Role of oxidative stress and caspase 3 in CD47-mediated neuronal cell death

CD47 or integrin-associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin-1 or the specific CD47-activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time-dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways.     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.     

Mechanism of basic fibroblast growth factor-induced cell death

Basic fibroblast growth factor (bFGF) is a potent mitogen for a number of different cell types. Its over-expression has been implicated in transformation and malignant progression. The use of bFGF to treat malignancy is therefore counterintuitive. However, recent studies have shown bFGF-induces cell death in some tumour types. This mini review will summarise the most recent findings on bFGF-induced cell death and discuss its potential mechanism of action.     

Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl‐1 during an extended mitosis requires the anaphase‐promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCF^Fbw7. Using live‐cell imaging, we show that the loss of Mcl‐1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine‐arginine (IR) C‐terminal tail regulates the manner in which Mcl‐1 engages with the APC/C, converting Mcl‐1 from a Cdc20‐dependent and checkpoint‐controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl‐1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl‐1 degradation, providing an improved strategy to kill cancer cells.     

Caspase 3, periodically expressed and activated at G2/M transition, is required for nocodazole-induced mitotic checkpoint

Caspases have been known for several years for their involvement in executing apoptosis, where unwanted or damaged cells are eliminated. Surprisingly, after analysis of the relevant data set from the Stanford microarray database, we noticed that the gene expression pattern for caspase 3, but not for caspase 1, 6, 7, 8, 9, or 10, undergoes periodic change in the HeLa cell cycle. In this study, we have demonstrated that caspase 3, but not other caspases, is upregulated and activated just prior to mitosis. Pretreatment of human hepatoma cells with a caspase 3 inhibitor z-DEVD-FMK, prior to the treatment with an antimicrotubule drug nocodazole, abrogates the mitotic arrest, suggesting that caspase 3 (or a caspase 3-like enzyme) might be involved in mitotic-spindle checkpoint. The studies not only characterize caspase 3 as a cell cycle-regulated protein, but also link the protein to nocodazole-dependent mitotic checkpoint, greatly expanding the understanding of caspase 3. These authors contributed equally.     

Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery

Autophagy is implicated in regulating cell death in activated T cells, but the underlying mechanism is unclear. Here, we show that inhibition of autophagy via Beclin 1 gene deletion in T cells leads to rampant apoptosis in these cells upon TCR stimulation. Beclin 1-deficient mice fail to mount autoreactive T-cell responses and are resistant to experimental autoimmune encephalomyelitis. Compared with Th17 cells, Th1 cells are much more susceptible to cell death upon Beclin 1 deletion. Cell death proteins are highly increased in Beclin 1-deficient T cells and inhibition of caspases and genetic deletion of Bim reverse apoptosis. In addition, p62/sequestosome 1 binds to caspase-8 but does not control levels of procaspase-8 or other cell death-related proteins. These results establish a direct role of autophagy in inhibiting the programmed cell death through degradation of apoptosis proteins in activated T cells.     

Caspase-3 mediated neuronal death after traumatic brain injury in rats

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.     

Caspase-3 mediated programmed cell death by a gold-stabilised peptide carbene

The synthesis of novel N-heterocyclic carbene complexes derived from a tripeptide ligand (L), containing non-natural amino acid, thiazolylalanine is described here. The peptide ligand was reacted with suitable precursors to generate gold and mercury carbene complexes. The plausible structures of both complexes were predicted by spectroscopic data and DFT calculations. The binding energy data was also analyzed to predict their stability. The gold carbene complex (1A), showed activity against MCF7 breast cancer cell line due to mitochondrial triggered caspase-3 mediated programmed cell death. Its internalization inside cells could be observed due to autofluorescence. This study affords a methodology for successful generation of peptide carbene complexes for their therapeutic potential.     

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.     

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.     

A cellular suicide strategy of plants: vacuole-mediated cell death

Programmed cell death (PCD) occurs in animals and plants under various stresses and during development. Recently, vacuolar processing enzyme (VPE) was identified as an executioner of plant PCD. VPE is a cysteine protease that cleaves a peptide bond at the C-terminal side of asparagine and aspartic acid. VPE exhibited enzymatic properties similar to that of a caspase, which is a cysteine protease that mediates the PCD pathway in animals, although there is limited sequence identity between the two enzymes. VPE and caspase-1 share several structural properties: the catalytic dyads and three amino acids forming the substrate pockets (Asp pocket) are conserved between VPE and caspase-1. In contrast to such similarities, subcellular localizations of these proteases are completely different from each other. VPE is localized in the vacuoles, while caspases are localized in the cytosol. VPE functions as a key molecule of plant PCD through disrupting the vacuole in pathogenesis and development. Cell death triggered by vacuolar collapse is unique to plants and has not been seen in animals. Plants might have evolved a VPE-mediated vacuolar system as a cellular suicide strategy.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor’s Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor’s Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

泛素/26S蛋白酶体系统介导的细胞程序化死亡

在一定的生理或者病理条件下,细胞为了自身发育或者抵御不良刺激,会采取细胞程序化死亡（programmed cell death,PCD）的方式结束生命。泛素/26S蛋白酶体系统（ubiquitin-26S proteasome system,UPS）作为生物体中重要的翻译后蛋白质调节系统,对PCD起着关键的调节作用。该文介绍UPS通过两条细胞凋亡信号转导通路以及天冬氨酸特异性半胱氨酸蛋白酶来调控PCD的研究进展。     

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.     

Extracellular ATP differentially modulates Toll-like receptor 4-mediated cell survival and death of microglia

The survival and death rates of inflammatory cells directly control their number and are substantially associated with the degree of inflammation. Microglia, key players in neuroinflammation, often cause excessive reactions implicated in neurological diseases. However, the mechanisms that determine microglial fate under pathological conditions remain to be elucidated. Here, we report that activation by lipopolysaccharide (LPS, a Toll-like receptor 4 ligand), an inflammation inducer, primarily promotes survival of microglia, but as its concentration is increased it induces cell death, resulting in decreased cell number. Moreover, extracellular ATP, which is released upon tissue damage, further enhanced the survival induced by a low LPS concentration and the death induced by a high LPS concentration. The survival-promoting effect of ATP was mimicked by non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), and also by the P2X(7) receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and was suppressed by the P2X(7) antagonists, Brilliant Blue G and A 438079. On the contrary, the death of LPS-activated microglia was not affected by adenosine 5'-O-(3-thiotriphosphate), but enhanced by adenosine, ATP breakdown product. Thus, extracellular ATP modulates microglial survival and death in different ways involving P2X(7) receptor activation and ATP degradation to adenosine, respectively. Such Toll-like receptor 4/purinergic signaling may provide a fine regulatory system of neuroinflammation through modulating the microglial cell number.     

Death effector domain-only polypeptides of caspase-8 and -10 specifically inhibit death receptor-induced cell death

Caspase-8 and -10 are thought to be involved in a signaling pathway leading to death receptor-mediated apoptosis. The prodomains of these caspases are known to form fibrous structures in the perinuclear region when overexpressed, though the meaning of the structures remains unclear. In a previous study we showed that the overexpressed caspase-8 or -10 prodomain (PDCasp8 or PDCasp10) did not induce cell death, and we hypothesized that these prodomains interfere with the receptor-mediated cell death signaling pathway. Indeed, in 293, HeLa and Jurkat cells, cell death mediated by agonistic anti-Fas antibody, TRAIL or overexpression of full-length caspase-8 was significantly inhibited by overexpression of PDCasp8 or PDCasp10 which colocalized with the Golgi complex and with overexpressed FADD. However, when about 20 amino acid residues were deleted from either terminus of the caspase-10 prodomain (amino acid residue 1 to 219), the ability to inhibit Fas-mediated cell death was lost. Interestingly, these deletion mutants also lost the ability to make fibrous structures and to bind FADD, suggesting that FADD binding is important for their function, and that PDCasp8 and PDCasp10 act as dominant-negative inhibitors.