Features Of Necrotic Cell Death Research Articles

Abstract 5995: How FDA-approved drug, Aprepitant, induces immunogenic death of cancer cells

Abstract Aprepitant is a long-standing FDA approved anti-nausea drug, recently re-purposed as an early-stage anticancer agent; how aprepitant kills cancer cells was not well studied. We find that aprepitant kills human and mouse cancer cells by hyperactivating the stress response pathway, the anticipatory unfolded protein response (a-UPR), thereby inducing immune cell activating necrotic cell death. Aprepitant is a small molecule neurokinin-1 receptor (NK-1R) antagonist widely used to prevent nausea and vomiting in cancer patients undergoing chemotherapy. Recently aprepitant has shown promise in mouse xenograft studies of colon, lung and pancreatic cancer and melanoma. Since aprepitant was thought to trigger release of calcium stored in the lumen of the endoplasmic reticulum, the step that we showed triggers hyperactivation of the a-UPR by ErSO family anticancer agents, we explored the role of the a-UPR in aprepitant action. Aprepitatnt robustly activates the PERK arm of the UPR increasing p-PERK and peIF2α, inhibiting protein synthesis. Notably, aprepitant treatment results in ATP depletion and activation of AMPK. Aprepitant induced rapid cell death was not blocked by apoptosis inhibitors or necroptosis inhibitors. In both standard 2-dimensional cell culture and 3-dimensional organoid culture aprepitant-treated breast cancer cells displayed typical features of necrotic cell death, including cell swelling, membrane rupture, and cytoplasmic vacuolization. Cell death by necrosis and membrane rupture leads to release of immune cell activating cell contents termed damage-associated molecular patterns (DAMPs). Aprepitant treatment induces release of the classic DAMPs, HMGB1 and ATP and medium from aprepitant-treated breast cancer cells enhances migration of differentiated human THP-1 macrophage. Thus aprepitant and other necrosis inducing therapies elicit immunogenic cell death (ICD) and may have the potential to enhance adjuvant immunotherapy. Genome-wide CRISPR screens with negative selection against aprepitant and other studies are being used to explore the pathways of aprepitant induced cell death and evaluate its therapeutic efficacy. Since FDA-approved aprepitant has been widely used for many years, identifying its pathway of action will both accelerate its clinical transition as an anticancer agent and help identify patients whose genetic profiles make them most likely to benefit from aprepitant therapy. Citation Format: Xinyi Dai, Junyao Zhu, Sofia Perry, Qianjin Jiang, David J. Shapiro. How FDA-approved drug, Aprepitant, induces immunogenic death of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5995.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.

Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Spatiotemporal Progression of Microcalcification in the Hippocampal CA1 Region following Transient Forebrain Ischemia in Rats: An Ultrastructural Study.

Calcification in areas of neuronal degeneration is a common finding in several neuropathological disorders including ischemic insults. Here, we performed a detailed examination of the onset and spatiotemporal profile of calcification in the CA1 region of the hippocampus, where neuronal death has been observed after transient forebrain ischemia. Histopathological examinations showed very little alizarin red staining in the CA1 pyramidal cell layer until day 28 after reperfusion, while prominent alizarin red staining was detected in CA1 dendritic subfields, particularly in the stratum radiatum, by 14 days after reperfusion. Electron microscopy using the osmium/potassium dichromate method and electron probe microanalysis revealed selective calcium deposits within the mitochondria of degenerating dendrites at as early as 7 days after reperfusion, with subsequent complete mineralization occurring throughout the dendrites, which then coalesced to form larger mineral conglomerates with the adjacent calcifying neurites by 14 days after reperfusion. Large calcifying deposits were frequently observed at 28 days after reperfusion, when they were closely associated with or completely engulfed by astrocytes. In contrast, no prominent calcification was observed in the somata of CA1 pyramidal neurons showing the characteristic features of necrotic cell death after ischemia, although what appeared to be calcified mitochondria were noted in some degenerated neurons that became dark and condensed. Thus, our data indicate that intrahippocampal calcification after ischemic insults initially occurs within the mitochondria of degenerating dendrites, which leads to the extensive calcification that is associated with ischemic injuries. These findings suggest that in degenerating neurons, the calcified mitochondria in the dendrites, rather than in the somata, may serve as the nidus for further calcium precipitation in the ischemic hippocampus.

Chrysophanic Acid Induces Necrosis but not Necroptosis in Human Renal Cell Carcinoma Caki-2 Cells.

Background:Chrysophanic acid, also known as chrysophanol, has a number of biological activities. It enhances memory and learning abilities, raises superoxide dismutase activity, and has anti-cancer effects in several model systems. According to previous reports, chrysophanic acid-induced cell death shares features of necrotic cell death. However, the molecular and cellular processes underlying chrysophanic acid-induced cell death remain poorly understood.Methods:Chrysophanic acid-induced cell death was monitored by cell viability assay and Annexin V-propidium iodide (PI) staining of renal cell carcinoma Caki-2 cells. The induction of intracellular reactive oxygen species (ROS) by chrysophanic acid and the suppression of ROS by anti-oxidants were evaluated by 2′,7′-dichlorofluorescin diacetate staining. The expression and phosphorylation of proteins that are involved in apoptosis and necroptosis were detected by immunoblotting.Results:The extent of chrysophanic acid-induced cell death was concentration and time dependent, and dead cells mainly appeared in the PI-positive population, which is a major feature of necrosis, upon fluorescence-activated cell sorting analysis. Chrysophanic acid-induced cell death was associated with the generation of intracellular ROS, and this effect was reversed by pretreatment with N-acetyl cysteine. Chrysophanic acid-induced cell death was not associated with changes in apoptotic or necroptotic marker proteins.Conclusions:The cell death induced by chrysophanic acid resembled neither apoptotic nor necroptotic cell death in human renal cell carcinoma Caki-2 cells.

Alternative Cell Death Mechanisms Determine Epidermal Resistance in Incompatible Barley-Ustilago Interactions

Programmed cell death is a key feature of epidermal plant immunity, which is particularly effective against biotrophic microbes that depend on living host tissue. The covered smut fungus Ustilago hordei establishes a compatible biotrophic interaction with its host plant barley. The maize smut U. maydis triggers a nonhost response in barley, which results in epidermal cell death. Similarly, Ustilago mutants being deleted for pep1, a gene encoding a secreted effector, are blocked upon host penetration. We studied the epidermal responses of barley to incompatible Ustilago strains. Molecular and cellular analyses were used to test the impact of Bax inhibitor-1 (BI-1), a suppressor of programmed cell death, on the barley nonhost resistance to U. maydis as well as Ustilago Δpep1 mutants. Overexpression of BI-1 resulted in partial break of barley nonhost resistance to U. maydis. By contrast, the epidermal cell death response triggered by pep1 deletion mutants was not impaired by BI-1. Hypersensitive-response-like cell death caused by U. maydis wild-type infection showed features of necrotic cell death, while Δpep1 mutant-induced host responses involved hallmarks of autophagy. Therefore, we propose that the mechanisms of epidermal cell death in response to different types of incompatible pathogens depend on spatial and temporal appearance of cell-death-triggering stimuli.

Abstract 2265: Embelin induces autophagy in ovarian cancer cells

Abstract Embelin is an active ingredient of traditional herbal medicine that exhibits cytotoxic effect and inhibits cell proliferation in various cancer cell types. It is a small molecular inhibitor of XIAP undergoing preclinical testing for its anticancer effects in many different cancer types. In this study we have investigated its role as a chemotherapeutic agent in a panel of ovarian cancer cell lines. Utilising the MTT assay and annexin staining we demonstrate that embelin exhibits a potent, dose dependent cytotoxic effect in human ovarian cancer cells. Surprisingly in contrast to the previously reported cytotoxicity of embelin in many other cancer cell types, treatment of ovarian cancer cells with embelin promoted autophagy and not apoptosis as evidenced by the death without caspase 3 activation and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Treatment of ovarian cancer cells with embelin demonsrnstrated autophagic hallmarks, including an increased number of autophagosomes were observed through acridine orange staining. Embelin also induced cell autophagy, as demonstrated by the punctuate distribution of GFP-LC3, as well as the LC3 cleavage and beclin1 activation. Red green fluorescence in acridine orange stained cells by flowcyometry further confirmed the autophagy action by embelin treatment. Our findings demonstrate a new death pathway underlying the cytotoxic effect of embelin, but also suggest that targeting autophagy could augment the anticancer activity, providing the framework for further development of embelin as a new chemotherapeutic agent for ovarian cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2265. doi:1538-7445.AM2012-2265

Abstract P042: Loss of Mcl-1 in the Heart Leads to Rapid Mitochondrial Dysfunction and Development of Heart Failure

Mcl-1 is an anti-apoptotic Bcl-2 family protein that is expressed at high levels in the heart. However, the functional importance of Mcl-1 in the heart has not been investigated. To study the function of Mcl-1 in cardiac myocytes, we generated inducible, myocyte-specific Mcl-1 knock-out mice using a tamoxifen-driven loxP/α-MHC-Cre-recombinase system. We found that deletion of Mcl-1 by tamoxifen injections resulted in rapid development of cardiac hypertrophy, pulmonary edema and cardiac dysfunction in adult mice. Although Mcl-1 is known to inhibit apoptosis in cells, Mcl-1 deficient hearts were TUNEL negative and showed no activation of caspase-3. The anti-apoptotic proteins Bcl-2 and Bcl-X L have also been reported to inhibit autophagy, but we found no increase in autophagy upon loss of Mcl-1. Western blot analysis of other Bcl-2 family members revealed that Bcl-2 was significantly increased in Mcl-1 deficient hearts, suggesting a compensatory mechanism. Interestingly, ultrastructural analysis revealed the presence of swollen mitochondria, ruptured cells, and intracellular contents in the extracellular space. These signs are characteristics of necrotic cell death. Mcl-1 deficient hearts also exhibited reduced mitochondrial respiration and significantly lower cellular ATP levels compared to wild type, suggesting that the loss of Mcl-1 impairs mitochondrial function. Moreover, mitochondria isolated from Mcl-1-/- hearts showed pronounced swelling at baseline that could be alleviated by polyethylene glycol-induced shrinkage. Calcium release experiments also showed higher baseline Ca 2+ levels in mitochondria from Mcl-1-/- hearts compared to wild type, suggesting that Mcl-1 deletion increases permeability to Ca 2+ . Our data suggest that loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial impairment and necrotic cell death. Thus, in addition to its anti-apoptotic role, Mcl-1 has an essential but unidentified role in maintaining mitochondrial function in cardiac myocytes.

Human dendritic cells efficiently phagocytose adenoviral oncolysate but require additional stimulation to mature

Oncolytic adenoviruses are emerging agents for treatment of cancer by tumor-restricted virus infection and cell lysis. Clinical trials have shown that oncolytic adenoviruses are well tolerated in patients but also that their antitumor activity needs improvement. A promising strategy toward this end is to trigger systemic and prolonged antitumor immunity by adenoviral oncolysis. Antitumor immune activation depends in large part on antigen presentation and T cell activation by dendritic cells (DCs). Thus, it is likely that the interaction of lysed tumor cells with DCs is a key determinant of such "oncolytic vaccination." Our study reveals that human DCs effectively phagocytose melanoma cells at late stages of oncolytic adenovirus infection, when the cells die showing preferentially features of necrotic cell death. Maturation, migration toward CCL19 and T cell stimulatory capacity of DCs, crucial steps for immune induction, were, however, not induced by phagocytosis of oncolysate, but could be triggered by a cytokine maturation cocktail. Therefore, oncolytic adenoviruses and adenoviral oncolysate did not block DC maturation, which is in contrast to reports for other oncolytic viruses. These results represent a rationale for inserting immunostimulatory genes into oncolytic adenovirus genomes to assure critical DC maturation. Indeed, we report here that adenoviral transduction of melanoma cells with CD40L during oncolysis triggers the maturation of human DCs with T cell stimulatory capacity similar to DCs matured by cytokines. We conclude that triggering and shaping DC-induced antitumor immunity by oncolytic adenoviruses "armed" with immunostimulatory genes holds promise for improving the therapeutic outcome of viral oncolysis in patients.

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.

Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by approximately 7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

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.

Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and devel- opment. (b-D-glucosyl)3 Yariv phenylglycoside (b-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with b-GlcY and observed inhibition of biomass increase in a culture medium with 50 lM b-GlcY. However, the growth was not inhibited by (a-D-galactosyl)3 Yariv phenylglycoside (a-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that b-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in b-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in b-GlcY- treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in b-GlcY- treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by b-GlcY. These data indicate that the b-GlcY-induced inhibition of Araucaria cell's growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.

Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Cadmium Induces Ca2+-Dependent Necrotic Cell Death through Calpain-Triggered Mitochondrial Depolarization and Reactive Oxygen Species-Mediated Inhibition of Nuclear Factor-κB Activity

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level.

VHA-8, the E subunit of V-ATPase, is essential for pH homeostasis and larval development in C. elegans

Vacuolar H +-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans.

Pathogenesis of hippocampal neuronal death after hypoxia–ischemia changes during brain development

Transient hypoxia–ischemia (HI) leads to delayed neuronal death in both mature and immature neurons but the underlying mechanisms are not fully understood. To understand whether the pathogenesis of HI-induced neuronal death is different between mature and immature neurons, we used a rat HI model at postnatal days 7 (P7), 15 (P15), 26 (P26) and 60 (P60) in order to investigate ultrastructural changes and active caspase-3 distribution in HI-injured neurons as a function of developmental age. In P7 pups, despite more than 95% of HI-injured neurons highly expressing active caspase-3, most of these active caspase-3-positive neurons revealed mixed features of apoptosis and necrosis (a chimera type) under electron microscopy (EM). Classical apoptosis was observed only in small populations of HI-injured P7 neurons. Furthermore, in rats older than P7, most HI-injured neurons displayed features of necrotic cell death under EM and, concomitantly, active caspase-3-positive neurons after HI declined dramatically. Classical apoptosis after HI was rarely found in neurons older than P15. In P60 rats, virtually all HI-injured neurons showed the shrinkage necrotic morphology under EM and were negative for active caspase-3. These results strongly suggest that pathogenesis of HI-induced neuronal death is shifting from apoptosis to necrosis during brain development.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Amyloid β(1–42) and its β(25–35) fragment induce in vitro phosphatidylcholine hydrolysis in bovine retina capillary pericytes

We describe the inhibitory effect of full-length Aβ(1–42) and Aβ(25–35) fragment of amyloid-β peptide on phosphatidylcholine (PtdCho) metabolism in bovine retina capillary pericytes. Cell cultures were incubated with Aβs for 24 h. Peroxidation indices (malondialdehyde and lactate dehydrogenase release) significantly increased after 20–50 μM Aβ(1–42) or Aβ(25–35) treatment. In addition, [Me- 3H]choline incorporation into PtdCho strongly decreased while either 3H-choline or 14C-arachidonic acid release from prelabeled cells increased, indicating PtdCho hydrolysis. The effect was very likely due to prooxidant action of both Aβ peptides. Reversed-sequence Aβ(35–25) peptide did not depress 3H-choline incorporation nor stimulate PtdCho breakdown. With addition of Aβs at low concentrations (2–20 μM) to pericytes, marked ultrastructural changes, well connected to metabolic alterations, emerged including shrinkage of cell bodies, retraction of processes, disruption of the intracellular actin network. Cells treated with higher concentrations (50–200 μM) displayed characteristics of necrotic cell death. The data suggest that: (a) Aβ(1–42) and Aβ(25–35) peptides may modulate phospholipid turnover in microvessel pericytes; (b) together with endothelial cells, pericytes could be the target of vascular damage during processes involving amyloid accumulation.

Is cisplatin-induced cell death always produced by apoptosis?

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a factor contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., 2000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by factors such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1210 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-2, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant keratinocytes transformed by H-ras oncogene, a high dose of cisplatin (312 microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a growing body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.