Necrotic Mode Of Cell Death Research Articles

Novel Porphyrin Conjugates with a Potent Photodynamic Antitumor Effect: Differential Efficacy of Mono‐ and Bis‐β‐cyclodextrin Derivatives In Vifro and In Vivo

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(beta-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(beta-CD)2. However, in vivo P(beta-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm(-2)). In contrast, P(beta-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(beta-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.

A Novel Targeted Therapy of Leydig and Granulosa Cell Tumors through the Luteinizing Hormone Receptor Using a Hecate-Chorionic Gonadotropin β Conjugate in Transgenic Mice

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)β conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGβ chain. Transgenic (TG) mice expressing the inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGβ conjugate for 3 weeks. Hecate-CGβ conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGβ treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGβ conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGβ conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Apoptotic pathways in ischemic acute renal failure

The study of cell death has emerged as an important and exciting area of research in cell biology. Although two kinds of cell death, apoptosis and necrosis, are recognized, one of the major advances in our understanding of cell death has been the recognition that the pathways traditionally associated with apoptosis may be very critical in the form of cell injury associated with necrosis. Renal tubular epithelial cell injury from ischemia has been generally regarded as a result of necrotic form of cell death. We briefly describe recent evidence indicating that pathways generally associated with apoptosis, including endonuclease activation, role of mitochondria and caspases, are important in renal tubular injury. It is likely that the cascades that lead to apoptotic or necrotic mode of cell death are activated almost simultaneously and may share some common pathways.

Combined action of extracellular signal-regulated kinase and p38 kinase rescues Molt4 T cells from nitric oxide-induced apoptotic and necrotic cell death

The mechanisms that regulate nitric oxide (NO)-induced apoptosis, especially in T cell apoptosis, are largely uncharacterized. Here, we report that protection from NO-induced cell death by phorbol 12-myristate 13-acetate (PMA) is dependent on both p38 and extracellular signal-regulated kinase (ERK) activation. Exposure of Molt4 cells to NO donor S-nitroso- N-acetyl- dl-penicillamine (SNAP) induced both apoptotic and necrotic modes of cell death along with a sustained increase in p38 kinase phosphorylation. However, the p38 inhibitor SB202190 only slightly protected Molt4 cells from NO toxicity. In contrast, PMA rapidly phosphorylated both p38 kinase and ERK, and the phosphorylation statuses were not altered in the presence of SNAP. Interestingly, although each mitogen-activated protein kinase (MAPK) inhibitor by itself had only a modest effect, the combination of inhibitors for both MAPKs almost completely abolished the protective effect of PMA. Furthermore, dominant negative or catalytically inactive variants that modulate p38 and ERK mimicked the effects of MAPK inhibitors. We located the action of p38 and ERK upstream of the p53/mitochondrial membrane potential loss and caspases cascade. Together, these findings suggest that the PMA-induced activations of ERK and p38 kinase are parallel events that are both required for inhibition of NO-induced death of Molt4 cells.

Flow Cytometry of Apoptosis

Application of flow cytometry to the study of cell death has three goals: identification and quantification of dead and dying cells; discrimination between apoptotic and necrotic modes of cell death; and elucidation of mechanisms involved in cell death. This massively detailed unit by a pioneer in the field brings together the most common flow cytometric methods for the study of apoptosis, covering a wide variety of apoptotic indices, from loss of membrane potential, caspase activation, and phosphatidyl exposure to DNA fragmentation and tissue transglutaminase activation. The authors also present their recently developed protocol, analogous to the FLICA approach for caspases, for the detection of serine proteases ('serpases'). The protocols are accompanied by extensive commentary discussion of applicability, strategic planning, problems, and pitfalls, plus a comprehensive list of references.

Ultrastructural changes in Neurospora cells undergoing senescence induced by kalilo plasmids

In N. crassa and N. intermedia, the kalilo plasmid triggers senescence by insertion into mitochondrial DNA. To investigate the cell death pathway induced by this plasmid, juvenile and senescent subcultures of several senescent strains were examined by light and transmission electron microscopy, and at the DNA level. There were no signs of apoptotic events, such as shrinkage of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. Instead, swollen mitochondria lacking cristae and containing amorphous inclusions, and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death.

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death.

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

Cytotoxicity and electron microscopy of cell death induced by goniothalamin.

The cytotoxicity of goniothalamin was found to be strong towards both cancerous (HGC-27, MCF-7, PANC-1, HeLa), and non-cancerous (3T3) cell lines, especially in cases of dividing cells. Drug exposure studies indicated that the cytotoxic action of goniothalamin was time- and dose-dependent. At the ultrastructural level, goniothalamin-induced cytotoxicity revealed a necrotic mode of cell death towards MCF-7 cells.