benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Neuroblastoma

Stenodactylin is a highly toxic plant lectin purified from the caudex of Adenia stenodactyla, with molecular structure, intracellular routing and enzyme activity similar to those of ricin, a well-known type 2 ribosome-inactivating protein. However, in contrast with ricin, stenodactylin is retrogradely transported not only in peripheral nerves but also in the central nervous system.. Stenodactylin properties make it a potential candidate for application in neurobiology and in experimental therapies against cancer. Thus, it is necessary to better clarify the toxic activity of this compound.. We investigated the mechanism of stenodactylin-induced cell death in the neuroblastoma-derived cell line, NB100, evaluating the implications of different death pathways and the involvement of oxidative stress.. Stenodactylin cytotoxicity was determined by evaluating protein synthesis and other viability parameters. Cell death pathways and oxidative stress were analysed through flow cytometry and microscopy. Inhibitors of apoptosis, oxidative stress and necroptosis were tested to evaluate their protective effect against stenodactylin cytotoxicity.. Stenodactylin efficiently blocked protein synthesis and reduced the viability of neuroblastoma cells at an extremely low concentration and over a short time (1 pM, 24 h). Stenodactylin induced the strong and rapid activation of apoptosis and the production of free radicals. Here, for the first time, a complete and long lasting protection from the lethal effect induced by a toxic type 2 ribosome-inactivating protein has been obtained by combining the caspase inhibitor Z-VAD-fmk, to either the hydrogen peroxide scavenger catalase or the necroptotic inhibitor necrostatin-1.. In respect to stenodactylin cytotoxicity, our results: (i) confirm the high toxicity to nervous cells, (ii) indicate that multiple cell death pathways can be induced, (iii) show that apoptosis is the main death pathway, (iv) demonstrate the involvement of necroptosis and (v) oxidative stress.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Catalase; Cell Death; Cell Line, Tumor; Humans; Imidazoles; Indoles; Lectins; N-Glycosyl Hydrolases; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species

To explore the role of MAPK signaling pathway in apoptosis and necroptosis induced by aluminum in SH-SY5Y cells.. To imitate neural cell death induced by aluminium, AlCl3 x 6H2O (4 mmol/L) was used to treat SH-SY5Y cells. Necrostatin-1 (Nec-1,60 μmol/L), the specific inhibitor for necroptosis, and zVAD-fmk (20 μmol/L), the specific inhibitor for apoptosis, were added into cultures for inhibiting the occurrence of necroptosis and apoptosis. CCK-8 was performed to measure cell viability, flow cytometry was used to test the difference of apoptosis rate and necrosis rate between groups, and western-blot was used to detect the change of MAPK protein.. Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, cell viabiligy of Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group decreaced (P < 0.05). Compared with Al(3+) exposed group, cell viability of Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased (P < 0.05). Necrotic rate and apoptotic rate in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group obviously increased compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group (P < 0.05). Compared with Al(3+) exposed group, necrotic and apaptotic rate of Al(3+) plus zVAD-fmk group and Al(3+) plus Nec-1 group were statistically significant decreased (P < 0.05). Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, expression of p-p38 in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased obviously (P < 0.05), and expression of p-ERK decreased significantly (P < 0.05). Compared with Al(3+) exposed group, expression of p-p38 decreased (P < 0.05), but p-ERK increased in Al(3+) plus Nec-1 group (P < 0.05).. The ERK and p38 MAPK signaling pathways are involved in aluminum-induced necroptosis in SH-SY5Y cells, but only ERK signaling pathway is involved in aluminum-induced apoptosis, and JNK signaling pathway is not involved in aluminum-induced cell death.

Topics: Aluminum; Aluminum Chloride; Aluminum Compounds; Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Cell Death; Cell Line, Tumor; Cell Survival; Chlorides; Humans; Imidazoles; Indoles; Mitogen-Activated Protein Kinase Kinases; Neuroblastoma; Signal Transduction

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Topics: Adamantane; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Bromodeoxyuridine; Caspase 3; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cinnamates; DNA Damage; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Models, Biological; Neuroblastoma; Propidium; Receptors, Death Domain; Tretinoin

The neurotoxins paraquat (PQ) and dopamine (DA or 6-OHDA) cause apoptosis of dopaminergic neurons in the substantia nigra pars compacta (SNpc), reproducing an important pathological feature of Parkinson's disease (PD). Secretogranin III (SCG3), a member of the multifunctional granin family, plays a key role in neurotransmitter storage and transport and in secretory granule biogenesis, which involves the uptake of exogenous toxins and endogenous "toxins" in neuroendocrine cells. However, the molecular mechanisms of neurotoxin-induced apoptosis in dopaminergic neurons and the role of SCG3-associated signaling pathways in neuroendocrine regulation are unclear. To address this, we used PQ- and DA-induced apoptosis in SH-SY5Y human dopaminergic cells as an in vitro model to investigate the association between SCG3 expression level and apoptosis. SCG3 was highly expressed in SH-SY5Y cells, and SCG3 mRNA and protein levels were dramatically decreased after PQ treatment. Apoptosis induced by PQ is associated with caspase activation and decreased SCG3 expression, and restoration of SCG3 expression is observed after treatment with caspase inhibitors. Overexpressed SCG3 in nonneuronal cells and endogenous SCG3 in SH-SY5Y cells are cleaved into specific fragments by recombinant caspase-3 and -7, but the fragments were not detected in PQ-treated SH-SY5Y cells. Therefore, SCG3 may be involved in apoptosis signal transduction as a caspase substrate, leading to loss of its original biological functions. In addition, SCG3 may be a pivotal component of the neuroendocrine pathway and play an important role in neuronal communication and neurotransmitter release, possibly representing a new potential target in the course of PD pathogenesis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 7; Caspase Inhibitors; Cell Line, Tumor; Chromogranins; Dopamine; Dopaminergic Neurons; Down-Regulation; HEK293 Cells; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurotoxins; Oxidopamine; Paraquat; Recombinant Fusion Proteins; RNA, Messenger; Substrate Specificity

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 μM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 μM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Calpain; Camptothecin; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Dipeptides; DNA Damage; DNA-Binding Proteins; Humans; Morpholines; Neuroblastoma; Protein Serine-Threonine Kinases; Purines; Pyrones; Roscovitine; Tumor Suppressor Proteins

Alpha-synuclein (ASYN) is central in Parkinson's disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson's disease. To elucidate the mechanism underlying wild type (WT) ASYN-mediated neurotoxicity, we have generated a novel Tet-Off SHSY-5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid-differentiated SHSY-5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non-apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo-inositol, an oligomer-stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non-apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN-induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.

Topics: alpha-Synuclein; Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Inositol; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Staurosporine; Time Factors; Transfection; Tretinoin

The aim of the present study was to determine whether phenoxazines such as 2-amino-4,4-alpha-dihydro-4alpha-phenoxazine-3-one (Phx-1) and 2-aminophenoxazine-3-one (Phx-3) may suppress the proliferation of human neuroblastoma cell line, NB-1 that is refractory to chemotherapeutic agents, inducing apoptosis through the activation of caspase pathway or not. Phx-1 and Phx-3 suppressed the proliferation of NB-1 cells extensively dependent on dose and time. The IC50 of Phx-1 and Phx-3 was about 20 microM and 0.5 microM, respectively, when the cells were treated with Phx-1 or Phx-3 for 72 h. Phx-1 and Phx-3 caused the mixed types of cell death-apoptosis and necrosis-in NB-1 cells, which was detected by flow cytometry. The induction of apoptosis/necrosis caused by these phenoxazines seemed to be correlated dominantly with the caspase independent pathway, because the increased activity of effector caspase 3/7 in NB-1 cells caused by 50 microM Phx-1 or 20 microM Phx-3 was completely cancelled by the addition of z-VAD-fmk, a pan-caspase inhibitor, but such phenoxazines-suppressed viability of NB-1 cells was not recovered to normal levels by this inhibitor. The results of this study demonstrate that Phx-1 and Phx-3 have antitumor activity against the neuroblastoma cell line, NB-1, though the IC50 was extremely low for Phx-3, inducing the mixed types of cell death, apoptosis and necrosis, caspase-independently.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Necrosis; Neuroblastoma; Oxazines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted great attention as a promising anti-cancer reagent. Recombinant soluble TRAIL (rsTRAIL) derivatives induce apoptosis in various cancer cells, but not in most normal cells. However, a number of cancerous cell types are resistant to TRAIL cytotoxicity, limiting its application in cancer therapy. In the present study, we report that actinomycin D (Act D) pretreatment increases apoptosis in human neuroblastoma SH-SY5Y cells treated with rsTRAIL. Both caspase-9 and caspase-7, but not caspase-3, were activated during the apoptosis process. z-VAD-fmk, a pan-caspase inhibitor, only partially suppressed apoptosis of the cells, suggesting that the Act D-enhanced apoptosis of SH-SY5Y occurred via caspase-dependent and -independent manners. In cells pretreated with Act D, we found decreased mitochondrial transmembrane potential, high levels of reactive oxygen species (ROS), and up-regulated apoptotic-inducing factor (AIF). Cell death was blocked in cells stably transfected with AIF-siRNA plasmid. Taken together, these data indicate that Act D sensitizes SH-SY5Y cells to rsTRAIL-induced apoptosis via caspase activation, impairment of the mitochondrial membrane, release of ROS, and up-regulation of AIF expression. This study provides a novel strategy for the therapy of malignant neuroblastoma resistant to rsTRAIL cytotoxicity.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Line, Tumor; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Protein Synthesis Inhibitors; Reactive Oxygen Species; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Transfection

In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.

Topics: Amino Acid Chloromethyl Ketones; Calcium; Calpain; Caspases; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Gliotoxin; Humans; Immunosuppressive Agents; Neurites; Neuroblastoma; Spectrin

Early onset familial Alzheimer's disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-induced apoptosis by interfering with E2F-1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild-type APP, APP with the Swedish mutation (APPswe), or double-mutated human APP and PS1 (APPswe/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 but was partially reversed by z-VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl-2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild-type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3'-OH kinase pathway reduced TUDCA protection against p53-induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53-induced apoptosis and modulates expression of Bcl-2 family.

Topics: Alzheimer Disease; Amino Acid Chloromethyl Ketones; Amyloid beta-Protein Precursor; Animals; Antiviral Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; In Situ Nick-End Labeling; Membrane Proteins; Mice; Mutation; Neuroblastoma; Presenilin-1; Presenilin-2; Proto-Oncogene Proteins c-bcl-2; Taurochenodeoxycholic Acid; Transfection; Tumor Suppressor Protein p53

Neuroblastoma is a pediatric tumor accounting for 15% of childhood cancer deaths and has a poor prognosis in children >1 year of age. We investigated the ability of apigenin, a nonmutagenic dietary flavonoid that has been shown to have antitumor effects in various tumor cell lines, to inhibit growth and induce apoptosis of the human neuroblastoma cell lines NUB-7, LAN-5, and SK-N-BE(2). Apigenin inhibited colony-forming ability and survival, and induced apoptosis of NUB-7 and LAN-5 cells. The presence of the C2-C3 double bond and the 4'-OH group on the flavonoid structure correlated with the growth-inhibitory potential of apigenin. Furthermore, apigenin inhibited NUB-7 xenograft tumor growth in anonobese diabetic/severe combined immunodeficiency mouse model, likely by inducing apoptosis. Apigenin did not inhibit survival of primary sympathetic neurons, suggesting that it is not toxic to nontransformed cells. The mechanism of action of apigenin seems to involve p53, as it increased the levels of p53 and the p53-induced gene products p21WAF1/CIP1 and Bax. Furthermore, apigenin (15-60 micromol/L) induced cell death and apoptosis of neuroblastoma cells expressing wild-type but not mutant p53. Apigenin increased caspase-3 activity and PARP cleavage, and Z-VAD-FMK, a broad-spectrum caspase-3 inhibitor, rescued NUB-7 cells from apigenin-mediated apoptosis indicating that apigenin induced apoptosis in acaspase-dependent manner. Overexpression of Bcl-X(L) rescued NUB-7 from apigenin-induced cell death, suggesting that Bax activity is important for the action of apigenin. Apigenin is thus a candidate therapeutic for neuroblastoma that likely acts by regulating a p53-Bax-caspase-3 apoptotic pathway.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apigenin; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Transplantation, Heterologous; Tumor Suppressor Protein p53

Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK inhibitory drugs. Exp. Cell Res. 295 9-24.], which depends on either Caspase 8 or Caspase 10 activation. However, neither Caspase 8 nor Caspase 10 is expressed in SH-SY5Y cells because of gene silencing. Upon Olomoucine or Roscovitine treatment, no re-expression of Caspase 8 or Caspase 10 was found. Therefore, in SH-SY5Y cells, this type of drugs is not triggering a canonical, Caspase 8/10-mediated, extrinsic apoptotic pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 10; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cyclin-Dependent Kinases; Enzyme Inhibitors; Humans; Jurkat Cells; Kinetin; Neuroblastoma; Purines; Roscovitine; Time Factors

Neuroblastoma is a highly heterogeneous tumor of young children. Although many advances have been made towards understanding the molecular mechanisms dictating the phenotypic heterogeneity, the prognosis of children with neuroblastoma, particularly of progressively growing variants, has remained dire. About 10% of neuroblastomas regress spontaneously, probably by apoptosis, while another 20% have amplified the MYCN gene resulting in a poor prognosis. In pursuit of identifying cell death-associated genes in neuroblastoma, we encountered the SCA2 gene, coding for ataxin-2, as an important player. Here, we report that enforced expression of wild-type ataxin-2, but not of mutant ataxin-2, sensitizes neuroblastoma cells for apoptosis. In line with this, higher levels of ataxin-2 were detected in apoptotic cells compared to nonapoptotic cells. Neuroblastoma tumors with amplified MYCN contain significantly less ataxin-2 protein than tumors without amplified MYCN. Collectively, our data suggest that ataxin-2 has an important role in regulating the susceptibility of neuroblastoma cells to apoptotic stimuli in vitro and in vivo.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Ataxins; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mutation; N-Myc Proto-Oncogene Protein; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Peptides; Proteins; Tumor Cells, Cultured; Up-Regulation

The inhibition of apoptosis is generally believed to be a major determinant of resistance to chemotherapy. However, recent findings have shown that caspase inhibitors do not protect cancer cells from death by cytotoxic agents, but may switch drug-induced apoptosis to an alternative 'default death'. The primary goals of this study were to determine the major characteristics of the 'default death' and the mechanism by which this switch is activated. For this purpose, we first investigated putative cell death modes induced by doxorubicin. Molecular markers associated with these death modes were utilized to identify the default death resulting from the inhibition of apoptosis. Our findings demonstrated that doxorubicin induced at least three distinct types of cell death, senescence, apoptosis and a type of necrosis, which were concentration dependent. Specific molecular markers such as p21/WAF1, activated caspase-3 and activated Akt were associated with these death modes. The pan-caspase inhibitor (Q-VD-OPH) greatly reduced doxorubicin-induced caspase-3 activation but did not protect cells against drug toxicity. The combination of doxorubicin and Q-VD-OPH caused an increased expression of p21/WAF1 and senescence -associated -beta-galactosidase activity, but did not alter Akt activation. Collectively, these findings suggest that the inhibition of apoptosis may lead to an increased expression of cell cycle inhibitors and cellular senescence.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Cell Death; Cellular Senescence; Cysteine Proteinase Inhibitors; Doxorubicin; Humans; Neuroblastoma; Tumor Cells, Cultured

Prion diseases are characterized by accumulation of misfolded prion protein (PrP(Sc)), and neuronal death by apoptosis. Here we show that nanomolar concentrations of purified PrP(Sc) from mouse scrapie brain induce apoptosis of N2A neuroblastoma cells. PrP(Sc) toxicity was associated with an increase of intracellular calcium released from endoplasmic reticulum (ER) and up-regulation of several ER chaperones. Caspase-12 activation was detected in cells treated with PrP(Sc), and cellular death was inhibited by overexpression of a catalytic mutant of caspase-12 or an ER-targeted Bcl-2 chimeric protein. Scrapie-infected N2A cells were more susceptible to ER-stress and to PrP(Sc) toxicity than non-infected cells. In scrapie-infected mice a correlation between caspase-12 activation and neuronal loss was observed in histological and biochemical analyses of different brain areas. The extent of prion replication was closely correlated with the up-regulation of ER-stress chaperone proteins. Similar results were observed in humans affected with sporadic and variant Creutzfeldt-Jakob disease, implicating for the first time the caspase-12 dependent pathway in a neurodegenerative disease in vivo, and thus offering novel potential targets for the treatment of prion disorders.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Caspase 12; Caspase Inhibitors; Caspases; Cell Survival; Cerebral Cortex; Creutzfeldt-Jakob Syndrome; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Mice; Mice, Inbred C57BL; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; PrPSc Proteins; Recombinant Proteins; Scrapie; Stress, Mechanical; Transfection; Tumor Cells, Cultured

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Caspase Inhibitors; Cell Differentiation; Enzyme Inhibitors; Humans; Neurites; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Tumor Cells, Cultured

Prion diseases are neurodegenerative pathologies characterized by the accumulation in the brain of a protease-resistant form of the prion protein (PrP(c)), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) maintains many PrP(Sc) characteristics. We investigated the intracellular signaling responsible for the PrP106-126-dependent cell death of SH-SY5Y, a cell line derived from a human neuroblastoma. In this cell line, PrP106-126 induced apoptotic cell death and caused activation of caspase-3, although the blockade of this enzyme did not inhibit cell death. The p38 MAP kinase blockers, SB203580 and PD169316, prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. Taken together, our data suggest that the p38 MAP kinase pathway can mediate the SH-SY5Y cell death induced by PrP106-126.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Mitogen-Activated Protein Kinases; Neuroblastoma; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Peptide Fragments; Prion Diseases; Prions; Pyridines; Tumor Cells, Cultured

Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Caspases; Cathepsin B; Cathepsin D; Cell Survival; Chromatin; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Leucine; Lysosomes; Neuroblastoma; Pepstatins; Tumor Cells, Cultured

Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aspartic Acid; Caspase Inhibitors; Caspases; Cytoplasm; Dose-Response Relationship, Drug; Enzyme Precursors; HeLa Cells; Humans; Kinetics; Neuroblastoma; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Research Design; Staurosporine; Tumor Cells, Cultured

We have performed differential screening to identify genes participating in NMDA-induced neuronal death. The gas1 (growth arrest-specific gene 1) gene, whose product is known to inhibit cell cycle progression, was induced in cultured corticohippocampal neurons committed to die after a brief exposure to NMDA. Overexpression of Gas1 in cultured hippocampal neurons and in human neuroblastoma NB69 cells produced a marked reduction in the number of viable cells. Furthermore, gas1 antisense oligodeoxynucleotide or antisense mRNA protected hippocampal neurons or NB69 cells from neuronal death. Importantly, Gas1-induced neuronal death was attenuated by coexpression of the human Bcl-2 protein or the baculoviral caspase inhibitor OpIAP2. While Gas1 does not directly interact with Bcl-2, OpIAP2 coimmunoprecipitates with Gas1. In addition, induction of gas1 also occurred in rat brain in two models of excitotoxicity: delayed neuronal death after intraperitoneal kainate injection and neuronal death in hippocampal slices after ischemia. These results indicate that Gas1 is induced by activation of glutamate receptors and is part of the gene expression program directing neuronal death after mild excitotoxic insults.

Topics: Amino Acid Chloromethyl Ketones; Animals; Caspase Inhibitors; Cell Cycle Proteins; Cell Death; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Gene Expression; GPI-Linked Proteins; Humans; Membrane Proteins; N-Methylaspartate; Neuroblastoma; Neurons; Neurotoxins; Proto-Oncogene Proteins c-bcl-2; Rats; Resting Phase, Cell Cycle; RNA, Antisense; RNA, Messenger; Staurosporine; Tumor Cells, Cultured

The clinical benefit of suicide gene therapy of tumors has been marginal, mostly due to the low gene transfer efficiency in vivo. The death-inducing ligand, TRAIL, effectively kills many tumor cell types, while sparing most normal tissues. We hypothesized that TRAIL may enhance HSV thymidine kinase/ganciclovir (TK/GCV) gene therapy of tumor cells by augmenting both target and bystander cell kill. Human SH-EP neuroblastoma cells expressing TK as well as bystander cells were effectively killed by apoptosis, and their clonogenicity was ablated following GCV. Human TRAIL enhanced TK/GCV-induced cell death and decreased clonogenicity of TK-expressing cells and also of bystander cells. Cooperation between TRAIL and TK/GCV depended both on caspase activation and on mitochondrial apoptogenic function because both the broad-spectrum caspase inhibitor zVAD.fmk and overexpression of Bcl-2 decreased enhancement of cell kill by TRAIL. Facilitation of TRAIL signalling by up-regulation of TRAIL receptors did not contribute to enhancement because cell surface expression of the agonistic TRAIL receptors 1 and 2 was not increased by TK/GCV. In conclusion, the concerted activation of caspases and the mitochondrial amplification of caspase activation by TK/GCV may explain the cooperative effect of TK/GCV and TRAIL on the kill of neuroblastoma cells. Because combined treatment also augmented the bystander cell kill, the addition of TRAIL may increase the efficacy of TK/GCV gene therapy of neuroblastoma.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Antiviral Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase Inhibitors; Caspases; Colony-Forming Units Assay; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Ganciclovir; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Humans; Membrane Glycoproteins; Neuroblastoma; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Tetrazolium Salts; Thiazoles; Thymidine Kinase; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

There is growing evidence that apoptotic mechanisms underlie the neurodegeneration leading to Parkinson's disease. 1-Methyl-4-phenylpyridinium ion (MPP(+)), the active metabolite of the parkinsonism-inducing drug MPTP, induced apoptosis in cultures of human SH-SY5Y neuroblastoma cells. Nuclear fragmentation, DNA laddering, and a 20% decrease in viability were seen after a 4-day incubation with 5 microM MPP(+). Cell viability decreased by 40% at 100 microM MPP(+), but the degree of apoptosis was not correlatively increased. The MPP(+)-induced apoptosis was completely prevented by the broad caspase inhibitor zVAD.fmk but not by the caspase-8 inhibitor IETD.fmk. Furthermore, MPP(+) had no effect on the levels of Fas or Fas-L, suggesting lack of activation of the Fas-L/Fas/caspase-8 pathway of apoptosis. There was no evidence of mitochondrial dysfunction at 5 microM MPP(+): No differences were seen in transmembrane potential or in cytochrome c release from controls. At 100 microM MPP(+), the mitochondrial potential decreased, and cytoplasmic cytochrome c and caspase-9 activation increased slightly. At both low and high concentrations of MPP(+), VDVADase and DEVDase activities increased. We conclude that MPP(+) can induce caspase-mediated apoptosis, which is prevented by caspase inhibition, at concentrations lower than those needed to trigger mitochondrial dysfunction and closer to those found in the brains of MPTP-treated animals.

Topics: 1-Methyl-4-phenylpyridinium; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochrome c Group; DNA Fragmentation; Dose-Response Relationship, Drug; Fas Ligand Protein; fas Receptor; Humans; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Osmolar Concentration; Parkinson Disease; Tumor Cells, Cultured

Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Caspase 10; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Decitabine; Dose-Response Relationship, Drug; Enzyme Inhibitors; GPI-Linked Proteins; Humans; Jurkat Cells; Membrane Glycoproteins; Methylation; Neuroblastoma; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 10c; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha

Apoptotic cell death and increased production of amyloid b peptide (Ab) are pathological features of Alzheimer's disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor Z-VAD and by Bcl-XL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPSW) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C-terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Ab42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Ab42 production of calsenilin/DREAM/KChIP3.

Topics: Amino Acid Chloromethyl Ketones; Amyloid beta-Peptides; Animals; Apoptosis; bcl-X Protein; Blotting, Western; Calcium; Calcium-Binding Proteins; Cells, Cultured; Cysteine Proteinase Inhibitors; Humans; Jurkat Cells; Kv Channel-Interacting Proteins; Membrane Proteins; Neuroblastoma; Oligonucleotides, Antisense; Peptide Fragments; Presenilin-2; Proto-Oncogene Proteins c-bcl-2; Rats; Repressor Proteins; Transfection

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cells, Cultured; Dopamine; Enzyme Inhibitors; Gene Transfer Techniques; Humans; Mesencephalon; Neurites; Neuroblastoma; Neurons; Oxidopamine; Protein Biosynthesis; Proteins; Rats; Tyrosine 3-Monooxygenase; X-Linked Inhibitor of Apoptosis Protein

In previous studies it has been shown that neural cells undergoing programmed cell death display strongly positive cytoplasmic immunoreactivity to polyclonal antibodies directed against a c-Jun N-terminal peptide. It was later found that c-Jun-like immunoreactivity in apoptosis was due to cross-reactivity with proteins other than c-JUN: We have analysed the biochemical counterpart of this property in neuroblastoma cell lines treated to induce apoptosis. Using the c-Jun/sc-45 antibody, several bands with apparent molecular masses distinct from c-Jun were detected in extracts in parallel with both the degree of apoptosis and the appearance of the cytoplasmic signal after immunostaining. c-Jun/sc-45 immunostaining was prevented by caspase inhibitors and did not require de novo protein synthesis. One of the antigens recognized by the c-Jun/sc-45 antibody was identified as seryl-tRNA synthetase. We provide evidence that seryl-tRNA synthetase is a substrate of caspase-3 in vitro and that the digested form turns highly immunoreactive towards the antibody. A carboxy-terminus epitope of the protein that constitutes a consensus site for caspase-3 is involved in c-Jun/sc-45 recognition. This epitope shares some amino acids with the peptide used as the immunogen and this could explain the cross-reactivity observed. In conclusion, we demonstrate here that cytoplasmic c-Jun/sc-45-like immunoreactivity specific to apoptosis is due to post-translational changes which occur in seryl-tRNA synthetase and probably also in other proteins as a consequence of caspase mediated proteolysis.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Antibody Specificity; Apoptosis; Caspase Inhibitors; Caspases; Cycloheximide; Enzyme Inhibitors; Epitopes; Humans; Immunohistochemistry; Immunosorbent Techniques; Microscopy, Fluorescence; Neuroblastoma; Peptide Fragments; Proto-Oncogene Proteins c-jun; Serine-tRNA Ligase; Tumor Cells, Cultured

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Biological Transport; Caspase 3; Caspase 9; Caspases; Cell Death; Cysteine Proteinase Inhibitors; Cytochrome c Group; Dopamine; Humans; Kinetics; Mitogen-Activated Protein Kinases; Neuroblastoma; Nomifensine; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Tumor Cells, Cultured

Neuroblastoma is the most common extracranial solid tumor of childhood. N-type neuroblastoma cells (represented by SH-SY5Y and IMR32 cell lines) are characterized by a neuronal phenotype. N-type cell lines are generally N-myc amplified, express the anti-apoptotic protein Bcl-2, and do not express caspase-8. The present study was designed to determine the mechanism by which N-type cells die in response to specific cytotoxic agents (such as cisplatin and doxorubicin) commonly used to treat this disease. We found that N-type cells were equally sensitive to cisplatin and doxorubicin. Yet death induced by cisplatin was inhibited by the nonselective caspase inhibitor z-Val-Ala-Asp-fluoromethylketone or the specific caspase-9 inhibitor N-acetyl-Leu-Glu-His-Asp-aldehyde, whereas in contrast, caspase inhibition did not prevent doxorubicin-induced death. Neither the reactive oxygen species nor the mitochondrial permeability transition appears to play an important role in this process. Doxorubicin induced NF-kappa B transcriptional activation in association with I-kappa B alpha degradation prior to loss of cell viability. Surprisingly, the antioxidant and NF-kappa B inhibitor pyrrolidine dithiocarbamate blocked doxorubicin-induced NF-kappa B transcriptional activation and provided profound protection against doxorubicin killing. Moreover, SH-SY5Y cells expressing a super-repressor form of I-kappa B were completely resistant to doxorubicin killing. Together these findings show that NF-kappa B activation mediates doxorubicin-induced cell death without evidence of caspase function and suggest that cisplatin and doxorubicin engage different death pathways to kill neuroblastoma cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Antioxidants; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Death; Child; Cisplatin; Doxorubicin; Enzyme Inhibitors; Flow Cytometry; Genes, Reporter; Humans; Neuroblastoma; NF-kappa B; Phenotype; Pyrrolidines; Thiocarbamates; Tumor Cells, Cultured

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochrome c Group; Death-Associated Protein Kinases; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Medulloblastoma; Microscopy, Fluorescence; Mitochondria; Mutation; Neuroblastoma; Oligopeptides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

Neuron death and neuron degeneration occur in the CNS during the course of aging. Although multiple cellular alterations transpire during the aging process, those that mediate age-associated neuron death have not been identified. Recent evidence implicates oxidative stress as a possible means of neuron death and neuron degeneration during aging. In the present study, we demonstrate a marked decrease in multicatalytic proteasome activity in the spinal cord of Fisher 344 rats at 12, 24 and 28 months, compared with spinal cord tissue from 3-week- and 3-month-old animals. Application of oxidative injury (FeSO(4)) or the lipid peroxidation product 4-hydroxynonenal decreases multicatalytic proteasome activity in a time- and dose-dependent manner in a motor neuron cell line. Loss of multicatalytic proteasome activity occurs before the loss of multicatalytic proteasome immunoreactivity, with FeSO(4)- and 4-hydroxynonenal-mediated decreases ameliorated by the application of a cell permeable form of the antioxidant glutathione. Application of multicatalytic proteasome inhibitors, but not inhibitors of lysosomal proteases, induced neuron death that was attenuated by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone or N-acetyl-Asp-Glu-Val-Asp-Cho (aldehyde). Together, these data suggest that multicatalytic proteasome inhibition occurs during aging of the spinal cord, possibly as the result of oxidative stress, and that multicatalytic proteasome inhibition may be causally related to neuron death.

Topics: Acetylcysteine; Aging; Aldehydes; Amino Acid Chloromethyl Ketones; Animals; Cell Death; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Glutathione; Iron; Lipid Peroxidation; Lysosomes; Mice; Motor Neurons; Multienzyme Complexes; Neuroblastoma; Oligopeptides; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Inbred F344; Reactive Oxygen Species; Spinal Cord; Tumor Cells, Cultured

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Caspases; Cell Cycle; Ceramides; Doxorubicin; Enzyme Activation; G1 Phase; Humans; Kinetics; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Neuroprotective Agents; Tumor Cells, Cultured

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fenretinide; Free Radicals; Humans; Mitochondria; Neuroblastoma; Oligopeptides; Poly(ADP-ribose) Polymerases; Receptors, Retinoic Acid; Tumor Cells, Cultured

Programmed cell death, or apoptosis, is a tightly regulated process mediated by selective cleavage of proteins by caspases, resulting in ordered destruction of the cell. In addition to structural proteins, proteins that mediate anti-apoptotic signal transduction are also substrates; their destruction eliminates potential futile attempts to escape execution. We asked whether cAMP response element binding protein (CREB), a transcription factor that mediates nerve growth factor (NGF) survival signals, is a target for caspases during apoptosis. CREB was specifically cleaved by caspases in neuroblastoma extracts, and in cells induced to undergo apoptosis by staurosporine. The destruction of CREB eliminates a key factor that could reverse apoptosis.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cyclic AMP Response Element-Binding Protein; Cytochrome c Group; Enzyme Inhibitors; Humans; Neuroblastoma; Neurons; Oligopeptides; PC12 Cells; Rats; Signal Transduction; Staurosporine; Transcription Factors; Tumor Cells, Cultured

We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Aspartic Acid; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Caspase 3; Caspases; Chlorobenzoates; Cysteine Endopeptidases; Enzyme Inhibitors; Humans; Mice; Neuroblastoma; Peptide Fragments; Recombinant Proteins; Staurosporine; Thapsigargin; Tumor Cells, Cultured