Activation Of Downstream Caspase-3 Research Articles

Protective Effects of Simvastatin on Endotoxin-Induced Acute Kidney Injury through Activation of Tubular Epithelial Cells' Survival and Hindering Cytochrome C-Mediated Apoptosis.

Increasing evidence suggests that apoptosis of tubular cells and renal inflammation mainly determine the outcome of sepsis-associated acute kidney injury (AKI). The study aim was to investigate the molecular mechanism involved in the renoprotective effects of simvastatin in endotoxin (lipopolysaccharide, LSP)-induced AKI. A sepsis model was established by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment. The severity of the inflammatory injury was expressed as renal damage scores (RDS). Apoptosis of tubular cells was detected by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL assay) (apoptotic DNA fragmentation, expressed as an apoptotic index, AI) and immunohistochemical staining for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL and survivin. We found that endotoxin induced severe renal inflammatory injury (RDS = 3.58 ± 0.50), whereas simvastatin dose-dependently prevented structural changes induced by LPS. Furthermore, simvastatin 40 mg/kg most profoundly attenuated tubular apoptosis, determined as a decrease of cytochrome C, caspase-3 expression, and AIs (p < 0.01 vs. LPS). Conversely, simvastatin induced a significant increase of Bcl-XL and survivin, both in the strong inverse correlations with cleaved caspase-3 and cytochrome C. Our study indicates that simvastatin has cytoprotective effects against LPS-induced tubular apoptosis, seemingly mediated by upregulation of cell-survival molecules, such as Bcl-XL and survivin, and inhibition of the mitochondrial cytochrome C and downstream caspase-3 activation.

Some Ototoxic Drugs Destroy Cochlear Support Cells Before Damaging Sensory Hair Cells

A wide variety of ototoxic drugs are capable of damaging the sensory hair cells in the mammalian cochlea resulting in permanent hearing loss. However, the toxic properties of these drugs suggest that some could potentially damage cochlear support cells as well. To test the hypothesis, we treated postnatal day three rat cochlear cultures with toxic doses of gentamicin, cisplatin, mefloquine, and cadmium. Gentamicin primarily destroyed the hair cells and disrupted the intercellular connection with the surrounding support cells. Gentamicin-induced hair cell death was initiated through the caspase-9 intrinsic apoptotic pathway followed by activation of downstream executioner caspase-3. In contrast, cisplatin, mefloquine, and cadmium initially damaged the support cells and only later damaged the hair cells. Support cell death was initiated through the caspase-8 extrinsic apoptotic pathway followed later by downstream activation of caspase-3. Cisplatin, mefloquine, and cadmium significantly reduced the expression of actin and laminin, in the extracellular matrix, leading to significant disarray of the sensory epithelium.

Treatment with Hydrogen-Rich Saline Delays Disease Progression in a Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and accumulating evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. The aim of this work was to investigate the effect of treatment with hydrogen molecule on the development of disease in mutant SOD1 G93A transgenic mouse model of ALS. Treatment of mutant SOD1 G93A mice with hydrogen-rich saline (HRS, i.p.) significantly delayed disease onset and prolonged survival, and attenuated loss of motor neurons and suppressed microglial and glial activation. Treatment of mutant SOD1 G93A mice with HRS inhibited the release of mitochondrial apoptogenic factors and the subsequent activation of downstream caspase-3. Furthermore, treatment of mutant SOD1 G93A mice with HRS reduced levels of protein carbonyl and 3-nitrotyrosine, and suppressed formation of reactive oxygen species (ROS), peroxynitrite, and malondialdehyde. Treatment of mutant SOD1 G93A mice with HRS preserved mitochondrial function, marked by restored activities of Complex I and IV, reduced mitochondrial ROS formation and enhanced mitochondrial adenosine triphosphate synthesis. In conclusion, hydrogen molecule may be neuroprotective against ALS, possibly through abating oxidative and nitrosative stress and preserving mitochondrial function.

Extract of Saccharina japonica induces apoptosis companied by cell cycle arrest and endoplasmic reticulum stress in SK-Hep1 human hepatocellular carcinoma cells.

Saccharina japonica is a family member of Phaeophyceae (brown macro-alga) and extensively cultivated in China, Japan and Korea. Here, the potential anti-cancer effect of n-hexane fraction of S. japonica was evaluated in SK-Hep1 human hepatocellular carcinoma cells. The N-hexane fraction reduced cell viability and increased the numbers of apoptotic cells in a both dose- and time-dependent manner. Apoptosis was activated by both caspase-dependent and independent pathways. The caspase-dependent cell death pathway is mediated by cell surface death receptors and activated caspase-8 amplified the apoptotic signal either through direct activation of downstream caspase-3 or pro-apoptotic proteins (Bad, Bax and Bak) subsequently leading to the release of cytochrome c. On the other hand, caspase-independent apoptosis appeared mediated by disruption of mitochondrial membrane potential and translocation of AIF to the nucleus where they induced chromatin condensation and/or large-scale DNA fragmentation. In addition, the n-hexane fraction induced endoplasmic reticulum (ER)-stress and cell cycle arrest. The results suggested that potential anti-cancer effects of n-hexane extract from S. japonica on SK-Hep1 cells.

Tributyltin induces oxidative damage, inflammation and apoptosis via disturbance in blood–brain barrier and metal homeostasis in cerebral cortex of rat brain: An in vivo and in vitro study

Tributyltin (TBT), a member of the organotin family, is primarily used for its biocidal activity. Persistent environmental levels of TBT pose threat to the ecosystem. Since neurotoxic influence of TBT remains elusive, we therefore, studied its effect on cerebral cortex of male Wistar rats. A single oral dose of Tributyltin-Chloride (TBTC) (10, 20, 30mg/kg) was administered and the animals were sacrificed on day 3 and day 7. Blood–brain barrier permeability remained disrupted significantly till day 7 with all the doses of TBTC. Pro-oxidant metal levels (Fe, Cu) were increased with a concomitant decrease in Zn. ROS generation was substantially raised resulting in oxidative damage (increased protein carbonylation and lipid peroxidation) with marked decline in tissue antioxidant status (GSH/GSSG levels). Protein expression studies indicated astrocyte activation, upregulation of inflammatory molecules (IL-6, Cox-2 and NF-κB) and simultaneous elevation in the apoptotic index (Bax/Bcl2). Neurodegeneration was evident by reduced neurofilament expression and increased calpain cleaved Tau levels.The in-vitro study demonstrated involvement of calcium and signaling molecules (p38), with downstream activation of caspase-3 and -8, and apoptotic cell death was evident by nuclear fragmentation, DNA laddering and Annexin V binding experiments. Ca2+ inhibitors (BAPTA-AM, EGTA, and RR) and free radical scavengers (NAC and biliprotein [C-PC]) increased cell viability (MTT assay), signifying specific roles of Ca2+ and ROS. Significance of p38 signaling was evaluated on pro-apoptotic proteins by using SB203580, a selective p38 inhibitor. Our data collectively illustrates that TBTC can disrupt BBB, induce oxidative stress, cause cell death and initiate neurodegeneration in rat brain.

Caffeic acid phenethyl ester protects against the dopaminergic neuronal loss induced by 6-hydroxydopamine in rats

Caffeic acid phenethyl ester (CAPE) is a botanical compound abundant in honeybees’ propolis. It has anti-inflammatory, antiviral, antioxidant, immunomodulatory and antitumor properties. Its beneficial effects against neurodegenerative diseases, including Parkinson’s disease, have also been suggested and some mechanisms have been proposed. Mitochondrial damage and oxidative stress are critical events in neurodegeneration. Release of cytochrome c from mitochondria to cytosol and the downstream activation of caspase-3 have been suggested as targets of the protective mechanism of CAPE. Most of the studies addressing the protective effect of CAPE have been performed in cell culture. This is the first study to demonstrate the protective effect of CAPE against the dopaminergic neuronal loss induced by 6-hydroxydopamine (6-OHDA) in rats. It also demonstrates, for the first time, the inhibitory effect of CAPE on mitochondrial permeability transition (MPT), a mediator of neuronal death that triggers cytochrome c release and caspase-3 activation. Scavenging of reactive oxygen species (ROS) and metal chelation was demonstrated in the brain-affected areas of the rats treated with 6-OHDA and CAPE. Additionally, we demonstrated that CAPE does not affect brain mitochondrial function. Based on these findings and on its ability to cross the blood–brain barrier, CAPE is a promising compound to treat Parkinson’s and other neurodegenerative diseases.

Differential role of IK and BK potassium channels as mediators of intrinsic and extrinsic apoptotic cell death

An important event during apoptosis is regulated cell condensation known as apoptotic volume decrease (AVD). Ion channels have emerged as essential regulators of this process mediating the release of K(+) and Cl(-), which together with osmotically obliged water, results in the condensation of cell volume. Using a Grade IV human glioblastoma cell line, we examined the contribution of calcium-activated K(+) channels (K(Ca) channels) to AVD after the addition of either staurosporine (Stsp) or TNF-α-related apoptosis-inducing ligand (TRAIL) to activate the intrinsic or extrinsic pathway of apoptosis, respectively. We show that AVD can be inhibited in both pathways by high extracellular K(+) or the removal of calcium. However, BAPTA-AM was only able to inhibit Stsp-initiated AVD, whereas TRAIL-induced AVD was unaffected. Specific K(Ca) channel inhibitors revealed that Stsp-induced AVD was dependent on K(+) efflux through intermediate-conductance calcium-activated potassium (IK) channels, while TRAIL-induced AVD was mediated by large-conductance calcium-activated potassium (BK) channels. Fura-2 imaging demonstrated that Stsp induced a rapid and modest rise in calcium that was sustained over the course of AVD, while TRAIL produced no detectable rise in global intracellular calcium. Inhibition of IK channels with clotrimazole or 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) blocked downstream caspase-3 activation after Stsp addition, while paxilline, a specific BK channel inhibitor, had no effect. Treatment with ionomycin also induced an IK-dependent cell volume decrease. Together these results show that calcium is both necessary and sufficient to achieve volume decrease and that the two major pathways of apoptosis use unique calcium signaling to efflux K(+) through different K(Ca) channels.

The yolk cell of the zebrafish blastula harbors functional apoptosis machinery.

We recently described the implication of the Bcl-2 related antiapoptotic Nrz protein during early zebrafish development. Nrz knock-down induces calcium-dependent cytoskeleton remodeling leading to margin constriction and premature embryo lethality. In the YSL, nrz knock-down embryos exhibit some typical features of apoptosis such as mitochondrial transmembrane potential loss and cytochrome c release. However, downstream caspase-3 activation has not been detected so far. Here, we report that the YSL contains fully functional apoptotic machinery that can activate caspase-3 following zBax ectopic expression. Furthermore, we present evidence that caspase-3 activation is actually detectable in nrz knock-down embryos when premature margin constriction is prevented.

The yolk cell of the zebrafish blastula harbors functional apoptosis machinery

We recently described the implication of the Bcl-2 related anti-apoptotic Nrz protein during early zebrafish development. Nrz knock-down induces calcium-dependent cytoskeleton remodeling leading to margin constriction and premature embryo lethality. In the YSL, nrz knock-down embryos exhibit some typical features of apoptosis such as mitochondrial transmembrane potential loss and cytochrome c release. However, downstream caspase-3 activation has not been detected so far. Here, we report that the YSL contains fully functional apoptotic machinery that can activate caspase-3 following zBax ectopic expression. Furthermore, we present evidence that caspase-3 activation is actually detectable in nrz knock-down embryos when premature margin constriction is prevented.

A Novel cyano derivative of 11‐Keto‐β‐Boswellic acid causes apoptotic death by disrupting PI3K/AKT/Hsp‐90 cascade, mitochondrial integrity, and other cell survival signaling events in HL‐60 cells

Intervention of apoptosis is a promising strategy for discovery of novel anti-cancer therapeutics. In this study, we examined the ability of a novel cyano derivative of 11-keto-β-boswellic acid, that is, butyl 2-cyano-3,11-dioxours-1,12-dien-24-oate (BCDD) to induce apoptosis in cancer cells. BCDD inhibited cell proliferation with 48 h IC(50) of 0.67 µM in HL-60, 1 µM in Molt4, and 1.5 µM in THP1 cells. The mechanism of cell death was investigated in HL-60 cells where it caused apoptosis by acting against several potential apoptosis suppressive targets. It inhibited phosphatidylinositol-3-kinase (PI3K)/AKT activity, NF-κB, Hsp-90, and survivin which may enhance the sensitivity of cells to apoptosis. Also, BCDD decreased the activity of Bid and Bax in cytosol, caused ΔΨ(mt) loss, releasing pro-apoptotic cytochrome c, SMAC/DIABLO leading to caspase-9-mediated down stream activation of caspase-3, ICAD, and PARP1 cleavage. Translocation of apoptotis-inducing factor (AIF) from mitochondria to the nucleus indicated some caspases-independent apoptosis. Though it upregulated DR-5 and caspase-8, the caspase inhibitor yet had no effect on apoptosis as against 75% inhibition by caspase-9 inhibitor. Attempts were made to examine any acclaimed role of AIF in the activation of caspase-8 using siRNA where it had no effect on caspase-8 activity while the Bax-siRNA inhibited caspase-3 activation suggesting predominance of intrinsic signaling. Our studies thus demonstrated that BCDD exerts multi-focal action in cancer cells while it required 10-fold higher the concentration to produce cytotoxicity in normal human PBMC and gingival cell line, and therefore, may find usefulness in the management of human leukemia.

Loss of Caspase-2-dependent Apoptosis Induces Autophagy after Mitochondrial Oxidative Stress in Primary Cultures of Young Adult Cortical Neurons

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.

Protein tyrosine phosphatase alpha regulates cell detachment and cell death profiles induced by nitric oxide donors in the A431 human carcinoma cell line

We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27BPTPα) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27BPTPα) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27BPTPα) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.

Impact of Polyunsaturated Fatty Acids on Oxidized Low Density Lipoprotein-Induced U937 Cell Apoptosis

Dietary supplements in polyunsaturated fatty acids (PUFA), particularly omega-3, are well known for their beneficial effects in preventing cardiovascular diseases (CVD). The aim of this study was to determine the role of PUFA on the modulation of apoptosis induced by hypochlorous acidoxidized LDL (HOCl-oxLDL) in U937 cells. We tested the effect of monocyte cell line U937 supplementation with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA) or oleic acid (OA) on the modulation of HOCl-oxLDL-induced apoptosis. First, we showed the incorporation of fatty acids in the cellular membrane in U937 cells. Then, we showed that both EPA and ARA exerted a pro-apoptotic effect through the intrinsic mitochondrial apoptotic pathway including the dissipation of mitochondrial membrane potential followed by cardiolipin depletion, the downstream activation of caspase-3 and the increase in DNA fragmentation. The pro-apoptotic effect of EPA or ARA was completely blocked in U937/Bcl-2 cells. A new mechanism of dietary supplements in PUFA with likely consequences in apoptosis could be suggested through the mitochondrial pathway in monocytes.

Some CLL Cells Bind Myosin-Exposed Apoptotic Cells. Exposure of Cytoplasmic Myosin Results From Transfer of Caspase-3 Dependent Cleavage Products to the Outer Cell Membrane

AbstractAbstract 3900Many monoclonal antibodies (mAbs) produced by B-cell chronic lymphocytic leukemia (CLL) cells bind a subset of apoptotic cells that expose intracellular myosin on the cell surface. CLL patients with mAbs that bind these myosin-exposed apoptotic cells (MEACs) have shorter overall survival. Thus, understanding the mechanism of formation of MEACs and how CLL cells interact with MEACs may help elucidate the pathogenesis of CLL.To test if formation of MEACs is part of general apoptotic mechanisms, apoptosis was induced in Jurkat T cells by either the intrinsic or extrinsic pathways. The intrinsic pathway was either achieved spontaneously by culturing at high cell density or induced by camptothecin (CPT) treatment. The extrinsic pathway was induced by Fas ligand (FasL) or anti-Fas mAb treatment. Apoptosis and myosin exposition were analyzed by flow cytometry. All four methods of apoptosis induction produced MEACs after prolonged incubation as detailed below. CPT, FasL or anti-Fas mAb incubation for 4 hrs induced significant apoptosis (43-58%) with a detectable fraction of MEACs (9-12%). After incubation for 16 hrs or longer, the majority of apoptotic cells were MEACs (61-89%). Similarly, spontaneous apoptosis produced more MEACs after longer incubation (20% on day 1 versus 59–69% on days 2–4). Both early apoptotic cells, which flip phosphatidylserine (PS) from the inner to outer membrane surface yet retain membrane integrity (AnnexinV+, 7-actinomycin D (7AAD)-), and late apoptotic cells, which become membrane permeable (AnnexinV+, 7AAD+), demonstrate a subpopulation of MEACs that increases with longer incubation times. In contrast, MEACs are not detectable in non-apoptotic cells (AnnexinV-, 7AAD-).Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator may be involved. Caspase-3 activation mediates apoptotic PS exposure and membrane permeability. Therefore, we tested a caspase-3 inhibitor, Z-DEVD-FMK, and found that it significantly reduced both apoptosis and MEAC formation. For example, Z-DEVD-FMK reduced FasL induced apoptosis and MEAC formation from 74 to 14% and from 57 to 10%, respectively. In contrast, caspase-1 inhibitor, Z-YVAD-FMK, had no effect. To test if intracellular myosin is transferred from the cytoplasm to the cell membrane surface during apoptosis, cytoplasmic and membrane protein extracts were prepared, isolated by ultracentrifugation, and blotted with anti-myosin antibody. Two protein bands of the size expected for caspase-3 cleaved myosin (149 and 94 kDa) appeared in membrane extracts of apoptotic cells, but not of live cells. A protein band of the size expected for full-length myosin (250 kDa) predominated in cytoplasmic extracts of live cells. Furthermore, Z-DEVD-FMK inhibited the formation of the 149 and 94 kDa myosin bands in membrane extracts as well as the formation of caspase-3 dependant PARP cleavage products; the same treatment did not alter CD3 membrane protein or GAPDH cytoplasmic protein levels.Taken together, these results suggest that both intrinsic and extrinsic apoptotic pathways produce MEACs at a later stage in apoptosis that involves the common downstream caspase-3 activation. In turn, myosin fragmentation occurs with subsequent exposure to the cell membrane, where the myosin fragments can serve as a potential neoantigen that may be recognized by some CLL mAbs. Because the mAbs we have used in these analyses were originally integral components of CLL surface membranes, we hypothesized that CLL cells could bind MEACs. Indeed, CLL cells binding to MEACs were visualized by confocal microscopy. To determine the functional consequences of such binding, analyses of the effects of MEAC binding on CLL cell survival in vitro are underway. Disclosures:No relevant conflicts of interest to declare.

Phosphomimetic Substitution of CytochromecTyrosine 48 Decreases Respiration and Binding to Cardiolipin and Abolishes Ability to Trigger Downstream Caspase Activation

Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

Mitochondrial autophagy protects against heat shock-induced apoptosis through reducing cytosolic cytochrome c release and downstream caspase-3 activation

Autophagy is an evolutionarily conserved process for bulk degradation of cytoplasmic components, including large molecules and organelles. It can either help to enhance or to resist apoptosis, depending on the circumstances. The mechanism of how autophagy impacts apoptosis and the subsequent cellular events upon heat shock remains unclear. In this study, we demonstrate for the first time that mitochondrial membrane permeability transition (MPT)-sensitive mitochondrial autophagy can protect against heat-induced apoptosis through reduction of cytosolic cytochrome c release and downstream caspase-3 activation. With confocal microscopy, it was revealed that as autophagosomes increased, mitochondrial content was mass decreased after heat shock. Detailed analysis shows that a single swelling mitochondrion could be entrapped into autophagosome. The depolarization of mitochondria preceded the mitochondrial loss, and both could be abolished by MPT inhibitor cyclosporine (CsA). In addition, along with the decrease of mitochondrial content, the level of total cytochrome c was also reduced, resulting in a reduction of its release to cytoplasm. When heat shock was combined with 3-methyladenine (3-MA), an inhibitor of autophagy, the mitochondrial loss and the reduction of total cytochrome c were both inhibited, and then caspase-3 activation and cell apoptosis were increased. Thus, it is reasonable to believe that, heat shock-induced cellular events can be modulated by controlling autophagy, and this may represent a novel approach to enhance the efficacy of hyperthermia.

Abstract B25: Apoptotic signaling in AML cells after therapy with the monoclonal antibody-therapeutic gemtuzumab ozogamicin

Abstract The aim of this study was to identify essential signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO). New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1–2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to a DNA damaging toxin, calicheamicin. GO enters the cell after binding to the CD33 antigen, preferentially expressed on myeloid cells, and cause DNA double strand breaks (DSB). We have previously demonstrated that activation of Bak and Bax is critical for GO-induced apoptotic signaling, followed by downstream mitochondrial depolarization and caspase-3 activation. As a continuation, we have now examined potential proapoptotic signaling events induced by GO upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. At the same time, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase 2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells. In conclusion, we demonstrate that GO, at clinically applicable concentrations (100–1000ng/ml), induce pro-apoptotic signaling which involve activation of caspase-2, and cleavage of the BH3-only protein Bid to the truncated, active protein tBid. Blocking caspase-2 activity reduced caspase-3 activation to only half. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B25.

Monte Carlo Simulation of Cell Death Signaling Predicts Large Cell-to-Cell Stochastic Fluctuations through the Type 2 Pathway of Apoptosis

Apoptosis, or genetically programmed cell death, is a crucial cellular process that maintains the balance between life and death in cells. The precise molecular mechanism of apoptosis signaling and the manner in which type 1 and type 2 pathways of the apoptosis signaling network are differentially activated under distinct apoptotic stimuli is poorly understood. Based on Monte Carlo stochastic simulations, we show that the type 1 pathway becomes activated under strong apoptotic stimuli, whereas the type 2 mitochondrial pathway dominates apoptotic signaling in response to a weak death signal. Our results also show signaling in the type 2 pathway is stochastic; the population average over many cells does not capture the cell-to-cell fluctuations in the time course (∼1–10h) of downstream caspase-3 activation. On the contrary, the probability distribution of caspase-3 activation for the mitochondrial pathway shows a distinct bimodal behavior that can be used to characterize the stochastic signaling in type 2 apoptosis and other similar complex signaling processes. Interestingly, such stochastic fluctuations in apoptosis signaling occur even in the presence of large numbers of signaling molecules.

The early phase of programmed cell death in Caco‐2 intestinal cells exposed to PTH

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. In a previous work we found that PTH promotes the apoptosis of human Caco-2 intestinal cells. In the current study, we demonstrate, for the first time, that stimulation of Caco-2 cells with PTH (10(-8) M) results in the dephosphorylation and translocation of pro-apoptotic protein Bad from the cytosol to mitochondria and release of cytochrome c and Smac/Diablo. The hormone also triggers mitochondria cellular distribution to the perinuclear region, morphological features consistent with apoptosis. PTH increases the enzymatic activity of caspase-3 (48 h) that is also evidenced from the appearance of its cleaved fragments in western blot experiments. Moreover, active caspase-3 is present in nucleus after PTH treatment. In addition, a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP), is degraded by 48 h of PTH treatment. Taken together, our results suggest that, in Caco-2 cells, the induction of apoptosis in response to PTH is mediated by translocation of mitochondria to the perinuclear region, dephosphorylation of Akt, dephosphorylation of Bad and its movement to the mitochondria and subsequent release of cytochrome c and Smac/Diablo which result in activation of downstream caspase-3.

Enhanced apoptosis following treatment with TRA-8 anti-human DR5 monoclonal antibody and overexpression of exogenous Bax in human glioma cells.

Specific activation of apoptosis in tumor cells offers a promising approach for cancer therapy. Induction of apoptosis leads to activation of specific proteases. Two major pathways for caspase activation in mammalian cells have been described. One apoptotic pathway involves members of the tumor necrosis factor family of cytokine receptors (eg death receptor 5 (DR5)). The other pathway is controlled by the Bcl-2 family of proteins. The purpose of this study was to investigate whether increased apoptosis occurs in human glioma cells following infection with a recombinant adenoviral vector encoding the human Bax gene under the control of human vascular endothelial growth factor (VEGF) promoter element (AdVEGFBax) in combination with an anti-human DR5 monoclonal antibody (TRA-8). Specific overexpression of exogenous Bax protein induced apoptosis and cell death in glioma cell lines, through activation of both caspase-8 and -9, leading to activation of downstream caspase-3. The relative sensitivity to AdVEGFBax for the glioma cell lines was U251MG>U373MG>U87MG>D54MG. The recently characterized TRA-8 monoclonal antibody induces apoptosis of most TRAIL-sensitive tumor cells by specific binding to DR5 receptors on the cellular membrane. TRA-8 induced rapid apoptosis and cell death in glioma cells, but did not demonstrate detectable cytotoxicity of primary normal human astrocytes. The efficiency of TRA-8-induced apoptosis was variable in different glioma cell lines. The relative sensitivity to TRA-8 was U373MG>U87MG>U251MG>D54MG. The combination of TRA-8 treatment and overexpression of Bax overcame TRA-8 resistance of glioma cells in vitro. Cell viability of U251MG cells was 71.1% for TRA-8 (100 ng/ml) alone, 75.9% for AdVEGFBax (5 MOI) alone and 41.1% for their combination as measured by MTS assay. Similar enhanced apoptosis results were obtained for the other glioma cell lines. In vivo studies demonstrated that the combined treatment significantly (P<0.05) suppressed the growth of U251MG xenografts and produced 60% complete tumor regressions without recurrence. These data suggest that the combination of TRA-8 treatment with specific overexpression of Bax using AdVEGFBax may be an effective approach for the treatment of human malignant gliomas.