Emodin inhibits TNF-alpha-induced human aortic smooth-muscle cell proliferation via caspase- and mitochondrial-dependent apoptosis

Vascular smooth-muscle cell (VSMC) proliferation plays a vital role in hypertension, atherosclerosis and restenosis. It has been reported that emodin, an active component extracted from rhubarb, can stop the growth of cancer cells; however, it is not known if emodin exerts similar anti-atherogenic effects in TNF-alpha treated human aortic smooth-muscle cells (HASMC). In this study, emodin treatment showed potent inhibitory effects in TNF-alpha-induced HASMC proliferation that were associated with induced apoptosis, including the cleavage of poly ADP-ribose polymerase (PARP). Moreover, inhibitors of caspase-3, -8 and -9 (Ac-DEVD-CHO, Z-IETD-FMK and Z-LEHD-FMK) efficiently blocked emodin-induced apoptosis in TNF-alpha treated HASMC. Therefore, emodin-induced cell death occurred via caspase-dependent apoptosis. Emodin treatment resulted in the release of cytochrome c into cytosol and a loss of mitochondrial membrane potential (DeltaPsi(m)), as well as a decrease in the expression of an anti-apoptotic protein (Bcl-2) and an increase in the expression of an a pro-apoptotic protein (Bax). Emodin-mediated apoptosis was also blocked by a mitochondrial membrane depolarization inhibitor, which indicates that emodin-induced apoptosis occurred via a mitochondrial pathway. Taken together, the results of this study showed that emodin inhibits TNF-alpha-induced HASMC proliferation via caspase- and a mitochondrial-dependent apoptotic pathway. In addition, these results indicate that emodin has potential as an anti-atherosclerosis agent.     

Overexpression of catalase in the mitochondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apoptosis

The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-alpha (TNF-alpha) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-alpha-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-alpha, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-alpha to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-alpha-induced apoptosis and toxicity produced by catalase overexpression. Addition of H(2)O(2) prevented TNF-alpha-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-alpha plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-alpha did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-alpha signaling was not altered by the overexpression of catalase, as activation of nuclear factor kappaB and AP-1 by TNF-alpha was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-alpha-induced apoptosis and activation of caspases by removal of H(2)O(2).     

Hyperthermia and tumour necrosis factor-alpha induced apoptosis via mitochondrial damage

Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.     

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.     

Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Reversal of TNF-alpha resistance by hyperthermia: role of mitochondria

The aim of this study is to examine the effect of hyperthermia on tumour necrosis factor-alpha (TNF-alpha) resistance in L929-11E cells. L929-11E is a TNF-alpha resistant variant derived from L929 cells, a commonly used model for TNF-alpha study. Based on the results from flow cytometry and Western blotting, hyperthermia (43 degrees C, 3 h) was found to induce apoptosis, mitochondrial potential (delta psi(m)) depolarization and release of cytochrome c in L929-11E cells. Similar responses were found in L929 cells when treated with TNF-alpha. Heating at 43 degrees C for 1 h did not significantly damage the mitochondria of L929-11E cells but partially reversed their resistance to TNF-alpha. When L929-11E cells were sequentially treated with heating (43 degrees C, 1 h) and TNF-alpha, a more severe damage in mitochondria was observed. Taken together, our results indicate (1) hyperthermia induced apoptosis in L929-11E cells via mitochondrial damages in a way very similar to the action of TNF-alpha in L929 cells, (2) hyperthermia could be used to overcome TNF-alpha resistance by altering mitochondrial activities and (3) L929-11E and its parental cells provide a useful model in elucidating the signalling linkage between TNF-alpha receptor and mitochondria.     

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (△Ψm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that △Ψm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P＜0.05) and cyclosporin A (CsA) could inhibit the collapse of △Ψm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P＜0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells

HL-60 cells treated by prostaglandin (PG) A₂ showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA₂-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA₂-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA₂, mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA₂. Finally, it was shown that PGA₂-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA₂ activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA₂ plays a pivotal role.     

Asbestos-induced alveolar epithelial cell apoptosis: role of mitochondrial dysfunction caused by iron-derived free radicals

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-inducedAEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (delta(psi)m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces delta(psi)m after a 4 h exposure period. Further, the delta(psi)m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell delta(psi)m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell delta(psi)m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.     

Disulfiram inhibits TNF-alpha-induced cell death

Disulfiram, a clinically employed alcohol deterrent, was recently discovered to inhibit caspase-3 and DNA fragmentation. Using LLC-PK1 cells and murine liver as models, we examined if the drug inhibited TNF-alpha-induced cell death. Disulfiram produced dose-dependent inhibition of TNF-alpha-induced cell death as well as caspase-3-like activity. Disulfiram retained 80% of its effect when added 4 h after TNF-alpha. Disulfiram protected the cells from cytokine-induced death for at least 6 days. The cells rescued by the drug preserved the ability to proliferate. The cells died spontaneously after exposure to TNF-alpha for just 70 min. Co-administration of 15 microM disulfiram and TNF-alpha for 70 min prior to their removal abolished TNF-alpha-induced killing, and this was associated with restoration of mitochondrial membrane potential and suppression of reactive oxygen species. Treatment of mice with TNF-alpha and D-galactosamine for 5 h markedly increased hepatic DNA fragmentation and caspase-3-like activity. Disulfiram at 0.6 mmol/kg abolished these effects. We conclude that disulfiram is a potent inhibitor of TNF-alpha-induced cell death in vitro. The underlying mechanisms include stabilization of mitochondrial membrane potential, suppression of reactive oxygen species, and inhibition of caspase-3-like activity. We further conclude that disulfiram inhibits DNA fragmentation in vivo in association with the blockade of caspase-3-like activity.     

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.     

Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.     

E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.     

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study.

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose ("chemical hypoxia"). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.     

C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and up-regulating FLIP

Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes (OLG) in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In the present study, we examined the possible involvement of the caspase-8 and Fas pathway in OLG apoptosis and the role of C5b-9 in this process. In a serum-free defined medium, OLG undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of cellular FLIP long isoform protein. The caspase-8 inhibitor Z-IETD-FMK inhibited cell death associated with differentiation in a dose-dependent manner. Exposure to C5b-9 induced an inhibition of caspase-8 activation, Bid cleavage, and a significant increase in expression of cellular FLIP long isoform. These C5b-9 effects were reversed by PI3K inhibitor LY294002. C5b-9 also down-regulated the expression of FasL and the Fas-induced apoptosis. These data suggest that C5b-9 through PI3K signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing.     

Caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells.

We reported previously that singlet oxygen, generated by irradiation of rose bengal with visible light, induced apoptosis in human promyelocytic leukemia HL-60 cells. However, the mechanism of apoptosis caused by this reactive oxygen species is unclear. In this study, we demonstrate that singlet oxygen induced caspase-3 activation and Z-DEVD-FMK, a caspase-3 inhibitor, blocked apoptosis induction, while caspase-1 activity was not detectable and the caspase-1 inhibitor Z-YVAD-FMK had a very limited effect on apoptosis. This suggests that the activation of caspase-3 by singlet oxygen is essential for the commitment of cells to undergo apoptosis. Further studies showed that singlet oxygen induced an increase in caspase-8 activity and a reduction in mitochondrial cytochrome c. Time course analysis indicated that the cleavage of caspase-8 precedes that of caspase-3. In addition, blockade of caspase-8 by Z-IETD-FMK inhibited cleavage of pro-caspase-3 and prevented loss of mitochondrial cytochrome c. These results suggest that caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis in HL-60 cells.     

Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently

Poly(L-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L-PLL, and high (27.4 kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine "scrambling" and loss of membrane integrity, but later (24 h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed.