The adenylate cyclase activator forskolin partially protects L929 cells against tumour necrosis factor-alpha-mediated cytotoxicity via a cAMP-independent mechanism

Recent reports indicate that cAMP-elevating agents can protect against cell death induced by many stimuli, including tumour necrosis factor-alpha (TNF-alpha). We investigated the ability of cAMP-elevating agents to modulate TNF-alpha-mediated cytotoxicity in L929 cells. Using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay and a DNA fragmentation assay as indicators of cell survival, we have shown that forskolin confers partial protection against TNF-alpha-mediated cytotoxicity and inhibits TNF-alpha-induced internucleosomal DNA fragmentation in L929 cells. The protection conferred by forskolin is cAMP-independent since 1,9-dideoxyforskolin (an adenylate cyclase-inactive analog) also protected against TNF-alpha, while both dibutyryl-cAMP and the cAMP-phosphodiesterase inhibitor theophylline were not protective. This is the first example (that we know of) of cAMP-independent cytoprotection by forskolin. We conclude that forskolin acts in a cAMP-independent manner, potentially at a site upstream of caspase-3 activation, to protect against TNF-alpha-mediated cytotoxicity in L929 cells, and that cAMP elevation, in general, does not confer protection against TNF-alpha-induced death in L929 cells. In addition, we observed that Cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor, protected L929 cells against TNF-alpha, underlining the importance of mitochondria in the cytotoxic process induced by TNF-alpha in L929 cells.     

A predominant apoptotic death pathway of neuronal PC12 cells induced by activated microglia is displaced by a non-apoptotic death pathway following blockage of caspase-3-dependent cascade.

Activated microglia have been implicated in the regulation of neuronal cell death. However, the biochemical mechanism for neuronal death triggered by activated microglia is still unclear. When treated with activated microglia, neuronal PC12 cells undergo apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation. Apoptotic bodies formed were subsequently phagocytosed by neighboring activated microglia. Pretreatment of the cells with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death. Although Bcl-2 overexpression in the cells caused the inhibition of caspase-3-like protease activity and DNA fragmentation and the effective interference of apoptosis induced by deprivation of trophic factors, it could not suppress the activated microglia-induced neuronal death. At the electron microscopic level, degenerating cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular-shaped masses, severely disintegrated perikarya, and marked vacuolation. Various protease inhibitors tested did not inhibit this cell death, whereas the radical oxygen species scavenger N-acetyl-L-cysteine significantly suppressed this death. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.     

Molecular characterization of cell death induced by a compatible interaction between Fusarium oxysporum f. sp. linii and flax (Linum usitatissimum) cells.

The cellular and molecular events associated with cell death during compatible interaction between Fusarium oxysporum sp. linii and a susceptible flax (Linum usitatissimum) cell suspension are reported here. In order to determine the physiological and molecular sequence of cell death of inoculated cells, reactive oxygen species (ROS) production, mitochondrial potential, lipoxygenase, DNase, protease and caspase-3-like activities, lipid peroxidation and secondary metabolite production were monitored. We also used microscopy, in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation assay. Cell death was associated with specific morphological and biochemical changes that are generally noticed in hypersensitive (incompatible) reaction. An oxidative burst as well as a loss of mitochondrial potential of inoculated cells, an activation of lipoxygenase and lipid peroxidation were noted. Enzyme-mediated nuclear DNA degradation was detectable but oligonucleosomal fragmentation was not observed. Caspase-3-like activity was dramatically increased in inoculated cells. Phenylpropanoid metabolism was also affected as demonstrated by activation of PAL and PCBER gene expressions and reduced soluble lignan and neolignan contents. These results obtained in flax suggest that compatible interaction triggers a cell death sequence sharing a number of common features with the hypersensitive response observed in incompatible interaction and in animal apoptosis.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Mitochondrial dysfunction in CD47-mediated caspase-independent cell death: ROS production in the absence of cytochrome c and AIF release

Ligation of CD47 by its natural ligand thrombospondin (TSP), or cross-linking by CD47 antibodies, triggers caspase-independent cell death in normal and leukemic cells. This kind of cell death is characterised by the cytoplasmic events of apoptosis including externalisation of phosphatidylserines and mitochondria swelling. We report herein selective mitochondrial changes in CD47-dependent cell death of T cells. After T cell stimulation via CD47, a rapid mitochondrial transmembrane potential (deltapsi(m)) disruption is accompanied by the production of reactive oxygen species (ROS) and phosphatidylserine exposure. Surprisingly, mitochondrial dysfunction does not induce cytochrome c or AIF release. Moreover, the dying cells do not exhibit caspase-3 activation and display intact nuclei without any large-scale, or oligonucleosomal DNA fragmentation. We conclude that DeltaPsi(m) loss and ROS production are an early step in CD47-dependent killing and neither cytochrome c, nor AIF are implicated in this new cell death pathway.     

Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture

When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.     

gamma-Linolenic acid and eicosapentaenoic acid induce modifications in mitochondrial metabolism, reactive oxygen species generation, lipid peroxidation and apoptosis in Walker 256 rat carcinosarcoma cells.

The polyunsaturated fatty acids gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) are cytotoxic to tumour cells. GLA inhibits Walker 256 tumour growth in vivo, causing alterations in mitochondrial ultrastructure and cellular metabolism. The objective of the present study was to investigate the mechanisms behind fatty acid inhibition of Walker 256 tumour growth under controlled in vitro conditions. At a concentration of 150 microM, both GLA and EPA caused a decrease in cell proliferation and an increase in apoptotic index. Increases in reactive oxygen species (ROS) and lipid peroxide production were identified, as well as alterations in energy metabolism and the deposition of large amounts of triacylglycerol in the form of lipid droplets. Mitochondrial respiratory chain complexes I+III and IV had significantly decreased activity and mitochondrial membrane potential was greatly diminished. Intracellular ATP concentrations were maintained at 70-80% of control values despite the decreased mitochondrial function, which may be in part due to increased utilisation of glucose for ATP generation. Cytochrome c release from mitochondria was found, as was caspase-3-like activation. DNA fragmentation in situ revealed many apoptotic events within the cell population. The mechanism(s) by which ROS and lipid peroxides induce apoptosis remains unclear, but the effects of GLA and EPA appear to involve the mitochondrial pathway of apoptosis induction leading to cytochrome c release, caspase activation, loss of mitochondrial membrane potential and DNA fragmentation.     

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.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G₀/G₁ phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G₀/G₁ phase and caspase-3 activation.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Hyperoxia-induced apoptosis does not require mitochondrial reactive oxygen species and is regulated by Bcl-2 proteins

Exposure of animals to hyperoxia results in lung injury that is characterized by apoptosis and necrosis of the alveolar epithelium and endothelium. The mechanism by which hyperoxia results in cell death, however, remains unclear. We sought to test the hypothesis that exposure to hyperoxia causes mitochondria-dependent apoptosis that requires the generation of reactive oxygen species from mitochondrial electron transport. Rat1a cells exposed to hyperoxia underwent apoptosis characterized by the release of cytochrome c, activation of caspase-9, and nuclear fragmentation that was prevented by the overexpression of Bcl-X(L.) Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice were resistant to hyperoxia-induced cell death. The administration of the antioxidants manganese (III) tetrakis (4-benzoic acid) porphyrin, ebselen, and N-acetylcysteine failed to prevent cell death following exposure to hyperoxia. Human fibrosarcoma cells (HT1080) lacking mitochondrial DNA (rho(0) cells) that failed to generate reactive oxygen species during exposure to hyperoxia were not protected against cell death following exposure to hyperoxia. We conclude that exposure to hyperoxia results in apoptosis that requires Bax or Bak and can be prevented by the overexpression of Bcl-X(L). The mitochondrial generation of reactive oxygen species is not required for cell death following exposure to hyperoxia.     

Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7beta-hydroxycholesterol-induced cell death: a morphological and biochemical study

On treatment with 7-ketocholesterol (7-keto) or 7beta-hydroxycholesterol (7beta-OH), which are major oxysterols in atherosclerotic plaques, the simultaneous identification of oncotic and apoptotic cells suggests that these compounds activate different metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase-9, -7, -8 activation, for Bid degradation mediating mitochondrial cytochrome c release, for cleavage of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmentation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. However, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7beta-OH-induced cell death characterized by staining with propidium iodide, loss of mitochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was independent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7beta-OH-induced apoptosis, that these oxysterols simultaneously activate caspase-3-dependent and/or -independent modes of cell death.