 24 h inhibitory concentration 50 (IC 50 ) of ZnO-NP for microglia is 6.6 μg/ml. Early events following ZnO-NP exposure involved increase in intracellular calcium level as well as reactive oxygen species (ROS). Neither of NADPH oxidase inhibitors, apocynin, (APO) and diphenyleneiodonium chloride (DPIC) were able to reduce the ROS level and rescue microglia from ZnO-NP toxicity. In contrary, N-acetyl cysteine (NAC) showed opposite effect. Exogenous supplementation of superoxide dismutase (SOD) reduced ROS significantly even beyond control level but partially rescued microglial viability. Interestingly, pyruvate supplementation rescued microglia near to control level. Following 10 h of ZnO-NP exposure, intracellular ATP level was measured to be almost 50 % to the control. ZnO-NP-induced ROS as well as ATP depletion both disturbed mitochondrial membrane potential and subsequently triggered the apoptotic pathway. The level of apoptosis-inducing proteins was measured by western blot analysis and found to be upregulated. Taken together, we have deciphered that ZnO-NP induced microglial apoptosis by NADPH oxidase-independent ROS as well as ATP depletion.

Histopathology and Functional Correlations in a Patient with a Mutation in RPE65, the Gene for Retinol Isomerase

PubMed Central

Rayborn, Mary E.; Li, Yong; Grossman, Gregory H.; Berson, Eliot L.; Hollyfield, Joe G.

2011-01-01

Purpose. Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. Methods. Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. Results. Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. Conclusions. The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed. PMID:21931134

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis1[W

PubMed Central

Kwon, Soon Il; Cho, Hong Joo; Kim, Sung Ryul; Park, Ohkmae K.

2013-01-01

A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD. PMID:23404918

Cell death in the pathogenesis of systemic lupus erythematosus and lupus nephritis.

PubMed

Mistry, Pragnesh; Kaplan, Mariana J

2017-12-01

Nephritis is one of the most severe complications of systemic lupus erythematosus (SLE). One key characteristic of lupus nephritis (LN) is the deposition of immune complexes containing nucleic acids and/or proteins binding to nucleic acids and autoantibodies recognizing these molecules. A variety of cell death processes are implicated in the generation and externalization of modified nuclear autoantigens and in the development of LN. Among these processes, apoptosis, primary and secondary necrosis, NETosis, necroptosis, pyroptosis, and autophagy have been proposed to play roles in tissue damage and immune dysregulation. Cell death occurs in healthy individuals during conditions of homeostasis yet autoimmunity does not develop, at least in part, because of rapid clearance of dying cells. In SLE, accelerated cell death combined with a clearance deficiency may lead to the accumulation and externalization of nuclear autoantigens and to autoantibody production. In addition, specific types of cell death may modify autoantigens and alter their immunogenicity. These modified molecules may then become novel targets of the immune system and promote autoimmune responses in predisposed hosts. In this review, we examine various cell death pathways and discuss how enhanced cell death, impaired clearance, and post-translational modifications of proteins could contribute to the development of lupus nephritis. Published by Elsevier Inc.

Technological advances in real-time tracking of cell death

PubMed Central

Skommer, Joanna; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

2010-01-01

Cell population can be viewed as a quantum system, which like Schrödinger’s cat exists as a combination of survival- and death-allowing states. Tracking and understanding cell-to-cell variability in processes of high spatio-temporal complexity such as cell death is at the core of current systems biology approaches. As probabilistic modeling tools attempt to impute information inaccessible by current experimental approaches, advances in technologies for single-cell imaging and omics (proteomics, genomics, metabolomics) should go hand in hand with the computational efforts. Over the last few years we have made exciting technological advances that allow studies of cell death dynamically in real-time and with the unprecedented accuracy. These approaches are based on innovative fluorescent assays and recombinant proteins, bioelectrical properties of cells, and more recently also on state-of-the-art optical spectroscopy. Here, we review current status of the most innovative analytical technologies for dynamic tracking of cell death, and address the interdisciplinary promises and future challenges of these methods. PMID:20519963

Live-cell visualization of gasdermin D-driven pyroptotic cell death.

PubMed

Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

2017-09-01

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Cyclooxygenase-2 (COX-2) Initiates Autophagy and Potentiates MPTP-Induced Autophagic Cell Death of Human Neuroblastoma Cells, SH-SY5Y: an Inside in the Pathology of Parkinson's Disease.

PubMed

Niranjan, Rituraj; Mishra, Kaushal Prasad; Thakur, Ashwani Kumar

2018-03-01

Cyclooxygenase-2 or COX-2 has been known to be crucial for Parkinson's disease (PD) pathogenesis; however, its exact role is still not known. We first time report that inhibition of COX-2 promotes 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP)-induced neuronal cell death via induction of autophagic mechanisms. We found that treatment with MPTP induced cell death of neuroblastoma cells SH-SY5Y in a dose dependent manner. Treatment of MPTP has also upregulated the expressions of autophagic proteins such as LC3, beclin, ATG-5, and p62. Interestingly, nimesulide, a preferential COX-2 inhibitor, further potentiated the MPTP-induced cell death of human neuroblastoma cells. Treatment of nimesulide with MPTP further potentiated expressions of p62, ATG-5, beclin-1, LC3 autophagic proteins. Furthermore, nimesulide with MPTP increased apoptotic protein cleaved caspase-3 and also induced expression of p53 gene. Interestingly, it was observed that Akt inhibitor significantly increased MPTP-induced cell death of neuroblastoma cells. However, (-) deprenyl, a monoamine oxidase B (MAO B) inhibitor, attenuated MPTP-induced autophagic response and protected cell death. The prior treatment with prostaglandin E2 protected against nimesulide induced-death of neuronal cells. This study confirms that neuroinflammation is associated to the autophagy and may be one of the main pathological mechanisms in Parkinson's disease and other inflammation-associated disorders.

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

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

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

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

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

PubMed

Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2015-10-01

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

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.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

PubMed

Chung, Kyung Min; Park, Hyunhee; Jung, Seonghee; Ha, Shinwon; Yoo, Seung-Jun; Woo, Hanwoong; Lee, Hyang Ju; Kim, Seong Who; Kim, Eun-Kyoung; Moon, Cheil; Yu, Seong-Woon

2015-10-01

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death. © 2015 AlphaMed Press.

Simultaneous Treatment with Statins and Aspirin Reduces the Risk of Prostate Cancer Detection and Tumorigenic Properties in Prostate Cancer Cell Lines

PubMed Central

Olivan, M.; Rigau, M.; Colás, E.; Garcia, M.; Montes, M.; Sequeiros, T.; Regis, L.; Celma, A.; Planas, J.; Placer, J.; Reventós, J.; de Torres, I.; Doll, A.; Morote, J.

2015-01-01

Nowadays prostate cancer is the most common solid tumor in men from industrialized countries and the second leading cause of death. At the ages when PCa is usually diagnosed, mortality related to cardiovascular morbidity is high; therefore, men at risk for PCa frequently receive chronic lipid-lowering and antiplatelet treatment. The aim of this study was to analyze how chronic treatment with statins, aspirin, and their combination influenced the risk of PCa detection. The tumorigenic properties of these treatments were evaluated by proliferation, colony formation, invasion, and migration assays using different PCa cell lines, in order to assess how these treatments act at molecular level. The results showed that a combination of statins and aspirin enhances the effect of individual treatments and seems to reduce the risk of PCa detection (OR: 0.616 (95% CI: 0.467–0.812), P < 0.001). However, if treatments are maintained, aspirin (OR: 1.835 (95% CI: 1.068–3.155), P = 0.028) or the combination of both drugs (OR: 3.059 (95% CI: 1.894–4.939), P < 0.001) represents an increased risk of HGPCa. As observed at clinical level, these beneficial effects in vitro are enhanced when both treatments are administered simultaneously, suggesting that chronic, concomitant treatment with statins and aspirin has a protective effect on PCa incidence. PMID:25649906

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.

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

PubMed

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

2015-07-01

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

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

PubMed

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

2010-05-15

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

Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

PubMed Central

Khan, Naveed Ahmed; Iqbal, Junaid

2015-01-01

In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

Programmed cell death as a defence against infection

PubMed Central

Jorgensen, Ine; Rayamajhi, Manira; Miao, Edward A.

2017-01-01

Eukaryotic cells can die from physical trauma, resulting in necrosis. Alternately, they can die via programmed cell death upon stimulation of specific signalling pathways. Here we discuss the utility of four cell death pathways in innate immune defence against bacterial and viral infection: apoptosis, necroptosis, pyroptosis and NETosis. We describe the interactions that interweave different programmed cell death pathways, which create complex signalling networks that cross-guard each other in the evolutionary arms race with pathogens. Finally, we describe how the resulting cell corpses — apoptotic bodies, pore-induced intracellular traps (PITs) and neutrophil extracellular traps (NETs) — promote clearance of infection. PMID:28138137

Neurotoxic injury pathways in differentiated mouse motor neuron–neuroblastoma hybrid (NSC-34D) cells in vitro—Limited effect of riluzole on thapsigargin, but not staurosporine, hydrogen peroxide and homocysteine neurotoxicity

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

Hemendinger, Richelle A., E-mail: richelle.hemendinger@carolinashealthcare.org; Carolinas Neuromuscular/ALS-MDA Center, Department of Neurology, Carolinas Medical Center, Charlotte, NC 28203; Armstrong, Edward J.

2012-01-15

The neuroblastoma–spinal motor neuron fusion cell line, NSC-34, in its differentiated form, NSC-34D, permits examining the effects of riluzole, a proven treatment for amyotrophic lateral sclerosis (ALS) on cell death induction by staurosporine (STS), thapsigargin (Thaps), hydrogen peroxide (H{sub 2}O{sub 2}) and homocysteine (HCy). These neurotoxins, applied exogenously, have mechanisms of action related to the various proposed molecular pathogenetic pathways in ALS and are differentiated from endogenous cell death that is associated with cytoplasmic aggregate formation in motor neurons. Nuclear morphology, caspase-3/7 activation and high content imaging were used to assess toxicity of these neurotoxins with and without co-treatment withmore » riluzole, a benzothiazole compound with multiple pharmacological actions. STS was the most potent neurotoxin at killing NSC-34D cells with a toxic concentration at which 50% of maximal cell death is achieved (TC{sub 50} = 0.01 μM), followed by Thaps (TC{sub 50} = 0.9 μM) and H{sub 2}O{sub 2} (TC{sub 50} = 15 μM) with HCy requiring higher concentrations to kill at the same level (TC{sub 50} = 2200 μM). Riluzole provided neurorescue with a 20% absolute reduction (47.6% relative reduction) in apoptotic cell death against Thaps-induced NSC-34D cell (p ≤ 0.05), but had no effect on STS-, H{sub 2}O{sub 2}- and HCy-induced NSC-34D cell death. This effect of riluzole on Thaps induction of cell death was independent of caspase-3/7 activation. Riluzole mitigated a toxin that can cause intracellular calcium dysregulation associated with endoplasmic reticulum (ER) stress but not toxins associated with other cell death mechanisms. -- Highlights: ► Calcium-dependent neurotoxins are potent cell death inducers in NSC-34D cells. ► Riluzole provides neurorescue against Thaps-induced NSC-34D cell death. ► Riluzole had no effect on neurotoxicity by STS, H{sub 2}O{sub 2} and Hcy. ► Riluzole reduces NSC-34D cell death independent of caspase-3/7 activation.« less

Preparation for global introduction of inactivated poliovirus vaccine: safety evidence from the US Vaccine Adverse Event Reporting System, 2000-12.

PubMed

Iqbal, Shahed; Shi, Jing; Seib, Katherine; Lewis, Paige; Moro, Pedro L; Woo, Emily J; Shimabukuro, Tom; Orenstein, Walter A

2015-10-01

Safety data from countries with experience in the use of inactivated poliovirus vaccine (IPV) are important for the global polio eradication strategy to introduce IPV into the immunisation schedules of all countries. In the USA, IPV has been included in the routine immunisation schedule since 1997. We aimed to analyse adverse events after IPV administration reported to the US Vaccine Adverse Event Reporting System (VAERS). We analysed all VAERS data associated with IPV submitted between Jan 1, 2000, and Dec 31, 2012, either as individual or as combination vaccines, for all age and sex groups. We analysed the number and event type (non-serious, non-fatal serious, and death reports) of individual reports, and explored the most commonly coded event terms to describe the adverse event. We classified death reports according to previously published body-system categories (respiratory, cardiovascular, neurological, gastrointestinal, other infectious, and other non-infectious) and reviewed death reports to identify the cause of death. We classified sudden infant death syndrome as a separate cause of death considering previous concerns about sudden infant syndrome after vaccines. We used empirical Bayesian data mining methods to identify disproportionate reporting of adverse events for IPV compared with other vaccines. Additional VAERS data from 1991 to 2000 were analysed to compare the safety profiles of IPV and oral poliovirus vaccine (OPV). Of the 41,792 adverse event reports submitted, 39,568 (95%) were for children younger than 7 years. 38,381 of the reports for children in this age group (97%) were for simultaneous vaccination with IPV and other vaccines (most commonly pneumococcal and acellular pertussis vaccines), whereas standalone IPV vaccines accounted for 0·5% of all reports. 34,880 reports were for non-serious events (88%), 3905 reports were for non-fatal serious events (10%), and 783 reports were death reports (2%). Injection-site erythema was the most commonly coded term for non-serious events (29%), and pyrexia for non-fatal serious events (38%). Most deaths (96%) were in children aged 12 months or younger; most (52%) had sudden infant death syndrome as the reported cause of death. The safely profiles of combined IPV and whole-cell pertussis vaccines, OPV and whole-cell pertussis vaccines, and OPV and acellular pertussis vaccines were similar. We noted no indication of disproportionate reporting of adverse events after immunisation with IPV-containing vaccines compared with other vaccines between 1990 and 2013. Fairly few adverse events were reported for the more than 250 million IPV doses distributed between 2000 and 2012. Sudden infant death syndrome reports after IPV were consistent with reporting patterns for other vaccines. No new or unexpected vaccine safety problems were identified for fatal, non-fatal serious, and non-serious reports in this assessment of adverse events after IPV. None. Copyright © 2015 Elsevier Ltd. All rights reserved.

Host-Cell Survival and Death During Chlamydia Infection

PubMed Central

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

2008-01-01

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

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

PubMed

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

2015-03-15

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

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

PubMed Central

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

2017-01-01

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

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

PubMed Central

Zille, Marietta; Karuppagounder, Saravanan S.; Chen, Yingxin; Gough, Peter J.; Phil, D.; Bertin, John; Finger, Joshua; Milner, Teresa A.; Jonas, Elizabeth A.; Ratan, Rajiv R.

2017-01-01

Background and Purpose Intracerebral hemorrhage (ICH) leads to disability or death with few established treatments. Adverse outcomes following ICH result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. Methods We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were employed. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. Results Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased following ICH in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by greater than 80% and had similar therapeutic windows in vitro. Conclusion Experimental ICH shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either one of these pathways can bring cells below that threshold to survival. PMID:28250197

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

PubMed

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

2016-06-02

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

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

Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

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

Bumpus, Namandje N., E-mail: nbumpus1@jhmi.edu

Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibitionmore » of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and 8-hydroxyefavirenz-mediated cell death. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz may be novel modulators of Bim.« less

Silicon does not mitigate cell death in cultured tobacco BY-2 cells subjected to salinity without ethylene emission.

PubMed

Liang, Xiaolei; Wang, Huahua; Hu, Yanfeng; Mao, Lina; Sun, Lili; Dong, Tian; Nan, Wenbin; Bi, Yurong

2015-02-01

Silicon induces cell death when ethylene is suppressed in cultured tobacco BY-2 cells. There is a crosstalk between Si and ethylene signaling. Silicon (Si) is beneficial for plant growth. It alleviates both biotic and abiotic stresses in plants. How Si works in plants is still mysterious. This study investigates the mechanism of Si-induced cell death in tobacco BY-2 cell cultures when ethylene is suppressed. Results showed that K2SiO3 alleviated the damage of NaCl stress. Si treatment rapidly increased ethylene emission and the expression of ethylene biosynthesis genes. Treatments with Si + Ag and Si + aminooxyacetic acid (AOA, ethylene biosynthesis inhibitor) reduced the cell growth and increased cell damage. The treatment with Si + Ag induced hydrogen peroxide (H2O2) generation and ultimately cell death. Some nucleus of BY-2 cells treated with Si + Ag appeared TUNEL positive. The inhibition of H2O2 and nitric oxide (NO) production reduced the cell death rate induced by Si + Ag treatment. Si eliminated the up-regulation of alternative pathway by Ag. These data suggest that ethylene plays an important role in Si function in plants. Without ethylene, Si not only failed to enhance plant resistance, but also elevated H2O2 generation and further induced cell death in tobacco BY-2 cells.

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

PubMed

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-07-15

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

24(S)-Hydroxycholesterol Induces Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis*

PubMed Central

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-01-01

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis. PMID:21613228

Transplanting hearts after death measured by cardiac criteria: the challenge to the dead donor rule.

PubMed

Veatch, Robert M

2010-06-01

The current definition of death used for donation after cardiac death relies on a determination of the irreversible cessation of the cardiac function. Although this criterion can be compatible with transplantation of most organs, it is not compatible with heart transplantation since heart transplants by definition involve the resuscitation of the supposedly "irreversibly" stopped heart. Subsequently, the definition of "irreversible" has been altered so as to permit heart transplantation in some circumstances, but this is unsatisfactory. There are three available strategies for solving this "irreversibility problem": altering the definition of death so as to rely on circulatory irreversibility, rather than cardiac; defining death strictly on the basis of brain death (either whole-brain or more pragmatically some higher brain criteria); or redefining death in traditional terms and simultaneously legalizing some limited instances of medical killing to procure viable hearts. The first two strategies are the most ethically justifiable and practical.

Does autophagy have a license to kill mammalian cells?

PubMed

Scarlatti, F; Granata, R; Meijer, A J; Codogno, P

2009-01-01

Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.

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

PubMed Central

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

2002-01-01

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

Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress

PubMed Central

Lee, Wei-Jiunn; Chien, Ming-Hsien; Chow, Jyh-Ming; Chang, Junn-Liang; Wen, Yu-Ching; Lin, Yung-Wei; Cheng, Chao-Wen; Lai, Gi-Ming; Hsiao, Michael; Lee, Liang-Ming

2015-01-01

The antiapoptotic and antiautophagic abilities of cancer cells constitute a major challenge for anticancer drug treatment. Strategies for triggering nonapoptotic or nonautophagic cell death may improve therapeutic efficacy against cancer. Curcumin has been reported to exhibit cancer chemopreventive properties. Herein, we report that curcumin induced apoptosis in LNCaP, DU145, and PC-3 cells but triggered extensive cytoplasmic vacuolation in PC-3M cells. Electron microscopic images showed that the vacuoles lacked intracellular organelles and were derived from the endoplasmic reticulum (ER). Moreover, curcumin-induced vacuolation was not reversed by an apoptosis- or autophagy-related inhibitor, suggesting that vacuolation-mediated cell death differs from classical apoptotic and autophagic cell death. Mechanistic investigations revealed that curcumin treatment upregulated the ER stress markers CHOP and Bip/GRP78 and the autophagic marker LC3-II. In addition, curcumin induced ER stress by triggering ROS generation, which was supported by the finding that treating cells with the antioxidant NAC alleviated curcumin-mediated ER stress and vacuolation-mediated death. An in vivo PC-3M orthotopic prostate cancer model revealed that curcumin reduced tumor growth by inducing ROS production followed by vacuolation-mediated cell death. Overall, our results indicated that curcumin acts as an inducer of ROS production, which leads to nonapoptotic and nonautophagic cell death via increased ER stress. PMID:26013662

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

PubMed

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-03-31

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

PubMed Central

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-01-01

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development. PMID:19279211

Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells.

PubMed

Palacios-Macapagal, Daphne; Connor, Jane; Mustelin, Tomas; Ramalingam, Thirumalai R; Wynn, Thomas A; Davidson, Todd S

2017-08-01

Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1-mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni -infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death. Copyright © 2017 by The American Association of Immunologists, Inc.

Glutathione in Cancer Cell Death

PubMed Central

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

2011-01-01

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

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

PubMed

Ishikawa, F; Ushida, K; Mori, K; Shibanuma, M

2015-01-22

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

In vivo delivery of miRNAs for cancer therapy: Challenges and strategies⋆

PubMed Central

Chen, Yunching; Gao, Dong-Yu; Huang, Leaf

2016-01-01

MicroRNAs (miRNAs), small non-coding RNAs, can regulate post-transcriptional gene expressions and silence a broad set of target genes. miRNAs, aberrantly expressed in cancer cells, play an important role in modulating gene expressions, thereby regulating downstream signaling pathways and affecting cancer formation and progression. Oncogenes or tumor suppressor genes regulated by miRNAs mediate cell cycle progression, metabolism, cell death, angiogenesis, metastasis and immunosuppression in cancer. Recently, miRNAs have emerged as therapeutic targets or tools and biomarkers for diagnosis and therapy monitoring in cancer. Since miRNAs can regulate multiple cancer-related genes simultaneously, using miRNAs as a therapeutic approach plays an important role in cancer therapy. However, one of the major challenges of miRNA-based cancer therapy is to achieve specific, efficient and safe systemic delivery of therapeutic miRNAs In vivo. This review discusses the key challenges to the development of the carriers for miRNA-based therapy and explores current strategies to systemically deliver miRNAs to cancer without induction of toxicity. PMID:24859533

Facilitation of trace metal uptake in cells by inulin coating of metallic nanoparticles

NASA Astrophysics Data System (ADS)

Santillán-Urquiza, Esmeralda; Arteaga-Cardona, Fernando; Torres-Duarte, Cristina; Cole, Bryan; Wu, Bing; Méndez-Rojas, Miguel A.; Cherr, Gary N.

2017-09-01

Trace elements such as zinc and iron are essential for the proper function of biochemical processes, and their uptake and bioavailability are dependent on their chemical form. Supplementation of trace metals through nanostructured materials is a new field, but its application raises concerns regarding their toxicity. Here, we compared the intracellular zinc uptake of different sources of zinc: zinc sulfate, and ZnO and core-shell α-Fe2O3@ZnO nanoparticles, coated or uncoated with inulin, an edible and biocompatible polysaccharide. Using mussel haemocytes, a well-known model system to assess nanomaterial toxicity, we simultaneously assessed zinc accumulation and multiple cellular response endpoints. We found that intracellular zinc uptake was strongly enhanced by inulin coating, in comparison to the uncoated nanoparticles, while no significant effects on cell death, cell viability, mitochondrial membrane integrity, production of reactive oxygen species or lysosome abundance were observed at concentrations up to 20 ppm. Since no significant increments in toxicity were observed, the coated nanomaterials may be useful to increase in vivo zinc uptake for nutritional applications.

Cell death induced by hydroxyapatite on L929 fibroblast cells.

PubMed

Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

2004-05-01

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

Physician Education: Apoptosis.

PubMed

Kataoka; Tsuruo

1996-01-01

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

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

PubMed

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

2016-05-03

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

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

PubMed Central

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

2016-01-01

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

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

Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death

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

Li, Liang, E-mail: 18710470987@163.com; Ye, Chaofei, E-mail: yechaofei001@163.com; Zhao, Rui, E-mail: 571828628@qq.com

MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of amore » series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death. - Highlights: • Expression of rapeseed MAPKKK4 induced ROS accumulation and cell death in leaves. • Cell death induced by MAPKKK4 is associated with membrane lipid peroxidation and DNA fragmentation. • MAPKKK4 interacts with MKK5 and MPK3. • MAPKKK4-induced ROS accumulation and cell death require downstream WIPK and SIPK. • MAPKKK4 is a novel MAPKKK modulating ROS accumulation and cell death.« less

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

PubMed

Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

2015-07-28

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury.

PubMed

Shi, Guodong; Liu, Yang; Liu, Tielong; Yan, Wangjun; Liu, Xiaowei; Wang, Yuan; Shi, Jiangang; Jia, Lianshun

2012-01-01

It is increasingly clear that microRNAs (miRNAs) play an important role in controlling cell survival. However, the functional significance of miRNAs in ischemic brain injury remains poorly understood. In the present study, we assayed the expression levels of miR-29b after ischemic brain injury, and defined the target genes and biological functions of miR-29b. We found that the miR-29b levels were significantly increased in rat brain after transient middle cerebral artery occlusion and neurons after oxygen-glucose deprivation. Moreover, ectopic expression of miR-29b promoted neuronal cell death, whereas its repression decreased cell death. Furthermore, we verified that miR-29b directly targeted and inhibited Bcl2L2 gene expression, and then increased neuronal cell death. Importantly, Bcl2L2 overexpression rescued neuronal cell death induced by miR-29b. These results suggest an important role of miR-29b in regulating neuronal cell death, thus offering a new target for the development of therapeutic agents against ischemic brain injury.

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

PubMed

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

2013-01-01

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

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

PubMed

Sun, Yamei; Bao, Qunchao; Xuan, Baoqin; Xu, Wenjia; Pan, Deng; Li, Qi; Qian, Zhikang

2018-07-01

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death. IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases. Copyright © 2018 American Society for Microbiology.

Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level.

PubMed

Yoo, Jae-Myung; Lee, Youn-Sun; Choi, Heon-Kyo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Oh, Seikwan; Yoo, Hwan-Soo

2005-03-01

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

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

PubMed Central

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

2017-01-01

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

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