Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells

Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C (PK-C) activation were observed during the apoptosis of C6 cells, and these events were inhibited by antioxidants and iron chelators without affecting BSO-induced GSH depletion. Furthermore, approximately 2 Mbp giant DNA fragments were observed in the BSO-treated cells. The giant DNA fragmentation were followed by approximately 30-700 kbp and then less than 100 kbp, including internucleosomal DNA fragmentations. Such serial DNA degradation was prevented by the antioxidants, the iron chelators, and the PK-C inhibitors. These results suggest that during apoptosis induced by GSH-depletion caused by BSO, reactive oxygen species endogenously produced cause lipid peroxidation and that the lipid peroxidation induced PK-C activation, processes which are thought to be involved in the giant DNA, high-molecular-weight DNA, and the internucleosomal DNA fragmentations.     

High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays

To continue elucidation of the biochemical and molecular pathways involved in the induction of apoptosis in granulosa cells (GC) of ovarian follicles destined for atresia, we characterized the occurrence and protease modulation of high and low molecular weight (MW) DNA fragmentation during rat GC death. Atresia of ovarian follicles, occurring either spontaneously in vivo or induced in vitro, was associated with both high MW and internucleosomal (low MW) DNA cleavage. Incubation of follicles in the presence of a putative irreversible and non-competitive inhibitor of caspase-1 (interleukin-1beta-converting enzyme or ICE), sodium aurothiomalate (SAM), completely prevented internucleosomal, but not high MW, DNA cleavage. As reported previously, morphological features of apoptosis (pyknosis, cellular condensation) and atresia (granulosa cell disorganization, oocyte pseudomaturation) remained detectable in SAM-treated follicles. The potential involvement of proteases in endonuclease activation was further analyzed in cell-free assays using nuclei from both GC (which autodigest their DNA) and HeLa cells (HC, which do not autodigest their DNA unless incubated with extracts prepared from other cell types). Crude cytoplasmic extracts prepared from GC induced both high MW and internucleosomal DNA cleavage in HC nuclei. The induction of low, but not high, MW DNA cleavage in HC nuclei by GC extracts was suppressed by pretreatment of the extracts with SAM or with any one of the serine protease inhibitors, dichloroisocoumarin (DCI), N-tosyl-L-leucylchloromethylketone (TLCK) or N-tosyl-L-phenylchloromethylketone (TPCK). Interestingly, SAM and DCI also prevented cation-induced low MW DNA fragmentation in GC nuclei; however, TLCK and TPCK were without effect. Our results support a role for cytoplasmic and nuclear serine proteases in the activation of the endonuclease(s) responsible for internucleosomal DNA cleavage during apoptosis.     

Pro- and Anti-Apoptotic Effects of an Inhibitor of Chymotrypsin-Like Serine Proteases

The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl –L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.     

Promoting effects of polyunsaturated fatty acids on chromosomal giant DNA fragmentation associated with cell death induced by glutathione depletion

Glutamate and buthionine sulfoximine (BSO) both reduce intracellular glutathione (GSH) concentration but by different mechanisms, and thereby induce cell death in C6 rat glioma cells. The effects of lipid peroxidation on chromosomal DNA damage during the GSH depletion-induced cell death were assessed. Polyunsaturated fatty acids (PUFA), such as arachidonic acid (AA), gamma-linolenic acid and linoleic acid enhanced lipid peroxidation, induced a loss of membrane integrity and consequently promoted 1-2 Mbp giant DNA fragmentation under both glutamate- and BSO-induced GSH-depletion. Treated C6 cells had 3'-OH termini in their DNA which were recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) analysis. Antioxidants capable of scavenging reactive oxygen species and lipid radicals and iron or copper scavengers inhibited both lipid peroxidation and 1-2 Mbp giant DNA fragmentation, consequently protecting against cell death under GSH depletion. These results suggest that GSH depletion induces lipid peroxidation and leads to 1-2 Mbp giant DNA fragmentation; and that PUFAs can promote giant DNA fragmentation and 3'-OH termini in chromosomal DNA enhancing lipid peroxidation of C6 cells.     

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.     

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP.1 cells differently dependent on both the concentration used and the apoptotic stimulus. TPCK (50 - 75 microM) induced both biochemical and ultrastructural changes characteristic of apoptosis, including proteolysis of poly (ADP-ribose) polymerase (PARP) and lamins together with formation of large kilobase pair fragments of DNA, particularly of 30 - 50 and 200 - 300 kilobase pairs in length but without internucleosomal cleavage of DNA. The induction of apoptosis by TPCK also involved the processing of CPP32 and Mch 3 to their catalytically active subunits. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE-like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concentrations of TPCK (5 - 20 microM) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 microM) inhibited cell death induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell survival, the second in facilitating internucleosomal cleavage of DNA and the third in the modulation of apoptosis induced by different apoptotic stimuli.     

Glutamate-induced Toxicity in Hippocampal Slices Involves Apoptotic Features and p38MAPK Signaling

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 μM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38^MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38^MAPK pathway.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.     

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

Reactive Oxygen Species-Mediated DNA Damage and Apoptosis in Human Skin Epidermal Cells After Exposure to Nickel Nanoparticles

Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.     

Polyunsaturated fatty acids promote 8-hydroxy-2-deoxyguanosine formation through lipid peroxidation under the glutamate-induced GSH depletion in rat glioma cells

It has been reported that glutamate decreased the intracellular glutathione (GSH) concentration and thereby induced cell death in C6 rat glioma cells. Polyunsaturated fatty acids such as arachidonic acid, gamma-linolenic acid, and linoleic acid enhanced lipid peroxidation promoting 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation under the glutamate-induced GSH-depletion. The enhancement of lipid peroxidation by polyunsaturated fatty acids was species-dependent. Some antioxidants capable of scavenging oxygen and lipid radicals and some iron or copper scavengers inhibited both the lipid peroxidation and the 8-OH-dG formation, consequently protecting against cell death induced by glutamate-induced GSH depletion. These results suggest that GSH depletion caused by glutamate induces lipid peroxidation and consequently 8-OH-dG formation and that polyunsaturated fatty acids enhance lipid peroxidation associated with mediated 8-OH-dG formation through a chain reaction.     

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.     

Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis

Micromolar concentrations of the flavonoid kaempferol were found to efficiently block cerebellar granule cell (CGC) death through low K+-induced apoptosis, as demonstrated by prevention of the activation of caspase-3, internucleosomal DNA fragmentation, and chromatin condensation, without a significant rise in intracellular free Ca2+ concentration. Half of the maximum protection against CGC apoptosis was attained with 8 +/- 2 microM kaempferol. Reactive oxygen species (ROS) were monitored with 2',7'-dichlorodihydrofluorescein diacetate. Quantitative analysis of intracellularly and extracellularly oriented ROS production up to 3 h from the onset of low K+-induced CGC apoptosis was carried out with acquired digital fluorescence microscopy images of CGC in culture plates using a CCD camera, and also with fluorescence measurements of resuspended CGCs. In both cases, nearly 90% of ROS production by CGCs during the early stages (up to 3 h) after induction of low-K+ apoptosis occurs at the plasma membrane. Kaempferol, at concentrations that blocked CGC apoptosis, has been found to be a particularly potent blocker of extracellularly oriented ROS production by CGCs, and to inhibit the ascorbate-dependent NADH oxidase and superoxide anion production activities of the neuronal plasma membrane redox chain.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis

Zebularine (Zeb) as a DNA methyltrasferase (DNMT) inhibitor has various cellular effects such as cell growth inhibition and apoptosis. In the present study, we evaluated the effects of Zeb on the growth and death of HeLa cervical cancer cells. Zeb inhibited the growth of HeLa cells with an IC(50) of approximately 130?μM at 72?h in a dose-dependent manner. DNA flow cytometric analysis indicated that Zeb induced an S phase arrest of the cell cycle, which was accompanied by the increased levels of cdk2 and cyclin A proteins. This agent also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (Ψ(m)), PARP-1 cleavage and the activation of caspase-3, -8 and -9. All of the tested caspase inhibitors significantly rescued some cells from Zeb-induced HeLa cell death. In relation to reactive oxygen species (ROS) and glutathione (GSH) levels, O (2) (?-) level was significantly increased in 100?μM Zeb-treated HeLa cells and caspase inhibitors reduced O (2) (?-) level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. In conclusion, this is the first report that Zeb inhibited the growth of HeLa cells via cell cycle arrest and apoptosis.     

Cytoskeletal disruption induces T cell apoptosis by a caspase-3 mediated mechanism

T cell apoptosis can be triggered by different mechanisms that lead to distinctive features such as cell shrinkage, membrane blebbing, phosphatidylserine externalization, and internucleosomal DNA fragmentation. Prevailing models for the induction of apoptosis place the cytoskeleton as a distal target of the death effector molecules ('executioners'). However, the cytoskeleton can also play a role in the induction of apoptosis as suggested by the finding that cytoskeletal disruption can induce apoptosis. The mechanism by which this occurs is unknown. Here, we report that T cell apoptosis by cytoskeletal disruption involves a protein synthesis-independent mechanism leading to up-regulation of caspase-3 protease activity and increased accessibility of active caspase-3 to its substrate. Thus, cytoskeleton integrity may regulate the subcellular compartmentalization of death effector molecules.