Loss Of Mitochondrial Transmembrane Potential Research Articles

A Mechanism of Virulence: Virulent Mycobacterium tuberculosis Strain H37Rv, but Not Attenuated H37Ra, Causes Significant Mitochondrial Inner Membrane Disruption in Macrophages Leading to Necrosis

Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48-72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca(2+) flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Deltapsi(m)) loss caused by mitochondrial permeability transition. Dissipation of Deltapsi(m) also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca(2+) loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Deltapsi(m) 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.

N-Methyl-DD-aspartate Blocks Activation of JNK and Mitochondrial Apoptotic Pathway Induced by Potassium Deprivation in Cerebellar Granule Cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.

Mitochondria-Regulated Death Pathway Mediates (DIPP-L-Leu)2-LLysOCH3- Induced K562 Cells Apoptosis.

(DIPP-L-Leu)2-L-LysOCH3 is a diisopropylphosphoryl dipeptide which is known to induce apoptosis of human leukemia K562 cells. The molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that (DIPP-L-Leu)2-L-LysOCH3-induced apoptosis is associated with cytosolic accumulation of cytochrome c, sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS) and elevation of intracellular Ca2+ concentration. A specific caspase assay reveals an increase in caspase-9 and caspase-3 activity but no change in caspase-8 activity. Immunofluorescence analysis indicates that (DIPP-L-Leu)2-L-LysOCH3 induced upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-2 and Bcl-x(L). These results suggest that the mitochondria-regulated death pathway mediates (DIPP-L-Leu)2-L-LysOCH3-induced K562 cells apoptosis.

Resveratrol induces apoptosis of human malignant B cells by activation of caspase-3 and p38 MAP kinase pathways

Red wine polyphenol, trans-resveratrol (trans-3,4′,5-trihydroxy stilbene), has potent chemopreventive effects against various tumors. In this study, we found for the first time that resveratrol rapidly induces S phase cell cycle arrest of human malignant B cells including myeloma cells in dose- and time-dependent manners, followed by S phase cell cycle arrest through ATM/Chk pathway. Resveratrol-induced apoptosis occurs in association with the activation of caspase-3 and the loss of mitochondrial transmembrane potentials. In addition, resveratrol induces the phosphorylation of p38 MAP kinase, and specific inhibition of p38 MAP kinase abolishes the resveratrol-induced apoptosis, indicating that activation of the p38 MAP kinase pathway is required for inducing apoptosis in malignant B cells. These results suggest that resveratrol may have potential as a novel therapeutic agent for the patients with B cell malignancies including multiple myeloma.

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl(2)) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (DeltaPsi m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1alpha) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl(2)-induced mitochondrial Delta Psi m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl(2)-induced HIF-1alpha protein accumulation. Moreover, inducible expression of HIF-1alpha gene dose not alter DFO and CoCl(2)-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl(2)-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1alpha-independent mechanisms, DFO and CoCl(2)-induced apoptosis involves different initiating signal pathways that remain to be investigated.

Functional studies of an HIV‐1 encoded glutathione peroxidase

In an alternate reading frame overlapping the viral envelope gene, HIV-1 has been shown to encoded a truncated glutathione peroxidase (GPx) module. Essential active site residues of the catalytic core regions of mammalian GPx sequences are conserved in the putative viral GPx (vGPx, encoded by the env-fs gene). Cells transfected with an HIV-1 env-fs construct show up to a 100% increase in GPx enzyme activity, and are protected against the loss of mitochondrial transmembrane potential and subsequent cell death induced by exogenous oxidants or mitochondrial reactive oxygen species. An intact vGPx gene was observed to be more common in HIV-1-infected long-term non-progressors, as compared to HIV-1 isolates from patients developing AIDS. An antioxidant/antiapoptotic protective role of the vGPx is also consistent with the observation that -1 frameshifting induced by the HIV-1 env-fs sequence AAAAAGA (which contains a potential "hungry" arginine codon, AGA) increases during arginine deficiency, which has been associated with increased oxidative stress. Under arginine-limited conditions, nitric oxide synthase generates superoxide, which rapidly combines with NO to form peroxynitrite, which can cause activated T-cells to undergo apoptosis. Thus, biosynthesis of the HIV-1 GPx as an adaptive response to low arginine conditions might delay oxidant-induced apoptotic cell death, providing an enhanced opportunity for viral replication.

Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations

To examine whether a relationship exists between loss of mitochondrial transmembrane potential and plasma membrane translocation of phosphatidylserine (PS) in subpopulations of human spermatozoa of men consulting for infertility. A tertiary institutional research center. Prospective observational study. Twelve infertile men and five fertile controls were compared. Sperm subpopulations were compared after density gradient separation. Mitochondrial membrane potential was measured with a cationic dye, translocation of PS was evaluated with Annexin-V binding, and motion parameters were assessed manually. In both the study and control groups and compared with the high-motility fraction, the low-motility fraction had significantly lower sperm motility and normal morphology, and significantly higher percentage of cells with disrupted mitochondrial membrane potential and plasma membrane PS translocation. There was a positive and significant correlation in both subfertile and control groups between the percentages of Annexin-V+ live cells and cells with mitochondrial membrane potential disruption (r = 0.82 and r = 0.85, respectively). The correlation of plasma membrane PS translocation and loss of mitochondrial membrane potential is suggestive of an early apoptosis phenotype, as is typically observed in somatic cells identified in sperm subpopulations with percentage of low-motile cells. We speculate that such changes might be used as diagnostic markers of sperm dysfunction(s) and that increased levels found in subfertile men might be indicators of reduced fertility potential.

Free radical-mediated neurotoxicity may be caused by inhibition of mitochondrial dehydrogenases in vitro and in vivo

We previously demonstrated that copper facilitated the formation of reactive oxygen species, and inhibited pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in animal models of Wilson’s disease in vivo. However, direct Cu 2+ toxicity has only been demonstrated for Wilson’s disease. We now hypothesize that inhibition of these mitochondrial dehydrogenases might also contribute to many other injuries and disorders that are reactive oxygen species-mediated. We have modeled reactive oxygen species-mediated injuries using inducers of reactive oxygen species such as hydrogen peroxide, ethacrynic acid or menadione, or another redox active metal (Cd 2+). Here we demonstrated that these toxic exposures were accompanied by an early marked reduction in both pyruvate dehydrogenase and α-ketoglutarate dehydrogenase activities, followed by a decrease in neuronal mitochondrial transmembrane potential and ATP, prior to murine cortical neuronal death. Thiamine (6 mM), and dihydrolipoic acid (50 μM), required cofactors for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (thiamine as thiamine pyrophosphate), attenuated the reactive oxygen species-induced reductions in these enzyme activities, as well as subsequent loss of mitochondrial transmembrane potential and ATP, and neuronal death. We next tested the effect of thiamine supplementation on an in vivo model of reactive oxygen species-mediated injury, transient middle cerebral artery occlusion, and reperfusion in rats. Oral or i.p. thiamine administration reduced the middle cerebral artery occlusion-induced infarct. These data suggest that reactive oxygen species-induced neuronal death may be caused in part by reactive oxygen species-mediated inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in vitro and in vivo, and that thiamine or dihydrolipoic acid may constitute potential therapeutic agents not just against Cu 2+ neurotoxicity, but may reduce neuronal degeneration in the broader range of diseases mediated by free radical stress.

NF-κB Protects Macrophages from Lipopolysaccharide-induced Cell Death: THE ROLE OF CASPASE 8 AND RECEPTOR-INTERACTING PROTEIN

Macrophages play a pivotal role in the pathogenesis of a variety of diseases. These studies were performed to characterize the mechanisms by which Toll-like receptor 4 (TLR4)-mediated NF-kappaB activation promotes resistance to cell death in macrophages. When NF-kappaB activation was inhibited by a super-repressor, IkappaBalpha, the TLR4 ligand lipopolysaccharide induced the activation of caspase 8, the loss of mitochondrial transmembrane potential (DeltaPsim), and apoptotic cell death in macrophages. The inhibition of caspase 8 activation suppressed DNA fragmentation but failed to protect macrophages against the loss of DeltaPsim and resulted in necrotic cell death. In contrast, the reduction of receptor-interacting protein 1 suppressed the loss of DeltaPsim and inhibited apoptotic cell death. Further, when caspase 8 activation was suppressed, the knock down of receptor-interacting protein inhibited the loss of DeltaPsim and necrotic cell death. These observations demonstrate that following TLR4 ligation by lipopolysaccharide, NF-kappaB is a critical determinant of macrophage life or death, whereas caspase 8 determines the pathway employed.

Bax activation and translocation to mitochondria mediate EGF-induced programmed cell death

The ErbB family of receptor tyrosine kinases is involved in the regulation of cell proliferation, differentiation and apoptosis. Previous studies indicate that cells expressing elevated levels of the EGFR and ErbB-2 undergo programmed cell death in response to EGF or other EGFR ligands. However, the detailed mechanisms of EGF-induced apoptosis are unclear. This report demonstrates that in the cells undergoing EGF-dependent apoptosis Bax changes its conformation and forms multimeric aggregates, which accumulate on the mitochondrial membrane. Bax activation and translocation to the mitochondria induces a loss of mitochondrial transmembrane potential and cell death. Also, during EGF-induced apoptosis there is downregulation of Bcl-xL, an anti-apoptotic protein. Expression of Bcl-xL in cells susceptible to EGF-dependent apoptosis prevents cell death. The data indicate that addition of EGF does not result in a significant release of cytochrome c from mitochondria and EGF-induced apoptosis is mainly caspase independent.

Antimelanoma Activity of Apoptogenic Carbonyl Scavengers

Therapeutic induction of apoptosis is an important goal of anticancer drug design. Cellular carbonyl stress mediated by endogenous reactive carbonyl species (RCS) such as glyoxal and methylglyoxal (MG) affects proliferative signaling and metastasis of human tumor cells. Recent research suggests that RCS produced constitutively during increased tumor cell glycolysis may be antiapoptotic survival factors and thus represent a novel molecular target for anticancer intervention. Here, we demonstrate the tumor cell-specific apoptogenicity of carbonyl scavengers, which act by covalently trapping RCS, against human (A375, G361, and LOX) and murine (B16) melanoma cell lines. A structure-activity relationship study identified nucleophilic carbonyl scavenger pharmacophores as the functional determinants of apoptogenic antimelanoma activity of structurally diverse agents such as 3,3-dimethyl-D-cysteine and aminoguanidine. Previous work has demonstrated that covalent adduction of protein-arginine residues in the mitochondrial permeability transition (MPT) pore and heat shock protein 27 by intracellular MG produced in tumor cell glycolysis inhibits mitochondrial apoptosis and enhances cancer cell survival. Indeed, in various melanoma cell lines, carbonyl scavenger-induced apoptosis was antagonized by pretreatment with the membrane-permeable RCS phenylglyoxal (PG). Carbonyl scavenger-induced apoptosis was associated with early loss of mitochondrial transmembrane potential, and cyclosporin A antagonized the effects of carbonyl scavengers, suggesting a causative role of MPT pore opening in carbonyl scavenger apoptogenicity. Consistent with RCS inhibition of mitochondrial apoptosis in melanoma cells, staurosporine-induced apoptosis also was suppressed by PG pretreatment. Our results suggest that carbonyl scavengers acting as direct molecular antagonists of RCS are promising apoptogenic prototype agents for antimelanoma drug design.

Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: A potential mechanism for loss of mitochondrial transmembrane potential and apoptosis

This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (DeltaPsim), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C2-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of DeltaPsim. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis.

Functional dissociation of ΔΨm and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Leukotriene B4 receptor inhibitor LY293111 induces cell cycle arrest and apoptosis in human anaplastic large-cell lymphoma cells via JNK phosphorylation

Anaplastic large-cell lymphoma (ALCL) is a heterogeneous lymphoma category in which a subset of cases carry the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. In this study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in ALCL cell lines. LY293111 inhibited proliferation of both ALK(+) and ALK(-) ALCL cell in a dose-dependent fashion and induced complete G(1)-S cell cycle arrest, which was accompanied by upregulation of p27 and downregulation of cyclin E. Pretreatment with LY293111 for 4 h resulted in profound inhibition of serum-induced phosphorylation of extracellular-regulated kinases-1 and 2 and Akt and a concomitant increase in the phosphorylation of the stress-activated kinase c-jun N-terminal kinases (JNK). Simultaneously, LY293111 induced caspase-dependent apoptosis via activation of the intrinsic pathway, including early loss of mitochondrial inner transmembrane potential and the production of reactive oxygen species (ROS), cleavage of caspases-9, -3, poly ADP-ribose polymerase (PARP) and X-linked inhibitor of apoptosis. The phospho-JNK inhibitor SP600125 partially protected Sup-M2 cells from LY293111-induced apoptosis, PARP cleavage and ROS generation, suggesting a role for JNK in LY293111-induced cell death. These results warrant further studies of LY293111 in ALCL.

Acrolein-induced cell death in PC12 cells: Role of mitochondria-mediated oxidative stress

Oxidative stress has been implicated in acrolein cytotoxicity in various cell types, including mammalian spinal cord tissue. In this study we report that acrolein also decreases PC12 cell viability in a reactive oxygen species (ROS)-dependent manner. Specifically, acrolein-induced cell death, mainly necrosis, is accompanied by the accumulation of cellular ROS. Elevating ROS scavengers can alleviate acrolein-induced cell death. Furthermore, we show that exposure to acrolein leads to mitochondrial dysfunction, denoted by the loss of mitochondrial transmembrane potential, reduction of cellular oxygen consumption, and decrease of ATP level. This raises the possibility that the cellular accumulation of ROS could result from the increased production of ROS in the mitochondria of PC12 cells as a result of exposure to acrolein. The acrolein-induced significant decrease of ATP production in mitochondria may also explain why necrosis, not apoptosis, is the dominant type of cell death. In conclusion, our data suggest that one possible mechanism of acrolein-induced cell death could be through mitochondria as its initial target. The subsequent increase of ROS then inflicts cell death and further worsens mitochondria function. Such mechanism may play an important role in CNS trauma and neurodegenerative diseases.

Black tea induces tumor cell apoptosis by Bax translocation, loss in mitochondrial transmembrane potential, cytochrome c release and caspase activation

Recently the anti-cancer role of black tea has gained immense importance. Nevertheless, the signaling pathways underlying black tea-induced tumor cell death are still unknown. Previously we reported that black tea induces Ehrlich's ascites carcinoma (EAC) cell apoptosis by changing the balance between pro-and anti-apoptotic proteins. It is now well accepted that many cell death pathways converge at the mitochondria to decrease mitochondrial transmembrane potential (MTP) thereby releasing apoptogenic proteins and resulting in the activation of effecter caspases responsible for the biochemical and morphological alterations associated with apoptosis. The role of pro-apoptotic protein, Bax, in initiating mitochondrial death cascade has also been established. Here we demonstrate that in culture black tea extract induces EAC apoptosis in a dose-dependent manner--with IC50 at 100 microg/ml. At this dose, intracellular Bax level increases in EAC followed by its translocation from cytosol to mitochondria resulting in loss in MTP. A search for the downstream pathway further reveals that black tea induces mitochondrial cytochrome c release and activates caspases 9 and 3 by 2 pathways, a) independent of and b) dependent on MTP loss. Interestingly, Black tea-induced death signal might probably be amplified through mitochondrial membrane depolarization via a feedback activation loop from caspase 3. All these findings indicate that black tea initiates mitochondrial death cascade in EAC cells and thereby results in EAC apoptosis.

Green Tea Component, Catechin, Induces Apoptosis of Human Malignant B Cells via Production of Reactive Oxygen Species

Green tea polyphenol, (-)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (-)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma. We investigated the effects of (-)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (-)-epigallocatechin-3-gallate-induced apoptosis. (-)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (-)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Deltapsim); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (-)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (-)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (-)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (-)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS. (-)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (-)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Cooperative Cytotoxicity of Proteasome Inhibitors and Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand in Chemoresistant Bcl-2-Overexpressing Cells

Bcl-2 overexpression is frequently detected in lymphoid malignancies, being associated with poor prognosis and reduced response to therapy. Here, we evaluated whether Bcl-2 overexpression affects the cytotoxic activity of proteasome inhibitors taken alone or in association with conventional anticancer drugs or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Jurkat cells engineered to overexpress Bcl-2 were treated with proteasome inhibitors (MG132, epoxomicin, and bortezomib), anticancer drugs (etoposide and doxorubicin), TRAIL, or combinations of these compounds. Cell death and loss of mitochondrial transmembrane potential were detected by flow cytometry. Cytosolic relocalization of cytochrome c and SMAC/Diablo, caspase cleavage, and Bcl-2 and Mcl-1 levels were determined by immunoblotting. Nuclear factor-kappaB inhibition was done by retroviral transduction with a dominant-negative mutant of IkappaBalpha. Bcl-2 overexpression results in significant inhibition of apoptosis in response to proteasome inhibitors, antiblastics, and TRAIL. Addition of TRAIL to proteasome inhibitors results in a synergistic cytotoxic effect in Bcl-2-overexpressing cells, whereas this result is not reproduced by the combination of proteasome inhibitors with antiblastic drugs. Importantly, proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of up-regulated Bcl-2. Bcl-2 cleavage to a fragment with putative proapoptotic activity and elimination of antiapoptotic Mcl-1 may both play a role in proteasome inhibitors-TRAIL cooperation. Conversely, nuclear factor-kappaB inhibition by proteasome inhibitors is per se insufficient to explain the observed synergy. Combined proteasome inhibitors and TRAIL overcome the apoptotic threshold raised by Bcl-2 and may prove useful in the treatment of chemoresistant malignancies with up-regulated Bcl-2.

Synergistic activation of JNK/SAPK induced by TNF-α and IFN-γ: Apoptosis of pancreatic β-cells via the p53 and ROS pathway

IFN-γ and TNF-α are major proinflammatory cytokines implicated in islet β-cell destruction, which results in type-1 diabetes; however, the underlying mechanism is not clear. Using pancreatic β-cell line MIN6N8 cells, co-treatment with TNF-α and IFN-γ, but neither cytokine alone, synergistically induced apoptosis, correlated with the activation of the JNK/SAPK, which resulted in the production of reactive oxidative species (ROS) and loss of mitochondrial transmembrane potential (ΔΨm). Additionally, cells transfected with wild-type JNK1 became more susceptible to apoptosis induced by TNF-α/IFN-γ through ROS production and loss of Δψm, while cascading apoptotic events were prevented in dominant-negative JNK1-transfected or JNK inhibitor SP600125-treated cells. As the antioxidant, N-acetyl-cysteine, failed to completely suppress apoptosis induced by TNF-α/IFN-γ, an additional pathway was considered to be involved. The level of p53 was significantly increased through synergistic activation of JNK by TNF-α/IFN-γ. Furthermore, the synergistic effect of TNF-α/IFN-γ on apoptosis and ROS production was further potentiated by the overexpression of wild-type p53, but not with mutant p53. This synergistic activation of JNK/SAPK by TNF-α/IFN-γ was also induced in insulin-expressing pancreatic islet cells, and increased ROS production and p53 level, which was significantly inhibited by SP600125. Collectively, these data demonstrate that TNF-α/IFN-γ synergistically activates JNK/SAPK, playing an important role in promoting apoptosis of pancreatic β-cell via activation of p53 pathway together with ROS.