Apoptosis-specific protein (ASP 45 kDa) is distinct from human Apg5, the homologue of the yeast autophagic gene apg5

We have examined whether the apoptosis-specific protein p45ASP and human Apg5 are identical proteins. Like p45ASP, myc-hApg5 cross-reacted with a c-Jun antibody and approximately 50% of myc-hApg5 was bound to a Triton X-100-insoluble fraction in HeLa cells. However, soluble myc-hApg5 was degraded during apoptosis induced by staurosporine or TNFalpha/cycloheximide whilst expression of soluble p45ASP was stabilised. Furthermore, myc-hApg5 degradation was blocked by the caspase inhibitor Boc-Asp(OMe)FMK whilst p45ASP expression was eliminated. Moreover, myc-hApg5 ( approximately 32 kDa) never assumed the size of p45ASP (45 kDa). It is therefore likely that p45ASP and human Apg5 are distinct proteins although they do share some common characteristics.     

Autophagonizer, a novel synthetic small molecule, induces autophagic cell death

Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.     

Necrotic,apoptotic and autophagic cell fates triggered by nanoparticles

Nanomaterials have gained a rapid increase in use in a variety of applications that pertain to many aspects of human life. The majority of these innovations are centered on medical applications and a range of industrial and environmental uses ranging from electronics to environmental remediation. Despite the advantages of NPs, the knowledge of their toxicological behavior and their interactions with the cellular machinery that determines cell fate is extremely limited. This review is an attempt to summarize and increase our understanding of the mechanistic basis of nanomaterial interactions with the cellular machinery that governs cell fate and activity. We review the mechanisms of NP-induced necrosis, apoptosis and autophagy and potential implications of these pathways in nanomaterial-induced outcomes.

Abbreviations: Ag, silver; CdTe, cadmium telluride; CNTs, carbon nanotubes; EC, endothelial cell; GFP, green fluorescent protein; GO, graphene oxide; GSH, glutathione; HUVECs, human umbilical vein endothelial cells; NP, nanoparticle; PEI, polyethylenimine; PVP, polyvinylpyrrolidone; QD, quantum dot; ROS, reactive oxygen species; SiO₂, silicon dioxide; SPIONs, superparamagnetic iron oxide nanoparticles; SWCNT, single-walled carbon nanotubes; TiO₂, titanium dioxide; USPION, ultra-small super paramagnetic iron oxide; ZnO, zinc oxide.     

Nano neodymium oxide induces massive vacuolization and autophagic cell death in non-small cell lung cancer NCI-H460 cells

Neodymium, a rare earth element, was known to exhibit cytotoxic effects and induce apoptosis in certain cancer cells. Here we show that nano-sized neodymium oxide (Nano Nd2O3) induced massive vacuolization and cell death in non-small cell lung cancer NCI-H460 cells at micromolar equivalent concentration range. Cell death elicited by Nano Nd2O3 was not due to apoptosis and caspases were not involved. Electron microscopy and acridine orange staining revealed extensive autophagy in the cytoplasm of the cells treated by Nano Nd2O3. Autophagy induced by Nano Nd2O3 was accompanied by S-phase cell cycle arrest, mild disruption of mitochondrial membrane potential, and inhibition of proteasome activity. Bafilomycin A1, but not 3-MA, induced apoptosis while inhibiting autophagy. Our results revealed a novel biological function for Nano Nd2O3 and may have implications for the therapy of non-small cell lung cancer.     

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of κB kinase (IKK)-β resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential ΔΨ_m and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.     

At the core of survival: autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell injury

Ischemic cell injury leads to cell death. Three main morphologies have been described: apoptosis, cell death with autophagy and necrosis. Their inherent dynamic nature, a point of no return (PONR) and molecular overlap have been stressed. The relationship between a defined cell death type and the severity of injury remains unclear. The functional role of autophagy and its effects on cell death onset is largely unknown. In this study we report a differential induction of cell death, which is dependent on the severity and duration of an ischemic insult. We show that mild ischemia leads to the induction of autophagy and apoptosis, while moderate or severe ischemia induces both apoptotic and necrotic cell death without increased autophagy. The autophagic response during mild injury was associated with an ATP surge. Real-time imaging and Fluorescence Resonance Energy Transfer (FRET) revealed that increased autophagy delays the PONR of both apoptosis and necrosis significantly. Blocking autophagy shifted PONR to an earlier point in time. Our results suggest that autophagic activity directly alters intracellular metabolic parameters, responsible for maintaining mitochondrial membrane potential and cellular membrane integrity. A similar treatment also improved functional recovery in the perfused rat heart. Taken together, we demonstrate a novel finding: autophagy is implicated only in mild injury and positions the PONR in cell death.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

Lysosomes and lysosomal cathepsins in cell death

Lysosomes are the key degradative compartments of the cell. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell's metabolism by participating in the degradation of heterophagic and autophagic material. Following the targeted lysosomal membrane's destabilization, the cathepsins can be released into the cytosol and initiate the lysosomal pathway of apoptosis through the cleavage of Bid and the degradation of the anti-apoptotic Bcl-2 homologues. Cathepsins can also amplify the apoptotic signaling, when the lysosomal membranes are destabilized at a later stage of apoptosis, initiated by other stimuli. However, the functional integrity of the lysosomal compartment during apoptosis enables efficient autophagy, which can counteract apoptosis by providing the energy source and by disposing the damaged mitochondria, which generate the ROS. Impairing autophagy by disabling the lysosome function is being investigated as an adjuvant therapeutic approach to sensitize cells to apoptosis-inducing agents. Destabilization of the lysosomal membranes by the lysosomotropic detergents seems to be a promising strategy in this context as it would not only disable autophagy, but also promote apoptosis through the initiation of the lysosomal pathway. In contrast, the impaired autophagy and lysosomal degradation linked with the increased oxidative stress underlie degenerative changes in the aging neurons. This further suggests that lysosomes and lysosomal cathepsins have a dual role in cell death. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Glucosamine induces autophagic cell death through the stimulation of ER stress in human glioma cancer cells

Autophagy can promote cell survival or death, but the molecular basis of its dual role in cancer is not well understood. Here, we report that glucosamine induces autophagic cell death through the stimulation of endoplasmic reticulum (ER) stress in U87MG human glioma cancer cells. Treatment with glucosamine reduced cell viability and increased the expression of LC3 II and GFP-LC3 fluorescence puncta, which are indicative of autophagic cell death. The glucosamine-mediated suppression of cell viability was reversed by treatment with an autophagy inhibitor, 3-MA, and interfering RNA against Atg5. Glucosamine-induced ER stress was manifested by the induction of BiP, IRE1α, and phospho-eIF2α expression. Chemical chaperon 4-PBA reduced ER stress and thereby inhibited glucosamine-induced autophagic cell death. Taken together, our data suggest that glucosamine induces autophagic cell death by inducing ER stress in U87MG glioma cancer cells and provide new insight into the potential anticancer properties of glucosamine.     

Downregulation of transferrin receptor surface expression by intracellular antibody

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4+/-2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.     

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.     

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.     

Hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death

NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.