cytochrome-c-t and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Topics: Animals; Apoptosis; Calcineurin; Cell Line; Cycloheximide; Cytochromes c; Estradiol; Estrogen Receptor beta; Isoproterenol; Leupeptins; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Pyrimidines; Rats; Signal Transduction

Oxidative stress-induced cell death is common in many neurological diseases. However, the role of poly(ADP-ribose) polymerase-1-induced cell death (parthanatos) has not been fully elucidated. Here, we found that hydrogen peroxide (H2O2) could lead to PARP-1 activation and apoptosis-inducing factor nuclear translocation in a concentration dependent manner. Iduna, as a novel regulator of parthanatos, was also induced by H2O2. Down-regulation of Iduna by genetic ablation promoted H2O2-induced cell damage. Up-regulation of Iduna reduced the loss of mitochondrial potential and ATP and NAD+ production, but did not affect the mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio, and Caspase-9/Caspase-3 activity. In contrast, overexpression of Iduna inhibited activation of PARP-1 and nuclear translocation of AIF. Further study showed that PARP-1 specific inhibitor, DPQ, blocked the protective effect of Iduna against H2O2-induced oxidative stress. Moreover, in the presence of proteasome inhibitor (MG-132) or ubiquitin E1 inhibitor (PYR-41), protective effect of Iduna was significantly weaken. These results indicate that Iduna acts as a potential antioxidant by improving mitochondrial function and inhibiting oxidative stress-induced parthanatos, and these protective effects are dependent on the involvement of ubiquitin-proteasome system.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2-Associated X Protein; Benzoates; Caspase 3; Caspase 8; Cell Line; Cytochromes c; Down-Regulation; Furans; Hydrogen Peroxide; Leupeptins; Membrane Potential, Mitochondrial; Mice; Mitochondria; NAD; Oxidative Stress; Poly(ADP-ribose) Polymerases; Pyrazoles; RNA Interference; RNA, Small Interfering; Ubiquitin-Protein Ligases

A growing body of evidence suggests the inhibition of NFkappaB as a strategy to induce cell death in tumor cells. In this work, we evaluated the effects of the pharmacological NFkappaB inhibitors BAY117082 and MG132 on leukemia cells apoptosis. BAY117082 and MG132 presented potent apoptotic effects compared to inhibitors of MAPKs, EGFR, PI3K/Akt, PKC and PKA signaling pathways. Non-tumor peripheral blood cells were insensitive to BAY117082 and MG132 apoptotic effects. BAY117082 and MG132-induced apoptosis was dependent on their ability to increase ROS as a prelude to mitochondria membrane potential (MMP) depolarization, permeability transition pore opening and cytochrome c release. Antioxidants blocked MG132 and BAY117082 effects on ROS, MMP and cell death. Although apoptotic markers as phosphatidylserine externalization, chromatin condensation and sub-G1 were detected in BAY117082-treated cells, caspases activation did not occur and apoptosis was insensitive to caspase inhibitors, suggesting a caspase-independent mechanism. In contrast, MG132 induced classical apoptosis through ROS-mitochondria and subsequent caspase-9/caspase-3 activation. At sub-apoptotic concentrations, BAY117082 and MG132 arrested cells in G2/M phase of the cell cycle and blocked doxorubicin-induced NFkappaB, which sensitized doxorubicin-resistant cells. Data suggest that the NFkappaB inhibitors MG132 and BAY117082 are potential anti-leukemia agents.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Caspases; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Jurkat Cells; K562 Cells; Leukemia; Leupeptins; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; NF-kappa B; Nitriles; Reactive Oxygen Species; Sulfones; Time Factors; U937 Cells

Proteasomal dysfunction and apoptosis are major hallmarks in the pathophysiology of Parkinson's disease (PD). PARK6 which is caused by mutations in the mitochondrial protein kinase PINK1 is a rare autosomal-recessively inherited disorder mimicking the clinical picture of PD. To investigate the cytoprotective physiological function of PINK1, we used primary fibroblasts from three patients homozygous for G309D-PINK1 as well as SHEP neuroblastoma cells stably overexpressing GFP-tagged wild type (wt) PINK1. Here we demonstrate that overexpression of wt PINK1 inhibits activation of Bax and release of cytochrome c, thereby diminishing caspase 9 processing and effector caspase activity after induction of proteasomal stress with the proteasome inhibitor (PI) MG132 in SHEP cells. Conversely, effector caspase activation induced by PIs, but not by the unrelated apoptotic stimulus staurosporine was potently enhanced in primary fibroblasts from homozygous PARK6 patients in comparison to those of heterozygous carriers or unaffected siblings. SHEP cells overexpressing wt PINK1 showed an elevated expression of the cytoprotective gene parkin, whereas PARK6 fibroblasts displayed significantly decreased expression of parkin in comparison to wild type control cells. Interestingly, overexpressed GFP-PINK1 was exclusively localized in the mitochondria of SHEP cells, but was redistributed to the cytoplasm under conditions of proteasomal stress. Our data indicate that PINK1 plays an important and specific physiological role in protecting cells from proteasomal stress, and suggest that PINK1 might exert its cytoprotective effects upstream of mitochondria engagement.

Topics: Analysis of Variance; Apoptosis; Blotting, Western; Cell Fractionation; Cysteine Proteinase Inhibitors; Cytochromes c; Cytoplasm; Fibroblasts; Flow Cytometry; Humans; Leupeptins; Microscopy, Confocal; Mitochondria; Parkinsonian Disorders; Proteasome Endopeptidase Complex; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Skin

The intracellular processing and the apoptotic activity of conjugates of oligoarginine and cytochrome c (Cyt c) were studied. Disulfide and thioether linked conjugates were prepared by coupling Cyt c to cysteinyl-nonaarginine, C(R)(9), through SPDP and SMPB cross-linkers, respectively. Internalization of the radiolabeled conjugates was measured, and biological activity via induction of apoptosis was determined using the annexin V and the acridine orange assays in HeLa cells. The internalization of both conjugates is increased when compared to that of Cyt c alone. However, the biological activity of the internalized Cyt c, indicated by apoptosis in HeLa cells, was expressed only in the thioether (SMPB) conjugate, but not the disulfide (SPDP) conjugate or free Cyt c. The addition of the proteasomal inhibitor MG132 increased the apoptotic activity of both the disulfide conjugate and free Cyt c, but not the thioether conjugate. Our results suggest that the intracellular cleavage of the linker in cell penetrating peptide conjugates is critical in determining the fate and activity of biologically degradable cargo molecules.

Topics: Acridine Orange; Annexin A5; Apoptosis; Arginine; Biological Transport; Butyrates; Cross-Linking Reagents; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Drug Carriers; HeLa Cells; Humans; Leupeptins; Oligopeptides; Succinimides

In several pathologic conditions, like cardiac ischemia/reperfusion, the sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are hallmarks. The present work aimed to investigate in cardiomyocytes which intracellular signalling pathways are altered by ADR redox ability. To mimic pathologic conditions, freshly isolated calcium tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (X/XO)]. ADR elicited a pro-oxidant signal with generation of reactive species, which was largely magnified by the ROS generating system. However, no change in cardiomyocytes viability was observed. The pro-oxidant signal promoted the translocation to the nucleus of the transcription factors, Heat shock factor-1 (HSF-1) and Nuclear factor-kappaB (NF-kappaB). In addition, proteasome activity was compromised in the experimental groups where the generation of reactive species occurred. The decrease in the proteasome activity of the ADR group resulted from its redox sensitivity, since the activity was recovered by adding the ROS scavenger, tiron. Proteasome inhibition seemed to elicit an increase in HSP70 levels. Furthermore, retention of mitochondrial cytochrome c and inhibition of caspase 3 activity were observed by X/XO incubation in presence or absence of ADR. In conclusion, in spite of all the insults inflicted to the cardiomyocytes, they were capable to activate intracellular responses that enabled their survival. These mechanisms, namely the pathways altered by catecholamine proteasome inhibition, should be further characterized, as they could be of relevance in the ischemia preconditioning and the reperfusion injury.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Active Transport, Cell Nucleus; Animals; Caspase 3; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA-Binding Proteins; Epinephrine; Free Radical Scavengers; Heat Shock Transcription Factors; HSP27 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; In Vitro Techniques; L-Lactate Dehydrogenase; Leupeptins; Male; Mitochondria, Heart; Myocytes, Cardiac; NF-kappa B; Oxidation-Reduction; Oxidative Stress; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Reactive Oxygen Species; Signal Transduction; Time Factors; Transcription Factors; Xanthine; Xanthine Oxidase

Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. MG-132, a proteasome inhibitor, suppresses cell proliferation and induces apoptosis in several PEL cell lines. Treatment of PEL cells with MG-132 results in downregulation of S-phase kinase protein 2 (SKP2) and accumulation of p27Kip1. Furthermore, MG-132 treatment of PEL cells causes Bax conformational changes, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosole. Such cytochrome c release results in sequential activation of caspases and apoptosis, while pretreatment of PEL cells with universal inhibitor of caspases, z-VAD-fmk prevents cell death induced by MG-132. Finally, our data demonstrated in PEL cells that MG-132 downregulates the expression of inhibitor of apoptosis proteins XIAP, cIAP1 and survivin. Altogether, these findings suggest that MG-132 is a potent inducer of apoptosis of PEL cells via downregulation of SKP2 leading to accumulation of p27Kip1, resulting in cell cycle arrest and apoptosis and strongly suggest that targeting the proteasomal pathway may provide a novel therapeutic approach for the treatment of PEL.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Leupeptins; Lymphoma, Primary Effusion; Microtubule-Associated Proteins; S-Phase Kinase-Associated Proteins; Survivin; X-Linked Inhibitor of Apoptosis Protein

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca(2+), calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.

Topics: Apoptosis; Apoptosis Inducing Factor; BH3 Interacting Domain Death Agonist Protein; Calcium Signaling; Calpain; Capsaicin; Caspases; Cell Line; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Enzyme Activation; Epithelial Cells; Heat-Shock Proteins; Humans; Leupeptins; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Protein Transport; Signal Transduction; Stress, Physiological; Time Factors; Transcription Factor CHOP; Tumor Suppressor Protein p53; Tunicamycin

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Cells, Cultured; Cytochromes c; GTP Phosphohydrolases; Humans; Leupeptins; Membrane Proteins; Mice; Mitochondria, Liver; Mitochondrial Membranes; Mutant Proteins; Peptides; Permeability; Protein Structure, Quaternary; Proto-Oncogene Proteins

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.

Topics: Apoptosis; Caspase 3; Cell Line; Cytochromes c; Cytosol; Heat-Shock Response; Hot Temperature; Leupeptins; Mitochondria; Nicotiana; Protease Inhibitors; Proteasome Endopeptidase Complex; Reactive Oxygen Species

Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either p53 or PUMA led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome c release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor-induced apoptosis in colon cancer cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Boronic Acids; Bortezomib; Caspase Inhibitors; Caspases; Cell Nucleus; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytochromes c; Humans; Leupeptins; Mice; Mitochondria; Proteasome Inhibitors; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

Proteasomal dysfunction has been implicated in neurodegenerative disorders and during aging processes. In frontotemporal dementias, corticobasal degeneration, and progressive supranuclear palsy, oligodendrocytes are specifically damaged. Application of proteasomal inhibitors to cultured oligodendrocytes is associated with apoptotic cell death. The present study was undertaken to investigate the death pathway activated in oligodendrocytes by proteasomal inhibition. Our data show that the proteasomal inhibitor MG-132 causes oxidative stress, as indicated by the upregulation of the small heat shock protein heme oxygenase-1 (HO-1) and the appearance of oxidized proteins. Activation of the mitochondrial pathway was involved in the apoptotic process. Mitochondrial membrane potential was disturbed, and cytochrome c was released from the mitochondria. Concomitantly, death-related caspases 3 and 9 were activated and poly(ADP-ribose)-polymerase cleavage occurred. MG-132-induced cell death, DNA-fragmentation, and caspase activation could be prevented by the broad caspase inhibitor zVAD-fmk. In contrast to oligodendrocytes, cultured astrocytes showed resistance to the treatment with proteasomal inhibitors and did not reveal cytotoxic responses. This was also observed in astrocytes differentiated in the presence of dibutyryl cyclic AMP. Hence, individual cells respond differently to proteasomal inhibition and the therapeutic use of proteasomal inhibitors, e.g. for the treatment of cancer or inflammatory diseases, needs to be carefully evaluated.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Astrocytes; Brain; Bucladesine; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Inhibitors; Heme Oxygenase-1; Leupeptins; Mitochondria; Mitochondrial Membranes; Neurodegenerative Diseases; Oligodendroglia; Oxidative Stress; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Wistar

Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds.

Topics: Acetylcysteine; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspases; Cell Survival; Complement Membrane Attack Complex; Complement System Proteins; Cysteine Proteinase Inhibitors; Cytochromes c; Gene Expression; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Leupeptins; Lymphocytes; Mitochondria; Mitochondrial Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA Interference

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 microM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso-N-acetyl-DL-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H2O2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.

Topics: Animals; Antioxidants; Cell Death; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Energy Metabolism; Enzyme Inhibitors; Free Radicals; Glutathione; Leupeptins; Mitochondria; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Oxidative Stress; PC12 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Superoxides

Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Boronic Acids; Bortezomib; Caspases; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytochromes c; Drug Resistance, Neoplasm; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Glycoproteins; Membrane Proteins; Mitochondrial Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.

Topics: Animals; Calcium; Calmodulin; Caspase 3; Caspases; Cell Count; Cell Death; Cell Size; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glutathione; Imidazoles; Leupeptins; Mitochondrial Diseases; PC12 Cells; Rats; Reactive Oxygen Species; Sulfonamides; Trifluoperazine

The effect of the depletion or oxidation of cellular GSH on cytotoxicity of MG132 was assessed. Viability loss and decrease in GSH contents in small cell lung cancer (SCLC) cells treated with MG132 was attenuated by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk). Thiol compounds (N-acetylcysteine and N-(2-mercaptopropionyl)glycine) and free radical scavengers reduced MG132-induced cell death. Antioxidants, including N-acetylcysteine, inhibited the MG132-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c and caspase-3 activation. Depletion of GSH due to buthionine sulfoxime did not affect the cell viability loss, ROS formation and GSH depletion due to MG132 in SCLC cells. A thiol oxidant monochloramine, p-chloromercuribenzoate and N-ethylmaleiamide also did not affect cytotoxicity of MG132. The results suggest that the toxicity of MG132 on SCLC cells is mediated by activation of caspase-8, -9 and -3. Removal of free radicals and recovery of GSH contents may attenuate MG132-induced apoptotic cell death. Nevertheless, depletion or oxidation of cellular GSH may not affect toxicity of MG132.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Survival; Cytochromes c; DNA Damage; Drug Interactions; Enzyme Activation; Glutathione; Humans; Leupeptins; Membrane Potentials; Mitochondria; Oxidation-Reduction; Tumor Cells, Cultured

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53