Induction Of Necrotic Cell Death Research Articles

Photodynamic therapy associating Photogem® and blue LED on L929 and MDPC‐23 cell culture

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem associated to blue LED (light-emitting diode) on L929 and MDPC-23 cell cultures, 30000 cells/cm2 were seeded in 24-well plates for 48 h, incubated with Photogem (10, 25 or 50 mg/l) and irradiated with an LED source (460+/-3 nm; 22 mW/cm2) at two energy densities (25.5 or 37.5 J/cm2). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet's post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT-induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.

Membrane-Bound Fas Ligand Requires RIP1 for Efficient Activation of Caspase-8 within the Death-Inducing Signaling Complex

The serine-threonine kinase RIP1 was originally identified through its ability to bind to the death domain of Fas (CD95). RIP1 has been shown to be recruited to the Fas death-inducing signaling complex (DISC) and is required for the induction of necrotic cell death. In this study, we show that in Jurkat T lymphocytes, RIP1 is also necessary for the most efficient activation of downstream caspases by Fas when treated with membrane-bound Fas ligand, but not with agonistic Abs or cross-linked soluble Fas ligand. RIP1 participates in the Fas-associated death domain protein-mediated recruitment of caspase-8 to the Fas receptor complex in a manner that promotes caspase-8 activation. Cross-linking Abs, such as CH11, bypass the requirement for RIP1 in caspase activation by initiating larger, though less efficient, DISC complexes, while membrane-bound Fas ligand initiates a smaller but more efficient DISC that functions, in part, by effectively incorporating more RIP1 into the complex. Consequently, RIP1 is likely a more integral part of physiological signaling through the Fas/CD95 receptor complex than previously recognized; at least when the signal is mediated by full-length membrane-bound FasL. Cross-linked soluble FasL, which also occurs physiologically, behaves similarly to the CH11 Ab, and may therefore be more likely to initiate nonapoptotic Fas signaling due to less RIP1 in the receptor complex. Thus, agonists that bind the same Fas receptor initiate mechanistically distinct pathways resulting in differential cytotoxicity.

The effect of glycosaminoglycan loss on chondrocyte viability: A study on porcine cartilage explants

Loss of glycosaminoglycan (GAG) is an early event in osteoarthritis. Recent findings showed increased cell death in arthritic cartilage and linkage with extracellular matrix degradation. The aim of this study was to analyze the direct effect of GAG loss on chondrocyte survival and cell death following mechanical injury. In full-thickness cartilage explants from porcine knee joints, GAG was depleted by digestion with chondroitinase ABC. Explants were subjected to single-impact mechanical injury. Cell viability and the types of cell death were analyzed by Live/Dead cell assay, staining for active caspase 3, and sensitivity to caspase inhibitor. GAG depletion did not directly lead to increased cell death. In chondroitinase ABC-treated explants, but not in control explants, mechanical injury caused an immediate reduction in cell viability (from 84.6% to 71.0%); the reduction was prominent in the superficial zone. This immediate cell death was not inhibited by the pancaspase inhibitor Z-VAD-FMK, suggesting cell necrosis. During subsequent culture, viability in these explants decreased further, to 50.5% on day 3. The second wave of cell death was reduced by the addition of Z-VAD-FMK in chondroitinase ABC-treated explants and was also associated with activation of caspase 3, suggesting apoptotic mechanisms of cell death. These results indicate that GAG loss alone does not directly lead to chondrocyte death. In response to mechanical injury, there is an immediate induction of necrotic cell death that is seen only in GAG-depleted explants and primarily in the superficial zone. During subsequent culture, cell death spreads via apoptotic mechanisms.

195 RELATIONSHIP BETWEEN CHONDROCYTE APOPTOSIS AND EXPRESSION OF CARTILAGE EXTRACELLULAR MATRIX MOLECULES

impact (2-14 MPa) mechanical injury in a mechanical testing device. GAG alternation was determined by safrain O and DMB assay and Cell viability was analyzed by live dead assay. The type of cell death was characterlized by immunohistochemistry using active caspase-3 and finally pan-caspase inhibitor Z-VADfmk was suspended in culture media and determine the prevention of apoptosis after injury. Results: GAG depletion did not lead to an increase in cell death directly when cartilage explants were observed for up to 2 weeks in culture. Mechanical injury in control explants did not cause an immediate change in cell viability, but viability decreased during the subsequent culture period to approximately 53.2% on day 3. In CABC-treated explants mechanical injury caused an immediate reduction in cell viability (from 84.6% to 71.0%). This immediate cell death was not inhibited by preincubation with the pan-caspase inhibitor Z-VADfmk, suggesting cell necrosis. During subsequent culture the viability in these explants decreased further to 50.5% on day 3. The second wave of cell death was almost completely inhibited by Z-VADfmk in CABC-treated explants. This second wave of cell death was also associated with activation of caspase-3, suggesting apoptotic mechanisms of cell death. When CABC-treated cartilage explants were subjected to mechanical injury, cell death was increased prominently in the superficial zone as compared to explants that were not treated with CABC. To determine mechanisms of increased cell death in CABC treated explants, strain measurements were performed. These results show that the same load (ie 8MPa) cause increased strain (40%) as compared to explants not treated with CABC. Conclusions: These results indicate that GAG loss alone does not directly lead to chondrocyte death. In response to mechanical injury there is an immediate induction of necrotic cell death that is seen only in GAG-depleted explants and prominent in the superficial zone. This appears to be the result of increased strain in response to mechanical load in the GAG-depleted explants. During subsequent culture cell death spreads via apoptotic mechanisms in CABC and non-CABC treated explants.

Cytokeratin-18 Is a Useful Serum Biomarker for Early Determination of Response of Breast Carcinomas to Chemotherapy

With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

TNF-Induced Activation of the Nox1 NADPH Oxidase and Its Role in the Induction of Necrotic Cell Death

Tumor necrosis factor (TNF) is an important cytokine in immunity and inflammation and induces many cellular responses, including apoptosis and necrosis. TNF signaling enables the generation of superoxide in phagocytic and vascular cells through the activation of the NADPH oxidase Nox2/gp91. Here we show that TNF also activates the Nox1 NADPH oxidase in mouse fibroblasts when cells undergo necrosis. TNF treatment induces the formation of a signaling complex containing TRADD, RIP1, Nox1, and the small GTPase Rac1. TNF-treated RIP1-deficient fibroblasts fail to form such a complex, indicating that RIP1 is essential for Nox1 recruitment. Moreover, the prevention of TNF-induced superoxide generation with dominant-negative mutants of TRADD or Rac1, as well as knockdown of Nox1 using siRNA, inhibits necrosis. Thus our study suggests that activation of Nox1 through forming a complex with TNF signaling components plays a key role in TNF-induced necrotic cell death.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.

Selective Induction of Necrotic Cell Death in Cancer Cells by β-Lapachone through Activation of DNA Damage Response Pathway

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells. We found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to β-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked β-lapachone-induced necrosis. Furthermore, necrotic cell death induced by β-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy

BopC Is a Novel Type III Effector Secreted by Bordetella bronchiseptica and Has a Critical Role in Type III-dependent Necrotic Cell Death

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages

In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor alpha. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.

Alteration of cytosolic free calcium homeostasis by SIN‐1: high sensitivity of L‐type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.

Phytoglobins can interfere with nitric oxide functions during plant growth and pathogenic responses: a transgenic approach

To investigate the possible role of the non-symbiotic plant hemoglobins (phytoglobins) in relation to nitric oxide (NO) functions and their presumable involvement in NO- or pathogenesis-induced necrosis, we have produced transgenic tobacco plants (HOT lines) overexpressing an alfalfa hemoglobin cDNA ( Mhb1) under the control of CaMV35S promoter. Upon treatment with active sodium nitroprusside (SNP), a widely used NO donor, the germination of seeds and development of seedlings were significantly less retarded in transgenic lines compared with the retardation of non-transformed seedlings. SNP-injection necrotized mature plant leaves of Mhb1-transformants to a lower extent than control leaves. Furthermore, infection of tobacco leaves either with Pseudomonas syringae pv. phaseolicola or Tobacco Necrosis Virus (TNV) resulted in reduced necrosis of mature transgenic plants. In response to bacterial infection, reactive oxygen species (ROS) and salicylic acid (SA) were produced at a higher level in transgenic HOT plants than in control ones. The presented experimental data support a conclusion that plant non-symbiotic hemoglobins are active functional partners in NO-dependent physiological responses such as alteration of plant growth and development as well as cell death and symptom generation after pathogen infection. The described experiments provide new insights to the role of phytoglobins in ROS-, NO- and SA-mediated cellular events during the induction of necrotic cell death.

Necrotic cell death induced by delta-aminolevulinic acid in mouse astrocytes. Protective role of melatonin and other antioxidants.

Accumulation of delta-aminolevulinic acid (ALA), as it occurs in acute intermittent porphyria (AIP), is the origin of an endogenous source of reactive oxygen species (ROS), which can exert oxidative damage to cell structures. In the present work we examined the ability of different antioxidants to revert ALA-promoted damage, by incubating mouse astrocytes with 1.0 mM ALA for different times (1-4 hr) in the presence of melatonin (2.5 mM), superoxide dismutase (25 units/mL), catalase (200 units/mL) or glutathione (0.5 mM). The defined relative index [(malondialdehyde levels/accumulated ALA) x 100], decreases with incubation time, reaching values of 76% for melatonin and showing that the different antioxidants tested can protect astrocytes against ALA-promoted lipid peroxidation. Concerning porphyrin biosynthesis, no effect was observed with catalase and superoxide dismutase whereas increases of 57 and 87% were obtained with glutathione and melatonin, respectively, indicating that these antioxidants may prevent the oxidation of porphobilinogen deaminase, reactivating so that the AIP genetically reduced enzyme. Here we showed that ALA induces cell death displaying a pattern of necrosis. This pattern was revealed by loss of cell membrane integrity, marked nuclear swelling and double labeling with annexin V and propidium iodide. In addition, no caspase 3-like activity was detected. These findings provide the first experimental evidence of the involvement of ALA-promoted ROS in the damage of proteins related to porphyrin biosynthesis and the induction of necrotic cell death in astrocytes. Interestingly, melatonin decreases the number of enlarged nuclei and shows a protective effect on cellular morphology.