cytochrome-c-t and Neuroblastoma

Synthetic retinoids such as fenretinide [N-(4-hydroxyphenyl)retinamide] induce apoptosis of neuroblastoma cells, act synergistically with chemotherapeutic drugs, and may provide opportunities for novel approaches to neuroblastoma therapy. Fenretinide-induced cell death of neuroblastoma cells is caspase dependent and results in the release of cytochrome c from mitochondria independently of changes in permeability transition. This is mediated by a signaling pathway characterized by the generation of reactive oxygen species (ROS) via 12-lipoxygenase (12-LOX), and an oxidative-stress-dependent induction of the transcription factor, GADD153 and the BCL2-related protein BAK. Upstream events of fenretinide-induced signaling involve increased levels of ceramide as a result of increased sphingomyelinase activity, and the subsequent metabolism of ceramide to gangliosides via glucosylceramide synthase and GD3 synthase. These gangliosides may be involved in the regulation of 12-LOX leading to oxidative stress and apoptosis via the induction of GADD153 and BAK. The targeting of sphingomyelinases or downstream effectors such as 12-LOX or GADD153 may present novel approaches for the development of more effective and selective drugs for neuroblastoma therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arachidonate 12-Lipoxygenase; CCAAT-Enhancer-Binding Proteins; Cell Death; Cell Line, Tumor; Cytochromes c; Fenretinide; Humans; Mitochondria; Models, Biological; Neuroblastoma; Oxidative Stress; Permeability; Reactive Oxygen Species; Retinoids; Signal Transduction; Transcription Factor CHOP; Transcription Factors

Mitochondrial dysfunction has been implicated in Parkinson's disease. Mic60 is a critical component of mitochondrial crista remodeling and participates in maintaining mitochondrial structure and function. This study investigated whether the carnosic acid (CA) of rosemary protects the mitochondria of SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) by regulating Mic60. Our results showed that CA pretreatment reversed the reduction in the Mic60 and citrate synthase proteins, as well as the protein induction of PKA caused by 6-OHDA. Moreover, Mic60 and PINK1 siRNAs blocked the ability of CA to lessen the release of mitochondrial cytochrome c by 6-OHDA. As shown by immunoprecipitation assay, in 6-OHDA-treated cells, the interaction of Mic60 with its phosphorylated threonine residue was decreased, but the interaction with its phosphorylated serine residue was increased. PINK1 siRNA and forskolin, a PKA activator, reversed these interactions. Moreover, forskolin pretreatment prevented CA from rescuing the interaction of PINK1 and Mic60 and the reduction in cytochrome c release and mitophagy impairment in 6-OHDA-treated cells. In conclusion, CA prevents 6-OHDA-induced cytochrome c release by regulating Mic60 phosphorylation by PINK1 through a downregulation of PKA. The regulation of Mic60 by CA can be considered as a protective mechanism for the prevention of Parkinson's disease.

Topics: Apoptosis; Cell Line, Tumor; Colforsin; Cytochromes c; Humans; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Oxidopamine; Parkinson Disease; Protein Kinases; RNA, Small Interfering

Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca

Topics: Apoptosis; Cytochromes c; Humans; Neuroblastoma; Neurodegenerative Diseases; Neurons

The developmental, epigenetic, and epidemiological studies on lead (Pb) toxicity have reported a strong connection between lead exposure and the progression of Alzheimer's disease (AD). The amyloid peptides were the main triggering elements, in the generation of extracellular plaques through which multiple cellular signaling events such as apoptosis and primarily oxidant-antioxidant balancing system will be affected, which leads to neuronal cell death. Our previous studies indicated that epigallocatechin gallate (EGCG), abundantly present in green tea, was found to be effective in alleviating the metal-induced neurotoxicity at the cellular level in terms of cell viability and apoptosis The aim of this study was to explore the protective mechanism of EGCG on the markers of oxidant-antioxidant system and mitochondria, which are involved in metal-induced neuronal cell death. Initially, the IC

Topics: Antioxidants; Apoptosis; Catechin; Cytochromes c; Humans; Lead; Mitochondria; Neuroblastoma; Oxidants; Oxidative Stress; Peptides

1,3,5-Tri-

Topics: Annexin A5; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cytochromes c; Humans; Hydrogen Peroxide; Lactate Dehydrogenases; Malondialdehyde; Mitogen-Activated Protein Kinases; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Quinic Acid; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

We investigated the effect of apelin-13 on the cellular model of AD, amyloid-β (Aβ) treated SH-SY5Y cells in rats.. The SH-SY5Y cells were pretreated with different doses of apelin-13 (1, 2.5, 5, and 10 μg/mL), half an hour before adding 50% Aβ treatment. After 24 h, cells were evaluated for survival, oxidative stress, mitochondrial calcium release, caspase-3, and cytochrome. Apelin-13 at the dose of 2.5 μg/mL protected against IC50 Aβ (p<0.001). Apelin-13 at doses of 1, 2.5, and 5 μg/mL showed protective effects against the reactive oxygen species (ROS) produced by Aβ (p<0.001). Apelin-13 at doses of 2.5 and 5 μg/mL reduced calcium release, caspase-3, and cytochrome. Apelin-13 prevented apoptosis, oxidative stress, and mitochondrial toxicity and can be a suitable option for treatment of AD. The appropriate treatment strategy for humans has to be investigated in future studies.

Topics: Actins; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Calcium; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Intercellular Signaling Peptides and Proteins; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Rats; Reactive Oxygen Species

Parkinson's disease (PD) is a destroying and prevalent neurodegenerative disease that is characterized by a progressive death of midbrain dopaminergic neurons. It is important to understand the possible neuroprotective effects of reagents that rescue the neurons from death and apoptosis. In this study, we investigated the effects of abscisic acid (ABA) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cell line as an

Topics: Abscisic Acid; Anilides; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Neurotoxicity Syndromes; Oxidopamine; Plant Growth Regulators; PPAR delta; PPAR gamma; Reactive Oxygen Species

Neuroblastoma is the main solid extracranial tumor of childhood. The amplification of N-myc oncogene (MYCN) and 1p deletion are the main molecular alterations. These features are what make treatment impossible, especially in high-risk patients with metastases.. Our study investigated the processes undergone by CHP-212 neuroblastoma cells, after being treated with Casiopeínas® (Cas) IIgly, IIIEa, and IIIia for 2, 10, and 24 h.. At 2 h, all the treatments Ied to apoptosis [defined by the presence of B-cell lymphoma 2 (BCL2), BCL2-associated X protein, cytochrome c, and caspase-3]. In addition, autophagy with specific molecules beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I (ratio >1). Later at 10 h, autophagy-associated proteins were observed, and at 24 h, only survival proteins nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), and extracellular signal-regulated kinases (ERK)2/ERK1>1 were found. Another relevant finding was the presence of caspase-10 throughout the study, especially in cells treated with CasIIgly and CasIIIEa.. These relationships indicate a possible mechanism of action of Casiopeínas on neuroblastoma.

Topics: Autophagy; Beclin-1; Cell Line, Tumor; Cell Survival; Coordination Complexes; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Humans; Microtubule-Associated Proteins; Neuroblastoma; Phenanthrolines; Proto-Oncogene Proteins c-bcl-2

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Nanoparticles; Neuroblastoma; Signal Transduction; Silicon Dioxide

Truncated apolipoprotein C-I is a post-translationally modified protein characterized by the loss of threonine and proline residues from the N-terminus of the mature peptide. The truncated peptide is involved in many physiological and pathological processes in vivo and is related to malignant diseases. The aim of the present study was to assess the effects of the truncated peptide on tumorigenesis in neuroblastoma. The truncated peptide was chemically synthesized, and a signal peptide was used as the negative control. The results of the CCK-8 assay showed that the truncated peptide selectively inhibited cell proliferation compared with the signal peptide, and inhibited migration and invasion as determined by wound healing and Transwell assays. Flow cytometry analysis demonstrated that the truncated peptide induced apoptosis and cell cycle arrest in the S phase. Bax, Bim, and tBid upregulation, and Bcl‑2 and Bcl‑xl downregulation were associated with permeabilization of the mitochondrial membrane, as detected by the JC-1 assay and the release of cytochrome c and apoptosis. Activation of caspase‑8 was associated with activation of cell death receptors such as the tumor necrosis factor receptor. PARP cleavage indicated apoptosis, and DNA damage was observed in the TUNEL assay. The results showed that the truncated apoC-I induced apoptosis in neuroblastoma by the extrinsic and intrinsic pathways. The anticancer effects were confirmed in vivo in a xenograft mouse model. In conclusion, the endogenous protein apoC-I may be a new promising therapeutic target to suppress tumor growth.

Topics: Animals; Apolipoprotein C-I; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; S Phase; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Alzheimer's disease (AD), a common protein misfolding progressive neurodegenerative disorder, is one of the most common forms of dementia. Amyloid precursor protein (APP) derived amyloid-β (Aβ) protein accumulate into interneuronal spaces and plays a crucial role in the disease progression and its pathology. The aggregated Aβ exerts its neurotoxic effects by inducing apoptosis and oxidative damage in neuronal cells.. We have investigated the effects of a synthesized Pro-Drug peptide (PDp) on Aβ1-40 induced cytotoxicity in human neuroblastoma SH-SY5Y cells, represents one of the most effective strategies in combating human AD.. Cells were treated with Aβ1-40 to induce cytotoxicity in the experimental model of AD to screen the inhibitory effect of PDp. Assays for cell viability, reactive oxygen species (ROS) generation, levels of intracellular free Ca2+ and expression of key apoptotic proteins were assessed by Western Blotting.. Our results showed that Aβ1-40 induces for 24h caused reduce cell viability, imbalance in Ca2+ homeostasis and increase in neuronal apoptosis in vitro. Treatment with PDp could effectively ameliorated Aβ1-40 induced neurotoxicity and attenuates ROS generation that mediates apoptotic signaling through Bcl-2, Bax, Caspase-3 activity and cytochrome c in the cells.. These findings suggested that PDp has potential role as a neuroprotective and therapeutic agent for combating human AD.

Topics: Amyloid beta-Peptides; Analysis of Variance; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Intracellular Fluid; L-Lactate Dehydrogenase; Neuroblastoma; Neurons; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Oxidative stress and mitochondrial dysfunction play key roles in Parkinson's disease (PD) initiation and progression. Ginsenosides are major compounds of Ginseng species and they are responsible for pharmacological activity. The aim of this study was to investigate the potential neuroprotective effects and mechanism of the major ginsenosides Rd and Re in rotenone-induced oxidative stress model in human neuroblastoma SH-SY5Y cells. Co-treatments with both ginsenosides inhibited the increased intracellular ROS production and by-products lipid peroxidation accumulation caused by rotenone. Moreover, these ginsenosides upregulated SOD and aconitase enzymes activities, and glutathione system; these antioxidant properties are related to Nrf2 induction and radical scavenger effect. Additionally, the results showed that both Rd and Re attenuated the extent of depolarization of mitochondrial membrane potential and restored calcium levels. Furthermore, these compounds prevented apoptosis by modulating Bax and Bcl-2 proteins and inhibiting cytochrome c release and caspase-3 activation. The ginsenoside Rd resulted to be more active than ginsenoside Re. These findings highlighted the efficacy of these compounds as neuroprotectant compounds for PD prevention and treatment through reducing oxidative stress, improving mitochondrial integrity and functions, and inhibiting apoptosis.

Topics: Apoptosis; Cell Line, Tumor; Cytochromes c; Ginsenosides; Glutathione; Humans; Mitochondria; Neuroblastoma; Oxidative Stress; Panax; Reactive Oxygen Species; Rotenone; Superoxide Dismutase

Parkinson's disease (PD) is the second most common neurodegenerative disease in humans. The hormone α-melanocyte-stimulating hormone (α-MSH) has been reported to be neuroprotective in previous studies. The aim of this study is to investigate the neuroprotective effects of α-MSH against the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Our results indicated that treatment with α-MSH in M17 cells attenuated MPP+-induced oxidative stress, embodied by exacerbated reactive oxygen species and protein carbonyls. In addition, we found that α-MSH could improve mitochondrial function in M17 cells through increasing the level of adenosine triphosphate and mitochondrial membrane potential. Furthermore, treatment with α-MSH restored the reduction of cell viability and the induction of lactate dehydrogenase release induced by α-MSH. Importantly, Hoechst staining results indicated that α-MSH treatment significantly reduces the number of apoptotic cells after treatment with MPP+. Mechanically, we found that α-MSH prevented apoptosis signals through reducing the level of cleaved caspase-3 and attenuating cytochrome c release. All these data imply that α-MSH produces a protective effect in PD. © 2015 IUBMB Life, 69(5):315-320, 2017.

Topics: 1-Methyl-4-phenylpyridinium; Adenosine Triphosphate; alpha-MSH; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Neurotoxicity Syndromes; Parkinson Disease; Reactive Oxygen Species

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP

Topics: Animals; Apoptosis; Autophagy; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Mice; Mitochondria; Neuroblastoma; Peptide Fragments; Prions; Recombinant Proteins; Ubiquitin-Protein Ligases

Topics: Cell Line, Tumor; Cell Survival; Cytochromes c; Down-Regulation; Flavanones; Humans; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Paraquat

This study demonstrates the protective potential of hydroxytyrosol (HT), an olive oil phenol, against methylmercury (MeHg)-induced neurotoxicity using IMR-32 human neuroblastoma cell line. HT inhibited MeHg-induced cytotoxicity and genotoxicity as confirmed by MTT, micronucleus, and comet assays. Cells preconditioned with HT showed reduction of MeHg-induced cellular oxidative stress along with the maintenance of glutathione, superoxide dismutase, glutathione-S-tranferase, and catalase. Fluorescence microscopy and DNA ladder assays indicated the inhibitory effect of HT against MeHg-induced apoptosis, which was further established by Western blotting. An effective concentration of 5 µM HT caused downregulation of p53, bax, cytochrome c, and caspase 3 and upregulation of prosurvival proteins including nuclear factor erythroid 2-related factor 2 (Nrf2) and metallothionein. This work indicates the cytoprotective potential of HT against MeHg-induced toxicity primarily by the lowering of oxidative stress, which may be endorsed to its antigenotoxic and antiapoptotic potential, in addition to its free radical scavenging ability. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1264-1275, 2016.

Topics: bcl-2-Associated X Protein; Caspase 3; Catalase; Cell Line, Tumor; Cell Survival; Comet Assay; Cytochromes c; DNA Fragmentation; Glutathione; Humans; Methylmercury Compounds; Microscopy, Fluorescence; Neuroblastoma; NF-E2-Related Factor 2; Oxidative Stress; Phenylethyl Alcohol; Superoxide Dismutase; Tumor Suppressor Protein p53

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, remains one of the most challenging types of cancer to treat. Therefore, the search for novel effective drugs for its treatment is essential. The present study used 10-hydroxycamptothecin (HCPT), which is a naturally occurring alkaloid anticancer agent extracted from the Chinese tree, Camptotheca acuminata, and has a strong anticancer activity in vitro and in vivo. HCPT is able to induce apoptosis in cells of various tumor types. However, few studies have been conducted on its efficacy in NB, and its apoptosis-inducing mechanism has not been elucidated. In the present study, the in vitro effects of HCPT on apoptosis in the human NB cell line, SMS-KCNR, and its underlying molecular mechanisms were investigated. Cell proliferation was measured by an MTT assay and apoptosis was measured using DAPI staining and flow cytometric analysis. In addition, western blot analysis was used to evaluate the apoptosis-associated signaling pathways. HCPT was observed to markedly inhibit cell proliferation and induce apoptosis in SMS-KCNR cells at a relatively low concentration (2.5-20 nM). DAPI staining revealed typical apoptotic feature, namely apoptotic body formation. The flow cytometric analysis revealed that the number of apoptotic cells increased from 20.89% (for 2.5 nM) to 97.66% (for 20 nM) following HCPT treatment for 48 h. Western blot analysis revealed that p53, cytoplasmic cytochrome c, cleaved caspase-3 and poly ADP-ribose polymerase (PARP) proteins were significantly upregulated, while the mitochondrial cytochrome c and pro-caspase-3 proteins were downregulated. However, the B-cell lymphoma 2 and Bcl-2-associated X proteins were unaffected. The results indicated that HCPT may inhibit proliferation and induce apoptosis in the SMS-KCNR cells. The possible mechanism of apoptosis induction is the p53-mediated mitochondrial apoptotic signaling pathway, which promotes cytochrome c release and induces apoptosis by activating caspase-3 and PARP. Our study provides experimental evidence for HCPT as a potent therapeutic drug in NB treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Caspase 3; Cell Proliferation; Cytochromes c; Humans; Neuroblastoma; Tumor Suppressor Protein p53

Here, we investigate the apoptotic effect of allicin, the predominant component of freshly crushed garlic, on neuroblastoma cells. In this paper, the authors have first assessed the effect of allicin on human neuroblastoma SK-N-SH cells and then investigated the underlying mechanism. The results indicate that allicin suppresses SK-N-SH cell growth in a dose-dependent and time-dependent manner and that 5 μmol/l of allicin leads to a significant increase in apoptotic rate with annexin-V/PI double staining. Western blot analysis shows that treatment with allicin-induced apoptosis through activation of caspases-3 and 9. Phosphorylation of p38 MAPK contributes to allicin-induced apoptosis upstream of caspase activation. Using p38 MAPK inhibitor, the authors discovered that p38 MAPK activation subsequently induces the release of cytochrome-c from mitochondria into the cytosol. Taken together, the results demonstrate that allicin can activate the p38 MAPK pathway, which leads to mitochondrial release of cytochrome-c, thus inducing SK-N-SH cell apoptosis. Overall, this study suggests that allicin may be used as one of the novel pharmacological treatment strategies in neuroblastoma.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disulfides; Enzyme Activation; Humans; Mitochondria; Neuroblastoma; p38 Mitogen-Activated Protein Kinases; Peripheral Nervous System Neoplasms; Sulfinic Acids

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bcl-2-Like Protein 11; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Evaluation, Preclinical; Female; Humans; Inhibitory Concentration 50; Mice; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Treatment Outcome; Xenograft Model Antitumor Assays

To study the protective effect of nuclear factor erythroid 2-related factor 2 (Nrf2) activation by curcumin against lead-induced toxicity and apoptosis in SH-SY5Y human neuroblastoma cells and its impact on expression of apoptosis-related proteins.. After the cells were treated with 0, 0.5, 1, 5, or 10 μmol/L curcumin for 24 hours, nucleoprotein was extracted and electrophoretic mobility shift assay was used to measure Nrf2-antioxidant responsive element (ARE) binding capacity. The optimal concentration of curcumin was figured out for treating cells. After pretreatment with 5 μmol/L curcumin for 24 hours, cells were exposed to lead acetate at different concentrations (0, 5, 25, and 125 μmol/L for control, low-dose, medium-dose, and high-dose groups). The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry, and Western blot were used to evaluate cell viability, apoptosis, and expression of apoptosis-related proteins, respectively.. Curcumin of 5 μmol/L caused significantly increased cell viability in low-, medium-, and high-dose groups exposed to lead acetate for 12 hours (98.42%±1.12% vs 92.92%±0.14%, P<0.05; 95.30%±1.17% vs 91.15%±0.67%, P<0.05; 94.50%±1.45% vs 85.98%±0.45%, P<0.05). Curcumin of 5 μmol/L also caused significantly increased cell viability in medium-and high-dose groups exposed to lead acetate for 24 hours (93.10%±1.63% vs 88.40%±4.13%, P<0.05; 90.13%±2.03% vs 83.63%±3.42%, P<0.05). The high-dose group had a significantly higher apoptotic rate than the control group 6.17%±1.31% vs 3.30%±0.53%, P<0.05). Curcumin of 5.0 μmol/L significantly reduced the apoptotic rate in the high-dose group (2.97%±0.15% vs 6.17%±1.31%, P<0.05). Exposure to lead acetate elevated the expression of Bax, cytochrome C, and caspase-3 and reduced Bcl-2 expression. Curcumin of 5.0 μmol/L significantly reduced the expression of Bax, cytochrome C, and caspase-3 in the high-dose group (P<0.05).. Nrf2 activation by curcumin has a protective effect against lead-induced toxicity and apoptosis in SH-SY5Y cells. The protective effect of Nrf2 against apoptosis may be associated with the regulation of apoptosis-related proteins.

Topics: Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Curcumin; Cytochromes c; Humans; Lead; Neuroblastoma; NF-E2-Related Factor 2

ABT-751 is a colchicine-binding site microtubule inhibitor. Fenretinide (4-HPR) is a synthetic retinoid. Both agents have shown activity against neuroblastoma in laboratory models and clinical trials. We investigated the antitumor activity of 4-HPR + the microtubule-targeting agents ABT-751, vincristine, paclitaxel, vinorelbine, or colchicine in laboratory models of recurrent neuroblastoma. Drug cytotoxicity was assessed in vitro by a fluorescence-based assay (DIMSCAN) and in subcutaneous xenografts in nu/nu mice. Reactive oxygen species levels (ROS), apoptosis, and mitochondrial depolarization were measured by flow cytometry; cytochrome c release and proapoptotic proteins were measured by immunoblotting. 4-HPR + ABT-751 showed modest additive or synergistic cytotoxicity, mitochondrial membrane depolarization, cytochrome c release, and caspase activation compared with single agents in vitro; synergism was inhibited by antioxidants (ascorbic acid, α-tocopherol). 4-HPR + ABT-751 was highly active against four xenograft models, achieving multiple maintained complete responses. The median event-free survival (days) for xenografts from 4 patients combined was control = 28, 4-HPR = 49, ABT-751 = 77, and 4-HPR + ABT-751 > 150 (P < 0.001). Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL) was significantly higher in 4-HPR + ABT-751-treated tumors than with single agents (P < 0.01) and was inhibited by ascorbic acid and α-tocopherol (P < 0.01), indicating that ROS from 4-HPR enhanced the activity of ABT-751. 4-HPR also enhanced the activity against neuroblastoma xenografts of vincristine or paclitaxel, but the latter combinations were less active than 4-HPR + ABT-751. Our data support clinical evaluation of 4-HPR combined with ABT-751 in recurrent and refractory neuroblastoma. Mol Cancer Ther; 15(11); 2653-64. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Disease Models, Animal; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fenretinide; Humans; Membrane Potential, Mitochondrial; Mice; Neoplasm Recurrence, Local; Neuroblastoma; Reactive Oxygen Species; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays

Amyloid beta peptide (Aβ) can cause neurotoxicity in Alzheimer's disease (AD). It evokes a cascade of oxidative damage to neurons. Pinocembrin (PCB), the most abundant flavonoid in propolis, has been proven to have neuroprotective effects in vivo and in vitro. In the present study, we investigated the neuroprotective effects of PCB on Aβ25-35-induced neurotoxicity. Exposure of SH-SY5Y cells to 25μM Aβ25-35 for 24h caused viability loss, apoptotic increase and reactive oxygen species (ROS) increase, pre-treatment with PCB for 4h significantly reduced the viability loss, apoptotic rate and attenuated Aβ-mediated ROS production. PCB strikingly inhibited Aβ25-35-induced mitochondrial dysfunctions, including lowered membrane potential, decreased Bcl-2/Bax ratio. In addition, PCB suppressed the release of cytochrome c and the cleavage of caspase-3. PCB treatment also resulted in an increase in Nrf2 protein levels and subsequent induction of heme oxygenase-1(HO-1) expression in SH-SY5Y cells. RNA interference-mediated knockdown of Nrf2 expression suppressed the PCB-induced HO-1 expression. Notably, we found that the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) markedly diminished the neuroprotective effect of PCB against Aβ-mediated neurotoxicity. Taken together, these results indicated that PCB protects SH-SY5Y cells from Aβ25-35-induced neurotoxicity through activation of Nrf2/HO-1 pathways. Thus, activation of Nrf2/HO-1 pathways and inhibition of mitochondria-dependent apoptosis together may protect cells from Aβ25-35-induceded neurotoxicity.

Topics: Amyloid beta-Peptides; Apoptosis; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Flavanones; Gene Expression Regulation, Neoplastic; Heme Oxygenase-1; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; NF-E2-Related Factor 2; Peptide Fragments; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection

Mitochondria play a key role in the process of lead (Pb)-induced impairment in nervous system. To further clarify the underlying mechanism of Pb neurotoxicity, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and cyclophilin D (CyPD), a component of MPT pore (MPTP), in Pb-induced mitochondrial apoptosis in nerve cells. In SH-SY5Y and PC12 cells, Cyclosporin A (CSA), a special inhibitor of CyPD, could alleviate cell death, lactate dehydrogenase (LDH) leakage and adenosine 5 triphosphate (ATP) decrease caused by PbAc. In the following experiments, we found PbAc increased the protein level of CyPD and induced MPT pore (MPTP) opening. When cells were pretreated with CSA to inhibit MPTP opening, the Pb-induced impairment of mitochondrial morphology (swelling and rupture) and the loss of mitochondria were attenuated. In addition, CSA obviously ameliorated the Pb-induced damage of mitochondrial function, such as reactive oxygen species (ROS) boost and mitochondrial membrane potential (MMP) collapse, as well as the release of cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) from mitochondria. These beneficial effects could finally result in cell survival under Pb-exposure conditions. Furthermore, scavenging ROS also significantly abrogated MPTP opening and attenuated Pb neurotoxicity. Therefore, we found that MPT played an important role in Pb-induced mitochondrial damage and, ultimately, cell death. Our results provided a potential strategy for inhibiting PbAc neurotoxicity. However, due to the high Pb concentrations used in this study further investigations at Pb concentrations closer to human exposure are needed to verify the results.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adenosine Triphosphate; Animals; Apoptosis Inducing Factor; Cell Line, Transformed; Cyclosporins; Cytochromes c; Humans; L-Lactate Dehydrogenase; Lead; Membrane Potential, Mitochondrial; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neuroblastoma; Neurons; Neuroprotective Agents; Neurotoxins; PC12 Cells; Rats; Reactive Oxygen Species

Compound MQA (1,5-O-dicaffeoyl-3-O-[4-malic acid methyl ester]-quinic acid) is a natural caffeoylquinic acid derivative isolated from Arctium lappa L. roots. This study aims to explore the neuroprotective effects of MQA against hydrogen peroxide (H

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Chlorogenic Acid; Cyclin D1; Cytochromes c; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; L-Lactate Dehydrogenase; Malondialdehyde; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Oxidants; Oxidative Stress; Quinic Acid; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson's disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP⁺), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP⁺-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP⁺-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP⁺-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.

Topics: 1-Methyl-4-phenylpyridinium; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Survival; Connexin 43; Cytochromes c; Herbicide Resistance; Herbicides; Humans; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Signal Transduction

p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 μmol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (~60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells.

Topics: Animals; Apoptosis; Autophagy; Biomarkers; Caspase 8; Cell Line, Tumor; Cell Shape; Coumaric Acids; Cytochromes c; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glutathione; Membrane Potential, Mitochondrial; Mice; Microtubule-Associated Proteins; Mitochondria; Neuroblastoma; Propionates; Reactive Oxygen Species; RNA, Messenger; Tumor Suppressor Protein p53; Up-Regulation

This study aimed to investigate the direct effects of advanced glycation end products (AGEs) on the mitochondrial structure and function of SH-SY5Y cells and the possible molecular mechanism(s) underlying mitochondria dysfunction by AGEs. SH-SY5Y cells were cultured in 400 μg/ml of AGE-bovine serum albumin (BSA) for 24 h, and changes in the mitochondrial function of SH-SY5Y cells were analysed as follows. Reactive oxygen species (ROS) were detected using 2',7'-dichlorodihydrofluorescein diacetate molecular probes. Mitochondrial membrane potential (ΔΨm) was determined by flow cytometry using fluorescent probes. The expression of cytochrome c (Cyt c) protein level was assessed by Western blotting. Mitochondrial structures were observed by transmission electron microscopy. Our results showed that AGE-BSA induced an increase in ROS levels, a decrease in mitochondrial ΔΨm, and the release of Cyt c from mitochondria in SH-SY5Y cells. The mitochondria of SH-SY5Y cells showed remarkable swelling and vacuolisation, but these changes were recovered after pretreatment with neutralising anti-receptor for advanced glycation end products (RAGE) antibody. Our results suggested that AGE-BSA induced mitochondrial dysfunction in SH-SY5Y cells through RAGE pathways. Thus, AGEs are potential mechanistic links between diabetes mellitus and Alzheimer's disease.

Topics: Cell Line, Tumor; Cytochromes c; Glycation End Products, Advanced; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species; Receptor for Advanced Glycation End Products; Serum Albumin, Bovine

Recent studies have demonstrated the role of indirect effect of radiation in neurodegeneration. However, the role of glial cells in neuroprotection against indirect effect of radiation is still not clear, although they are known to protect neurons under stress conditions in central nervous system. Our study showed that indirect effect of radiation increased the oxidative stress that further enhances the expression of key apoptotic proteins and leads to neuronal cell death. We also investigated the indirect effect of radiation on neuronal cells in the presence of glial cells in a transwell co-culture system, while our analysis was focused on neuronal cells. Irradiated cell-conditioned medium (ICCM) was used as source of indirect radiation and neuroprotective effect was analyzed by various endpoints. It was observed that ICCM-induced reactive oxidative species level was significantly reduced in SH-SY5Y cells co-cultured with glial U87 cells, which might help to maintain the integrity of mitochondrial membrane potential. Increased levels of antioxidant enzyme superoxide dismutase and antioxidant glutathione were observed in SH-SY5Y cells co-cultured with glial U87 cells. Moreover, it was also observed that co-culture with glial cells inhibits the expression of ICCM-induced apoptotic proteins, i.e. Bax, cytochrome c, and caspase-3 in SH-SY5Y cells. Hence, it can be speculated that in co-culture system glial cells may protect the neuronal SH-SY5Y cells by reducing the ICCM-induced oxidative stress and apoptotic death.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Survival; Coculture Techniques; Culture Media, Conditioned; Cytochromes c; Dose-Response Relationship, Drug; Gamma Rays; Glioma; Glutathione; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Neuroblastoma; Oxidative Stress; Protective Agents; Reactive Oxygen Species; Superoxide Dismutase

High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro.. In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells.. SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release.. Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclophilins; Cyclosporine; Cytochromes c; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; Nanoparticles; Neuroblastoma; Peptidyl-Prolyl Isomerase F; Phosphatidylserines; Reactive Oxygen Species; Saposins

Neuroblastoma is a neuroendocrine tumour derived from neural crest cells and it remains a major therapeutic challenge in pediatric oncology. As response rates to chemotherapy are low, surgery remains the only effective treatment but since many tumors have metastasized at the time of diagnosis, curative surgery is rarely achieved. Consequently, a substantial need for new therapeutic options emerges. Quercetin a flavonoid, has been reported to lower the risk of several cancers. This study was designed to investigate its effects on apoptosis induction in the N2a, a mouse neuroblastoma cell line. The cell viability was determined by dimethyl thiazolyl tetrazolium bromide assay and diamidino-2-phenylindole staining was performed to confirm the apoptosis. The gene expression of bcl-w, p53, p27 and protein expression of caspases (3 and 9), bax, cytochrome-c were studied. This in vitro outcome suggests that quercetin can be used as a potent anti-cancer drug in future.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Gene Expression; Indoles; Mice; Neuroblastoma; Polymerase Chain Reaction; Proteins; Quercetin; RNA, Messenger; Tumor Suppressor Protein p53

Prion diseases are a group of infectious neurodegenerative diseases characterized by neuronal death and degeneration. Human leukocyte antigen-B-associated transcript 3 (BAT3) is an important apoptosis regulator. We therefore investigated the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106-126-induced apoptosis.. BAT3 and prion protein were overexpressed in Hela, Neuro2A, or primary neuronal cells by transfection with BAT3-HA or PRNP-EGFP expression plasmids and their relationship studied by immunofluorescence and Western blotting. The effect of BAT3 on PrP106-126-induced cytotoxicity and apoptosis was detected by the CCK-8 assay and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The expression of cytochrome c and Bcl-2 was examined by Western blotting.. BAT3 interacted with prion protein and enhanced PrP expression. After PrP106-126 peptide treated, BAT3 was transported from the nucleus to cytoplasm, increased cell viability, and protected neurons from PrP106-126-induced apoptosis through stabilizing the level of Bcl-2 protein and inhibiting the release of cytochrome c to cytoplasm.. Our present data showed a novel molecular mechanism of PrP106-126-induced apoptotic process regulation through the overexpression of BAT3, which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Green Fluorescent Proteins; Humans; Models, Biological; Molecular Sequence Data; Neuroblastoma; Neurons; Peptide Fragments; Protein Transport; Proteins; PrPC Proteins; Rats; Rats, Sprague-Dawley

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase-3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl-xl and Bcl-2 protein levels. Over-expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2AC ) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen-activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over-expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA-induced cell death. Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability. We found that PTPA located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines by decreasing mitochondrial membrane potential, which leads to translocation of Bax and a simultaneous release of Cyt C. In the cells with tau over-expression, PTPA knockdown inactivated PP2A to phosphorylate tau to avoid cell apoptosis which induced by PTPA knockdown.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Cytoplasm; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice; Mitochondria; Neuroblastoma; Phosphoprotein Phosphatases; Phosphorylation; Sincalide; tau Proteins

The sex steroid hormone 17β-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H(2)O(2)-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H(2)O(2)-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H(2)O(2) treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB-cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERβ) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERβ activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H(2)O(2) toxicity.

Topics: Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Globins; HeLa Cells; Humans; Hydrogen Peroxide; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Neuroglobin; RNA Interference; RNA, Small Interfering; Transfection; Up-Regulation

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.

Topics: Apoptosis; Azoles; bcl-2-Associated X Protein; Buthionine Sulfoximine; Cell Line, Tumor; Cell Survival; Coculture Techniques; Cytochromes c; Cytosol; Ethacrynic Acid; Glioblastoma; Glutathione; Humans; Isoindoles; Mitochondria; Neuroblastoma; Neurons; Organoselenium Compounds; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

Topics: Apoptosis; Caspases; Cell Differentiation; Cell Line, Tumor; Child, Preschool; Cytochromes c; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Infant; Mitochondria; N-Myc Proto-Oncogene Protein; Neoplasm Regression, Spontaneous; Neoplasm Staging; Nervous System Neoplasms; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Prognosis; Signal Transduction; Survival Analysis; Xenograft Model Antitumor Assays

Streptococcus pneumoniae causes the most severe form of the bacterial meningitis which is the major cause of bacterial meningitis. Virulence factors produced by S. pneumoniae have been known to contribute significantly to the disease process. ClpP protease (ClpP) which is essential for virulence and survival under stress conditions in S. pneumoniae was examined for the ability to induce apoptosis and the mechanism of the induction of apoptosis in human neuron-like cells, SK-N-SH neuroblastoma cells. ClpP inhibited cell growth and induced apoptosis in SK-N-SH cells. Treatment with ClpP resulted in hypodiploid DNA contents, increased Bax/Bcl-2 ratio and induction of reactive oxygen species (ROS) production. The release of cytochrome c from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was not observed in ClpP-treated cells. In addition, pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum caspase inhibitor, could not rescue apoptotic cells from ClpP toxicity. Coincidently, caspase-3 and -8 activation and cleavage of PARP were not detected. Moreover, caspase independent apoptosis-inducing factor (AIF) was released from mitochondria and translocated to the nucleus in response to ClpP. We also found that ClpP treatment resulted in the increase of p53 activity and cytoplasmic p53 levels were increased by ClpP, suggesting that functional activation of p53 is intact despite increased cytoplasmic accumulation. Taken together, these data suggest that ClpP contributes to neuronal damage in meningitis and provide further insight into the mechanisms underlying action of pneumococcal virulence factors during bacterial pathogenesis.

Topics: Apoptosis; Bacterial Proteins; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; Cytochromes c; Endopeptidase Clp; Humans; Mitochondria; Neuroblastoma; Reactive Oxygen Species; Serine Endopeptidases; Signal Transduction; Streptococcus pneumoniae; Tumor Suppressor Protein p53

2,2',4,4'-tetrabromodiphenyl ether (PBDE-47)-elicited neurotoxicity is associated with neural apoptosis; however the underlying mechanisms remain unclear. To investigate whether the mitochondrial p53 pathway is involved in neuronal apoptosis induced by PBDE-47 and to correlate DNA hypomethylation with p53 activation, human neuroblastoma (SH-SY5Y) cells were treated with different concentrations of PBDE-47 (1, 5, 10 μmol/L) for 24h in vitro. The apoptosis and ultrastructural alterations in cells, levels of p53, Bcl-2, Bax, cytochrome c (Cyt c), caspase-3 and methylation status of p53 promoter were determined. Hoechst 33258 staining and transmission electron microscopy analysis showed that PBDE-47 induced SH-SY5Y cells apoptosis characterized by the typical apoptotic morphological changes. In addition, PBDE-47 activated the p53-dependent mitochondrial apoptotic pathway as evidenced by up-regulation of p53 and Bax, down-regulation of Bcl-2 and Bcl-2/Bax ration, enhancement of Cyt c release from mitochondria into the cytosol, activation of caspase-3 as well as ultrastructural abnormalities of mitochondria. However, no obvious decrease in p53 promoter methylation levels was observed in any of the treatment groups by bisulfite genomic sequencing. Collectively, these results suggest that the mitochondrial p53 pathway is involved in PBDE-47-induced SH-SY5Y cells apoptosis, nevertheless p53 promoter hypomethylation may not be implicated in this process.

Topics: Apoptosis; Base Sequence; Caspase 3; Cell Line, Tumor; Cytochromes c; Halogenated Diphenyl Ethers; Humans; Methylation; Mitochondria; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

To screen, identify and verify the serum specific protein of neuroblastoma in a tumor-bearing murine model and speculate about the source of cytochrome C.. NB cells (KP-N-NS) were inoculated subcutaneously into nude mice. And the sera samples of tumor-bearing mice (observation group, n = 14) and controls (control group, n = 25) were collected at 4 weeks to screen for and identify differentially expressed proteins. The method of surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was employed to screen the serum specific protein of neuroblastoma between two groups. Then serum protein targets were purified by high-performance liquid chromatography (HPLC) and identified by LC-MS/MS (LTQ).. By comparing protein peaks among two sera groups, we identified the peak (11 609) showing significant differential expression between two groups. The expression of peak (11 609) was 3338.4 ± 1410.9 in observation group and 59.8 ± 40.7 in control group. This peak was identified as murine cytochrome C.. Cytochrome C is a serum specific protein for neuroblastoma tumor and it comes from the apoptosis of non-tumor cells.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cytochromes c; Female; Mice; Mice, Nude; Neuroblastoma; Peptide Mapping

Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Cell Survival; Copper; Cytochromes c; Ditiocarb; Enzyme Activation; Humans; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Cisplatin induces apoptosis through different pathways. The intrinsic apoptotic pathway is mediated by mitochondria, which, as a result of cisplatin treatment, undergo morphological alterations. The aim of this study was to investigate cisplatin-induced mitochondrial functional and morphological long-term effects in neuroblastoma B50 rat cells. To this purpose, we followed evaluated different several apoptotic markers by means of flow cytometry, confocal and electron microscopy and western blotting techniques. We applied different treatment protocols based on the incubation of the neuroblastoma B50 rat cells with 40 μM cisplatin: (i) for 48 h and harvesting of the cells at the end of the treatment; (ii) further recovery in drug-free medium for 7 days post-treatment; (iii) conditions as in (ii) followed by re-seeding in normal medium and growth for a further 4 days. We observed apoptosis induction after the first treatment and after the recovery from cell death after long-term culture in drug-free medium. Interestingly, the latter phenomenon was characterized by mitochondrial elongation and mitochondrial protein rearrangement. In recovered and re-seeded cells, mitochondrial equilibrium moved toward fusion, possibly protecting cells from apoptosis.

Topics: Actins; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA, Mitochondrial; Drug Resistance, Neoplasm; Flow Cytometry; GTP Phosphohydrolases; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial Dynamics; Neuroblastoma; Neurons; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Rats; Sirtuin 1; Time Factors

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.

Topics: Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Camptothecin; Caspase 3; Cell Line, Tumor; Cytochromes c; DNA Damage; Doxorubicin; Etoposide; Gene Expression Regulation; Glycogen Synthase Kinase 3; Humans; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Protein Multimerization; Protein Transport; Signal Transduction; Tumor Suppressor Protein p53

The purpose of this study was to investigate the anti-tumor effects of the isatin in vitro and in vivo. Human neuroblastoma cells (SH-SY5Y) were exposed to isatin at various concentrations (0, 50, 100, 200 μmol/l) for 48 h. Bcl-2 and Bax mRNA were analyzed via RT-PCR. Bcl-2, Bax, the inhibitor of caspase-activated DNase (ICAD) and cytochrome c protein were analyzed via western blot. Apoptosis, caspase-9, 3 activation and mitochondrial depolarization were assayed by flow cytometry. SH-SY5Y cells were injected into the right side of the mouse armpit. When the neoplasm was detected, the nude mice were randomly divided into four groups and received an injection of DMEM (negative control), 25 or 50mg/kg isatin, or cyclophosphamide (positive control). The inhibitory effects of isatin on the murine xenograft were determined using a growth curve and Bcl-2 and Bax mRNA and protein were studied using RT-PCR and western blot, respectively. The results showed that apoptosis of SH-SY5Y cells was induced by isatin. Furthermore, Bcl-2 expression was decreased and the ratio of Bcl-2 to Bax was significantly decreased by isatin. The mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c into the cytosol was increased after treatment with isatin. Simultaneously, caspase-9, 3 was activated, followed by degradation of ICAD, a caspase-3 substrate. Finally, tumor xenograft growth was markedly suppressed and a decrease was found in Bcl-2 and Bax expression in vivo. These results suggest that isatin can induce apoptosis and inhibit the growth of neuroblastoma cells via the mitochondrial pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Female; Humans; Isatin; Mice; Mice, Nude; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tumor Burden; Xenograft Model Antitumor Assays

Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Blood-Brain Barrier; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cytochromes c; DNA Fragmentation; Endothelial Cells; Humans; Lignans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred ICR; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tight Junctions

Sorafenib is a multikinase inhibitor that is approved for use against renal cell and hepatocellular carcinoma. We found that sorafenib potently induced cell death in human neuroblastoma cells. To understand the molecular basis of sorafenib-mediated cell death in human SH-SY5Y cells, we performed a temporal quantitative proteome analysis. The results showed significant quantitative changes of 193 unique proteins. Bioinformatics-assisted pathway analysis of the regulated proteins revealed that mitochondrial proteins, especially components of the electron transport chain and the mitochondrial ribosomes, were significantly affected upon exposure to sorafenib. The observed down-regulation of the respiratory chain complex I (NADH dehydrogenase) was accompanied with loss of mitochondrial transmembrane potential (Δψm) and complete impairment of complex I enzyme activity. The destabilization of complex I subunits was consistent, rapid, and independent of caspase activation as well as Bcl-2 overexpression. This study provides an overview of the molecular machinery driving sorafenib-mediated cell death in neuroblastoma cells and suggests that sorafenib could be a potential therapeutic drug for the treatment of neuroblastoma.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzenesulfonates; Cell Line, Tumor; Cell Shape; Cytochromes c; Down-Regulation; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mitochondria; NADH Dehydrogenase; Neuroblastoma; Niacinamide; Phenylurea Compounds; Protein Interaction Maps; Proteolysis; Proteome; Pyridines; Reactive Oxygen Species; Signal Transduction; Sorafenib; Superoxide Dismutase

Neuroblastoma (NB) is one of the most common malignant solid tumors of childhood. It is still not clear whether the apoptosis of tumor cells or the non-tumor cells contributes to the increase of concentration of cytochrome c (Cyt c) in the serum of the cancer patients. The aim of this research was to identify the source of the Cyt c in the serum when the tumor grows up by subcutaneous inoculation of human NB cells into nude mice.. We subcutaneously inoculated human NB cells (KP-N-NS) into nude mice and collected the sera of tumor-bearing mice (n = 14) and control mice (n = 25) 4 weeks later in order to screen for and identify differentially expressed proteins in the serum. Differentially expressed proteins in the serum were screened by surface-enhanced laser desorption/ionization-time-of-flight (SELDI-TOF) mass spectrometry.. The relative intensity of a protein having a mass-to-charge ratio (m/z) of 11 609 was 3338.37 ± 3410.85 in the tumor group and 59.84 ± 40.74 in the control group, indicating that the expression level of this protein in the tumor group was 55.8 times higher than that in the control group. Serum proteins were separated and purified by high-performance liquid chromatography (HPLC). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to produce peptide mass fingerprints (PMFs). Spectrum analysis and a database search revealed that the highly expressed protein (m/z = 11 605.4) from the serum of tumor-bearing mice was the mouse Cyt c.. Increased concentration of Cyt c in the serum of tumor-bearing nude mice might be partially attributed to the secretion of this protein by non-tumor cells.

Topics: Animals; Apoptosis; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytochromes c; Female; Humans; Mice; Mice, Nude; Neuroblastoma; Tandem Mass Spectrometry; Xenograft Model Antitumor Assays

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP(+)), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Blotting, Western; Cell Line, Tumor; Cytochromes c; Electron Transport Complex II; Gene Expression; Humans; Membrane Potential, Mitochondrial; Membrane Transport Proteins; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Mitochondrial Uncoupling Proteins; Neuroblastoma; Oxygen Consumption; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Topics: Activating Transcription Factor 6; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Cell Line, Tumor; Cytochromes c; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Membrane Proteins; Mice; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor CHOP; Up-Regulation

We investigated the mechanisms underlying the protective effects of loganin against hydrogen peroxide (H(2)O(2))-induced neuronal toxicity in SH-SY5Y cells. The neuroprotective effect of loganin was investigated by treating SH-SY5Y cells with H(2)O(2) and then measuring the reduction in H(2)O(2)-induced apoptosis using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays. Following H(2)O(2) exposure, Hoechst 33258 staining indicated nuclear condensation in a large proportion of SH-SY5Y cells, along with an increase in reactive oxygen species (ROS) production and an intracellular decrease in mitochondria membrane potential (MMP). Loganin was effective in attenuating all the above-stated phenotypes induced by H(2)O(2). Pretreatment with loganin significantly increased cell viability, reduced H(2)O(2)-induced LDH release and ROS production, and effectively increased intracellular MMP. Pretreatment with loganin also significantly decreased the nuclear condensation induced by H(2)O(2). Western blot data revealed that loganin inhibited the H(2)O(2)-induced up-regulation of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3, increased the H(2)O(2)-induced decrease in the Bcl-2/Bax ratio, and attenuated the H(2)O(2)-induced release of cytochrome c from mitochondria to the cytosol. Furthermore, pretreatment with loganin significantly attenuated the H(2)O(2)-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results suggest that the protective effects of loganin against H(2)O(2)-induced apoptosis may be due to a decrease in the Bcl-2/Bax ratio expression due to the inhibition of the phosphorylation of JNK, p38, and ERK 1/2 MAPKs. Loganin's neuroprotective properties indicate that this compound may be a potential therapeutic agent for the treatment of neurodegenerative diseases.

Topics: Apoptosis; Blotting, Western; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Hydrogen Peroxide; Iridoids; L-Lactate Dehydrogenase; Membrane Potentials; Neuroblastoma; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinases; Reactive Oxygen Species

The present study investigates the protective effects of water extract of ginseng (Panax ginseng C.A. Meyer) against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced cytotoxicity in SH-SY5Y human neuroblastoma cells and explores the underlying mechanisms. The approach may be used for screening therapeutic agents for degenerative disorders such as Parkinson's disease.. SH-SY5Y human neuroblastoma cells were used to analyze the protective effects of water extract of ginseng (WEG) against multiple parameters such as MPP(+)-induced viability, oxidative injury, expression of Bax, Bcl-2, cytochrome c and cleaved caspase-3.. WEG exerted inhibitory effect on cell death, overproduction of ROS, elevated Bax/Bcl-2 ratio, release of cytochrome c and activation of caspase-3 expression in MPP(+)-treated SH-SY5Y cells.. WEG exhibited significant protective effects against MPP(+)-induced cytotoxicity in SH-SY5Y cells possibly through the suppression of ROS generation and the inhibition of mitochondria-dependent apoptotic pathway.

Topics: 1-Methyl-4-phenylpyridinium; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; Drug Evaluation, Preclinical; Enzyme Inhibitors; Herbicides; Humans; Mitochondria; Neuroblastoma; Neurotoxins; Panax; Parkinson Disease; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

It was previously shown that [Pt(O,O'-acac)(γ-acac)(DMS)] induces apoptosis in various cancer cells and exerts antimetastatic responses in vitro. In rats, [Pt(O,O'-acac)(γ-acac)(DMS)] reaches the central nervous system in quantities higher than cisplatin causing less excitotoxicity. The aim of the present paper was to investigate whether [Pt(O,O'-acac)(γ-acac)(DMS)] is able to exert cytotoxic effects on SH-SY5Y human neuroblastoma cell line, and to study the intracellular transduction mechanisms underlying these effects. Here we have demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] was more effective than cisplatin in provoking apoptosis characterized by: (a) mitochondria depolarization, (b) decrease of Bcl-2 expression and increase of BAX expressions with cytosol-to-mitochondria translocation, (c) activation of caspase-7 and -9 and (d) generation of reactive oxygen species (ROS). [Pt(O,O'-acac)(γ-acac)(DMS)] provoked the activation of the following signalling kinases that were interacting with each other: PKC-δ and -ɛ, ERK1/2, p38MAPK, JNK1/2, NF-κB, c-src and FAK. We found that ROS generated by NADPH oxidase was responsible for the [Pt(O,O'-acac)(γ-acac)(DMS)]-mediated PKC-δ and -ɛ activation and consequential phosphorylation of all MAPKs. [Pt(O,O'-acac)(γ-acac)(DMS)]-induced mitochondrial apoptosis was blocked when p38MAPK and JNK1/2 were inhibited, whilst the effects on Bax/Bcl-2 mRNA and protein levels were blocked inhibiting NF-κB. NF-κB nuclear translocation was blocked inhibiting MEK1/2 activity. In addition to the induction of apoptosis [Pt(O,O'-acac)(γ-acac)(DMS)] downregulated pro-survival pathway. Survival inhibition started from mitochondrial ROS generation which induced c-src, FAK and Akt activation. In conclusion, our results suggest that [Pt(O,O'-acac)(γ-acac)(DMS)] may be considered a promising compound for the treatment of neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of this compound.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cytochromes c; Humans; Mitochondria; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Organoplatinum Compounds; Phosphorylation; Rats; Reactive Oxygen Species; Signal Transduction

Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 μM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 μM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 μM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

Topics: Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Glutathione; Humans; Neuroblastoma; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue; Ubiquinone

Mangiferin (MGN), a C-glucosylxanthone was investigated for its ability to protect against methylmercury (MeHg) induced neurotoxicity by employing IMR-32 (human neuroblastoma) cell line. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and clonogenic cell survival assays confirmed the efficacy of MGN supplementation in attenuating MeHg-induced cytotoxicity. Pre-treatment with MGN significantly (p<0.01) inhibited MeHg-induced DNA damage (micronuclei, olive tail moment and % tail DNA) thereby demonstrating MGN's antigenotoxic potential. Also, pre-treatment with MGN significantly reduced MeHg-induced oxidative stress, intra-cellular Ca(2+) influx and inhibited depolarization of mitochondrial membrane. MGN pre-treated cells demonstrated a significant (p<0.05) increase in the GSH and GST levels followed by a significant (p<0.05) decrease in malondialdehyde (MDA) formation. In addition, inhibition of MeHg induced apoptotic cell death by MGN was demonstrated by microscopic, Annexin-V FITC and DNA fragmentation assays and further confirmed by western blot analysis. The present findings indicated the protective effect of MGN against MeHg induced toxicity, which may be attributed to its anti-genotoxic, anti-apoptotic and anti-lipid peroxidative potential plausibly because of its free radical scavenging ability, which reduced the oxidative stress and in turn facilitated the down-regulation of mitochondrial apoptotic signalling pathways.

Topics: Annexin A5; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Survival; Comet Assay; Cytochromes c; Cytokinesis; Cytoprotection; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Free Radical Scavengers; Humans; Inhibitory Concentration 50; Intracellular Space; Membrane Potential, Mitochondrial; Metallothionein; Methylmercury Compounds; Micronucleus Tests; Necrosis; Neuroblastoma; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tubulin; Tubulin Modulators; Tumor Stem Cell Assay; Xanthones

Luteolin, a dietary flavonoid, induces apoptosis in various types of cancer cells. However, its role in neuroblastoma and the underlying mechanisms remain to be elucidated. In the present study, we investigated the molecular mechanisms of the anti-cancer effect of luteolin in Neuro-2a mouse neuroblastoma cells. Luteolin induced apoptotic cell death and activation of caspase-12, -9, and -3, and knockdown of caspase-12 by siRNA transfection reduced luteolin-induced cell death. Luteolin also induced expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated proteins (GRP) 94 and 78, cleavage of ATF6α, and phosphorylation of eIF2α. CHOP knockdown or ER stress inhibitor, 4-phenylbutyric acid, reduced luteolin-induced cell death. These results suggest involvement of ER stress in luteolin-induced neuroblastoma cell death. We then showed that luteolin induced accumulation of reactive oxygen species and that the anti-oxidant N-acetylcysteine reduced luteolin-induced cell death and expression of CHOP and GRP78. We also demonstrated rapid reduction of mitochondrial membrane potential by luteolin, and N-acetylcysteine, as well as 4-phenylbutyric acid or CHOP siRNA transfection ameliorated luteolin-induced late loss, but not early loss of mitochondrial membrane potential. Finally, we showed that luteolin induced activation of mitogen-activated protein kinases such as JNK, p38, and ERK, and inhibitors of mitogen-activated protein kinases reduced luteolin-induced cell death and CHOP expression, as well as mitochondrial Bax translocation and cytochrome c release. Collectively, our results suggest that luteolin induces apoptosis through ER stress and mitochondrial dysfunction in Neuro-2a mouse neuroblastoma cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Luteolin; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitogen-Activated Protein Kinases; Neuroblastoma; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Transcription Factor CHOP

Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma.. Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined.. Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR).. Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cytochromes c; Disease-Free Survival; Female; Fenretinide; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Neuroblastoma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

Topics: Analysis of Variance; Animals; Antioxidants; Apoptosis; Caspase 3; Cell Cycle; Cell Proliferation; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Female; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Photosensitizing Agents; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Pyrimidinones; Rats; Rats, Sprague-Dawley; Serine; Signal Transduction; Thioctic Acid; Time Factors; Zinc

Bcl-2 family proteins regulate mitochondrial apoptosis downstream of diverse stressors. Cancer cells frequently deregulate Bcl-2 proteins leading to chemoresistance. We have optimized a platform for solid tumors in which Bcl-2 family resistance patterns are inferred. Functional mitochondria were isolated from neuroblastoma (NB) cell lines, exposed to distinct BH3-domain peptides, and assayed for cytochrome c release. Such BH3 profiles revealed three patterns of cytochrome c response. A subset had a dominant NoxaBH3 response implying Mcl1 dependence. These cells were more sensitive to small molecules that antagonize Mcl1 (AT-101) than those that antagonize Bcl-2, Bcl-xL and Bcl-w (ABT-737). A second subset had a dominant BikBH3 response, implying a Bcl-xL/-w dependence, and was exquisitely sensitive to ABT-737 (IC(50) <200 nM). Finally, most NB cell lines derived at relapse were relatively resistant to pro-death BH3 peptides and Bcl-2 antagonists. Our findings define heterogeneity for apoptosis resistance in NB, help triage emerging Bcl-2 antagonists for clinical use, and provide a platform for studies to characterize post-therapy resistance mechanisms for NB and other solid tumors.

Topics: Apoptosis; Biphenyl Compounds; Cell Line; Cell Line, Tumor; Cytochromes c; Gossypol; Humans; Immunoblotting; Immunoprecipitation; Mitochondria; Neuroblastoma; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, alpha-tocopheryl succinate (alpha-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that alpha-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, alpha-TOS stimulates rapid Ca(2+) entry into the cells with subsequent accumulation of Ca(2+) in mitochondria-a prerequisite step for MPT induction. Alteration of mitochondrial Ca(2+) buffering capacity was observed as early as 8 hr after incubation with alpha-TOS, when no activation of Bax was yet detected. Ca(2+) accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca(2+) uptake significantly mitigated the apoptotic response. Importantly, Ca(2+)-induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria.

Topics: alpha-Tocopherol; Animals; Antioxidants; Calcium; Caspase 3; Cell Membrane Permeability; Cells, Cultured; Cytochromes c; Humans; Hydrogen Peroxide; Jurkat Cells; Male; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neuroblastoma; Oxidants; Permeability; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tocotrienols; Vitamin E

It has been a puzzle why the tangle-bearing neurons in Alzheimer's disease (AD) brain do not die preferentially of apoptosis even though they are actually challenged by multiple proapoptotic factors. Recently, we have reported that phosphorylation of tau can antagonize apoptosis induced by exogenous apoptotic inducers. Amyloid-beta (Abeta), a recognized endogenous proapoptotic factor, is significantly increased in the AD brains, however, it is not known whether tau could abate the Abeta-potentiated apoptosis. Here, we observed that the cells bearing high level of Abeta were more vulnerable than the controls to H2O2-induced apoptosis, and this effect of Abeta was associated with decrease of Bcl-2, elevation of Bax and cytosolic cytochrome-c, as well as activation of caspase-3, suggesting that Abeta could potentiate the oxidant-induced cell apoptosis with involvement of mitochondria-caspase-3 pathway. More importantly, we also found that expression of tau that became hyperphosphorylated could reduce the Abeta-potentiated apoptosis with simultaneous preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity, implying that overexpression of tau that became hyperphosphorylated can attenuate the Abeta-potentiated cell apoptosis through mitochondria-caspase-3 pathway. These findings provide an explanation of the chronic nature of neurodegeneration of neurons with neurofibrillary pathology of abnormal hyperphosphorylated tau in AD and related tauopathies.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Mice; Neuroblastoma; Peptide Fragments; Signal Transduction; tau Proteins; Transfection

The formation of neurofibrillary tangles, mainly composed of hyperphosphorylated tau protein, is a hallmark in the brain of human tauopathies, including Alzheimer's disease (AD). Although neurons bearing neurofibrillary tangles are constantly exposed to various apoptotic stimuli, they do not appear to preferentially die by apoptosis. The underlying mechanism for such resistance to apoptosis remains elusive. Previously, we studied the role of tau phosphorylation in apoptosis and found that tau hyperphosphorylation by glycogen synthase kinase-3 (GSK-3) rendered cells more resistant to apoptosis. In this study, we show that the overexpression of tau without any exogenous activation of kinases also confers increased resistance to apoptosis in both N2a cells and in a tau transgenic mouse model. Mechanistically, the overexpression of tau was associated with a reduced p53 level, decreased release of cytochrome C from mitochondria, and inhibition of caspases-9/-3. Additionally, a decreased phosphorylation and increased nuclear translocation of beta-catenin were also detected in N2a/tau cells, and knockdown of beta-catenin eliminated the anti-apoptotic effect of tau. Furthermore, tau was spontaneously hyperphosphorylated upon overexpression and by staurosporine treatment. The phosphorylation level of p53 decreased upon tau overexpression, and a more profound reduction of the phosphorylated p53 was detected when the cells were treated with lithium and roscovitine, inhibitors of GSK-3 and cyclin-dependent kinase-5 (Cdk-5). These results suggest that the overexpression of tau, which may be hyperphosphorylated by endogenous GSK-3 and Cdk-5, is anti-apoptotic by mechanisms involving modulation of multiple anti-apoptotic factors, including beta-catenin and p53-mitochondria-caspase-mediated apoptotic pathways.

Topics: Animals; Annexin A5; Apoptosis; beta Catenin; Camptothecin; Caspase 3; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Green Fluorescent Proteins; Hippocampus; Humans; Maytansine; Mice; Mice, Transgenic; Mutation; Neuroblastoma; RNA, Small Interfering; Staurosporine; Subcellular Fractions; tau Proteins; Time Factors; Transfection; Tumor Suppressor Protein p53

Diclofenac, a nonsteroidal anti-inflammatory drug, induces apoptosis on the neuroblastoma cell line SH-SY5Y through a mitochondrial dysfunction, affecting some antioxidant mechanisms. Indeed, the time- and dose-dependent increase of apoptosis is associated to an early enhancement of the reactive oxygen species (ROS). Mitochondrial superoxide dismutase (SOD2) plays a crucial role in the defence against ROS, thus protecting against several apoptotic stimuli. Diclofenac decreased the protein levels and the enzymatic activity of SOD2, without any significant impairment of the corresponding mRNA levels in the SH-SY5Y extracts. When cells were incubated with an archaeal exogenous thioredoxin, an attenuation of the diclofenac-induced apoptosis was observed, together with an increase of SOD2 protein levels. Furthermore, diclofenac impaired the mitochondrial membrane potential, leading to a release of cytochrome c. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis of SH-SY5Y cells and point to a possible role of SOD2 in this process.

Topics: Antioxidants; Apoptosis; Cell Line, Tumor; Cytochromes c; Diclofenac; Gene Expression Regulation, Neoplastic; Humans; Intracellular Space; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Protein Transport; Reactive Oxygen Species; RNA, Messenger; Subcellular Fractions; Superoxide Dismutase; Thioredoxins

To study the effect of necrostatin (Nec-1) on apoptosis induced by aluminum (Al), and approach the mechanism.. Neural cell death model was made by 4 mmol/L Al treated neuroblastoma cells (SH-SY5Y). Cell viabilities were detected at different concentrations of Al and/or Nec-1. Hoechst 33342/PI double staining was used to observe apoptosis and (or) necrosis that were quantified by flow cytometry using Annexin V/PI double staining. Apoptotic pathway was tested by activities of Caspase-3, Caspase-8 and Caspase-9. In addition, the expression of NF-kappa B and Cyt-c was measured by immunocytochemistry.. Cell viabilities were significantly decreased with the increasing concentrations of Al (P < 0.05), which could be significantly upregulated by 60 micromol/L Nec-1 (P < 0.05) and were correlated with the concentrations of Nec-1 (P < 0.05, P < 0.01). Apoptosis and necrosis were observed under fluorescent microscope and quantified by flow cytometry, which suggested an increasing trend of apoptotic and necrotic rates (P < 0.05, P < 0.01). Whereas, Nec-1 could not only decrease the necrotic rate but also apoptotic rate as well (P < 0.05, P < 0.01). Data of Nec-1 on caspases activities showed that Nec-1 could not affect Caspase-9 activity (P > 0.05) and Cty-c protein expression as well (P > 0.05). However, Nec-1 could reduce Caspase-8 activity significantly (P < 0.05, P < 0.01) and increase NF-kappa B protein expression (P < 0.05, P < 0.01) and finally decrease Caspase-3 activity (P < 0.05).. Nec-1 could reduce cell apoptosis induced by Al, through Caspase-8 pathway, and up-regulate the expression of NF-kappa B protein.

Topics: Aluminum; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Imidazoles; Indoles; Neuroblastoma; NF-kappa B

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X(L) and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death.

Topics: Analgesics, Opioid; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cytochromes c; Dose-Response Relationship, Drug; Electron Transport; Electron Transport Complex II; Humans; Methadone; Mice; Mice, Knockout; Mitochondria, Liver; Mitochondrial Proton-Translocating ATPases; Necrosis; Neuroblastoma; Protein Transport; Rats; Reactive Oxygen Species

The etiology of Parkinson's disease (PD) is not completely defined, although environmental factors (for example, exposure to the herbicide paraquat [PQ]) and genetic susceptibility (such as DJ-1 mutations that have been associated with an autosomal-recessive form of early-onset PD) have been demonstrated to contribute. Alterations in macroautophagy have been described in the pathogenesis of this neurodegenerative disease. We have established a model system to study the involvement of the DJ-1 protein in PQ-induced autophagy. When we transfected cells exposed to PQ with DJ-1-specific siRNA, we observed an inhibition of the autophagic events induced by the herbicide, as well as sensitization additive with PQ-induced apoptotic cell death and exacerbation of this cell death in the presence of the autophagy inhibitor 3-methyladenine. These results suggest, for the first time, an active role for DJ-1 in the autophagic response produced by PQ, opening the door to new strategies for PD therapy.

Topics: Adenine; Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Herbicides; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Models, Biological; Neuroblastoma; Oncogene Proteins; Paraquat; Protein Deglycase DJ-1; Reactive Oxygen Species; RNA Interference

Hsp20 is chaperone protein that is highly and constitutively expressed in the brain, cardiac tissue and many other organs. Recently, it is well established that Hsp20 can enhance cardiac function and render cardioprotection. However, the potential benefits of Hsp20 and its phosphorylation form action on ischemic stroke and its underlying mechanism(s) are largely unknown.To investigate whether Hsp20 exerts protective effects in vitro ischemia/reperfusion (I/R) injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation (OGD) and reoxygenation. Expression mRNA and protein levels of Hsp20 were strongly downregulated in mouse N2A cells at the 0-hour and 6-hour recovery time points following 4 hours of OGD, and returned to basal level 12 and 24 hours after OGD treatment. The ratio of phosphorylated to total Hsp20 protein was not significantly affected by OGD treatment at the 0-hour and 6-hour recovery time points following 4 hours of OGD. However, markedly higher serine phosphorylation of Hsp20 was observed 12 and 24 hours after OGD treatment. Furthermore, overexpression of Hsp20 reduced OGD-induced apoptosis by reducing the release of cytochrome c from mitochondria to cytosol. However, blockade of Hsp20 phosphorylation at Ser16 abrogated this anti-apoptotic effect.Our data demonstrate that increased Hsp20 expression in mouse N2A neuroblastoma cells protects against I/R injury, resulting in reduced apoptosis, which is by reducing the release of cytochrome c from mitochondria to cytosol. Phosphorylation of Ser16 plays an important role in its protective effect. Thus, Hsp20 may constitute a new therapeutic target for cerebral ischemic diseases.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cytochromes c; Gene Expression Regulation, Neoplastic; Glucose; Green Fluorescent Proteins; HSP20 Heat-Shock Proteins; Hypoxia; Mice; Mitochondria; Mutation; Neuroblastoma; Phosphorylation; RNA, Messenger; Time Factors

13-cis-Retinoic acid (13-cis-RA) is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immunoblotting. Pretreatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC(99)) increased from clinically achievable levels to nonachievable levels, greater than 5-fold (cisplatin) to greater than 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxicity for neuroblastoma in vitro, mediated at least in part via the antiapoptotic Bcl-2 family of proteins.

Topics: Alkylating Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cisplatin; Cytochromes c; Cytoprotection; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Isotretinoin; Melphalan; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Nitrophenols; Piperazines; Protective Agents; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Signal Transduction; Sulfonamides; Topoisomerase Inhibitors; Tumor Suppressor Protein p53

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Autoradiography; Benzoquinones; Caspase 3; Cell Line, Tumor; Copper; Cytochromes c; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; NAD; Neuroblastoma; Phenanthrolines; Phosphorus Isotopes; Salsoline Alkaloids; Tetrahydroisoquinolines; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine 3-Monooxygenase

The critical processes of mitochondrial fission and fusion are regulated by members of the dynamin family of GTPases. Imbalances in mitochondrial fission and fusion contribute to neuronal cell death. For example, increased fission mediated by the dynamin-related GTPase, Drp1, or decreased fusion resulting from inactivating mutations in the OPA1 GTPase, causes neuronal apoptosis and/or neurodegeneration. Recent studies indicate that post-translational processing regulates OPA1 function in non-neuronal cells and moreover, aberrant processing of OPA1 is induced during apoptosis. To date, the post-translational processing of OPA1 during neuronal apoptosis has not been examined. Here, we show that cerebellar granule neurons (CGNs) or neuroblastoma cells exposed to pro-apoptotic stressors display a novel N-terminal cleavage of OPA1 which is blocked by either pan-caspase or caspase-8 selective inhibitors. OPA1 cleavage occurs concurrently with mitochondrial fragmentation and cytochrome c release in CGNs deprived of depolarizing potassium (5K condition). Although a caspase-8 selective inhibitor prevents both 5K-induced OPA1 cleavage and mitochondrial fragmentation, recombinant caspase-8 fails to cleave OPA1 in vitro. In marked contrast, either caspase-8 or caspase-3 stimulates OPA1 cleavage in digitonin-permeabilized rat brain mitochondria, suggesting that OPA1 is cleaved by an intermembrane space protease which is regulated by active caspases. Finally, the N-terminal truncation of OPA1 induced during neuronal apoptosis removes an essential residue (K301) within the GTPase domain. These data are the first to demonstrate OPA1 cleavage during neuronal apoptosis and they implicate caspases as indirect regulators of OPA1 processing in degenerating neurons.

Topics: Animals; Apoptosis; Brain; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cells, Cultured; Cerebellum; Cytochromes c; Digitonin; GTP Phosphohydrolases; Humans; Mitochondria; Neuroblastoma; Neurons; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Staurosporine

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in stress-induced transcriptional activation and has been implicated in chemoresistance of cancers. In the present study, we investigated the role of NF-kappaB in inducible chemoresistance of neuroblastoma. Doxorubicin, VP16 and the cytotoxic ligand TRAIL trigger NF-kappaB activation, whereas cisplatin and taxol have no impact on NF-kappaB activity. Specific inhibition of NF-kappaB activation by overexpression of dominant-negative mutant IkappaBalpha-super-repressor does not alter cell death upon doxorubicin or VP16 treatment, although it prevents doxorubicin- or VP16-mediated NF-kappaB activation. By comparison, inhibition of TRAIL-stimulated NF-kappaB activation by IkappaBalpha-superrepressor or the small molecule NF-kappaB inhibitor BMS-345541 significantly enhances TRAIL-induced apoptosis, pointing to an antiapoptotic function of NF-kappaB in TRAIL-mediated apoptosis. Analysis of signaling pathways reveals that NF-kappaB inhibition prevents TRAIL-triggered up-regulation of Mcl-1, promoting TRAIL-induced cytochrome c release and activation of caspases. Accordingly, knockdown of Mcl-1 by RNA interference significantly enhances TRAIL-induced apoptosis and also increases sensitivity of neuroblastoma cells to CD95- or chemotherapy-induced apoptosis. In conclusion, NF-kappaB regulates apoptosis in a stimulus-specific manner in neuroblastoma cells and confers protection against TRAIL-induced apoptosis. By demonstrating that NF-kappaB inhibition sensitizes neuroblastoma cells for TRAIL-induced apoptosis, our findings have important implications. Thus, NF-kappaB inhibitors may open new perspectives to potentiate the efficacy of TRAIL-based protocols in the treatment of neuroblastoma.

Topics: Apoptosis; Cell Culture Techniques; Cell Death; Cytochromes c; Humans; Luciferases; Neuroblastoma; NF-kappa B; Retroviridae; RNA Interference; RNA, Catalytic; TNF-Related Apoptosis-Inducing Ligand

Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

Topics: Aldehydes; Analysis of Variance; Animals; Antimanic Agents; bcl-2-Associated X Protein; Brain; Cell Line, Tumor; Central Nervous System Stimulants; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Electron Transport Complex IV; Gene Expression Regulation; Humans; Lithium; Male; Methamphetamine; Mitochondria; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Organic Chemicals; Oxygen Consumption; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-jun; Rats; Rats, Inbred WKY; RNA, Small Interfering; Time Factors; Transfection; Tyrosine 3-Monooxygenase; Valproic Acid

To determine the involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in cytoprotection, we subjected N2-A cells to oxygen-glucose deprivation followed by reoxygenation (H-R). Following H-R insults, H(2)O(2) production was increased while cell viability declined, which was accompanied by loss of mitochondrial membrane potential (MMP), cytochrome c release, caspases 9 and 3 activation, poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis. Rosiglitazone up to 5 microM protected cell viability, normalized MMP, and prevented apoptotic signals. The protective effect of rosiglitazone was abrogated by GW9662, a PPAR-gamma antagonist, or a specific PPAR-gamma small interference RNA (siRNA) but not a control scRNA. PPAR-gamma overexpression alone was effective in maintaining MMP and preventing apoptosis and its protective effect was also abrogated by PPAR-gamma siRNA or GW9662. To elucidate the mechanism by which PPAR-gamma protects MMP and prevents apoptosis, we analyzed Bcl-2, Bcl-xl, and phosphorylated Bad (p-Bad). H-R suppressed them. Rosiglitazone or PPAR-gamma overexpression restored them via PPAR-gamma. Rosiglitazone or PPAR-gamma overexpression preserved phosphorylated Akt and 3-phosphoinositide-dependent kinase-1 (PDK-1) in a PPAR-gamma dependent manner. These results indicate that ligand-activated PPAR-gamma protects N2-A cells against H-R damage by enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Anilides; Animals; Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Caspase 3; Caspase 9; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytoprotection; Dose-Response Relationship, Drug; Glucose; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroblastoma; Phosphorylation; Poly(ADP-ribose) Polymerases; PPAR gamma; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; RNA Interference; RNA, Small Interfering; Rosiglitazone; Thiazolidinediones; Time Factors; Transfection; Up-Regulation

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.

Topics: Animals; Apoptosis; Cardiolipins; Cell Line, Tumor; Cloning, Molecular; Cross-Linking Reagents; Cytochromes c; HeLa Cells; Humans; Lipids; Mice; Neuroblastoma; Neurons; Oxidative Stress; Parkinson Disease; Peroxidases; RNA, Small Interfering; Synucleins

Fibril deposit formation of amyloid beta-protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Abeta oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Abeta small oligomers and fibrillar aggregates to simulate the action of diffusible Abeta oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Abeta. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Abeta peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.

Topics: Amyloid beta-Peptides; Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Neuroblastoma; Peptide Fragments; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Protein Transport; Signal Transduction; Substrate Specificity; Time Factors

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

Arachidonic acid (AA)-induced apoptosis of human neuroblastoma SK-N-SH cells was characteristic of elevation of intracellular Ca(2+) concentration ([Ca(2+)]i), ROS generation, activation of 38 MAPK and JNK and loss of mitochondrial membrane potential (DeltaPsim). Subsequent modulation of Bcl-2 family members and cytochrome c release accompanied with activation of caspase-9 and -3 were involved in the death of SK-N-SH cells. BAPTA-AM (Ca(2+) chelator) pretreatment rescued viability of AA-treated cells through abolishing phosphorylation of p38 MAPK and JNK, DeltaPsim loss and ROS generation. N-Acetylcysteine (ROS scavenger) pretreatment reduced the dissipation of DeltaPsim, but insignificantly affected AA-induced p38 MAPK and JNK activation. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) attenuated mitochondrial depolarization, degradation of Bcl-2/Bcl-xL, and mitochondrial translocation of Bax. Transfection of specific siRNA proved that p38alpha MAPK and JNK1 were involved in modulating Bcl-2 family proteins. Taken together, our data suggest that the cytotoxicity of AA toward SK-N-SH cells is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca(2+)-evoked activation of p38alpha MAPK and JNK1.

Topics: Apoptosis; Arachidonic Acid; Calcium; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 8; Neuroblastoma; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Transfection

Gold(III) porphyrin 1a is a new class of anticancer drug, which inhibits cell proliferation of wide range of human cancer cell lines and induces apoptosis in human nasopharyngeal carcinoma cells. However, the underlying signalling mechanism by which gold(III) porphyrin 1a modifies the intracellular apoptosis pathways in tumour cells has not been explained in detail in neuroblastoma cells.. Cell proliferation and apoptosis were determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V binding, respectively. Western blot assay was used to detect proteins involved in apoptotic and Akt pathways. In vivo tumour growth was assessed by inoculating tumour cells to nude mice subcutaneously, and gold(III) porphyrin 1a was administrated intravenously.. This study assessed the antitumour effect and mechanism of gold(III) porphyrin 1a on neuroblastoma in vitro and in vivo. Gold(III) porphyrin 1a displayed a growth inhibition and induction of apoptosis in neuroblastoma cells effectively in vitro, which was accompanied with release of cytochrome c and Smac/DIABLO and caspases activation. Further studies indicated that gold(III) porphyrin 1a inhibited X-linked inhibitor of apoptosis (XIAP). However, we found that gold(III) porphyrin 1a can induce a survival signal, Akt activation within minutes and could last for at least 24 h. To further confirm association between activation of Akt and the effectiveness of gold(III) porphyrin 1a, neuroblastoma cells were treated with API-2, an Akt-specific inhibitor. API-2 sensitised cells to gold(III) porphyrin 1a-induced apoptosis and growth inhibition.. These results suggested that Akt may be considered as a molecular 'brake' that neuroblastoma cells rely on to slow down gold(III) porphyrin 1a-induced apoptosis and antiproliferation. Gold(III) porphyrin 1a is a mitochondrial apoptotic stimulus but also activates Akt, suggesting an involvement of Akt in mediating the effectiveness to growth inhibition and apoptosis by gold(III) porphyrin 1a and that inhibition of Akt can enhance the anticancer activity of gold(III) porphyrin 1a in neuroblastoma.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Metalloporphyrins; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondrial Proteins; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins c-akt; Xenograft Model Antitumor Assays

Hydrogen peroxide, as other reactive oxygen species (ROS) produced during redox processes, induces lipid membrane peroxidation and protein degeneration causing cell apoptosis. ROS are recently considered as messengers in cell signalling processes, which, through reversible protein disulphide bridges formation, activate regulatory factors of cell proliferation and apoptosis. Disulphide bridges formation is catalysed by sulphydryl oxidase enzymes.. The neuroprotective effect of ALR protein (Alrp), a sulphydryl oxidase enzyme, on H(2)O(2)-induced apoptosis in SH-SY5Y cells has been evaluated.. Cell viability, flow cytometric evaluation of apoptotic cells, fluorescent changes of nuclear morphology, immunocytochemistry Alrp detection, Western blot evaluation of mitochondrial cyt c release and mitochondrial swelling were determined.. Alrp prevents the H(2)O(2)-induced cell viability loss, apoptotic cell death and mitochondrial swelling in SH-SY5Y cells in culture.. The data demonstrate that Alrp improves SH-SY5Y cells survival in H(2)O(2)-induced apoptosis. It is speculated that this effect could be related to the Alrp enzymatic activity.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochrome Reductases; Cytochromes c; Humans; Hydrogen Peroxide; Mitochondria; Mitochondrial Swelling; Neuroblastoma; Neurons; Oxidants; Oxidoreductases Acting on Sulfur Group Donors; Proteins; Rats; Recombinant Proteins

The proteasome inhibitor model of Parkinson's disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined.. We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)-Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways.. Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP-Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using L-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine.. The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD.

Topics: Acetylcysteine; Apoptosis; bcl-2-Associated X Protein; Cell Death; Cell Line, Tumor; Cell Survival; Coloring Agents; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria, Liver; Neuroblastoma; Reactive Oxygen Species; Recombinant Fusion Proteins; Tetrazolium Salts; Thiazoles

The beta-adrenoceptor blockers exhibit a well-characterized anti-apoptotic property in the heart and kidney while less is known about the effect of this class of drugs on neuronal apoptosis. We studied the effects of three beta-adrenoceptor blockers propranolol (1-(isoproplyamino)-3-(naphthalene-1-yloxy)propan-2-ol), atenolol (2-[4-[2-hydroxy-3-(1-methylethylamino)propoxyl]phenyl]ehanamide), and ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), against staurosporine-induced apoptosis in SH-SY5Y human neuroblastoma cells. Staurosporine increased caspase 3-like activity, DNA fragmentation, PARP cleavage, and the number of TUNEL positive cells consistent with the induction of apoptosis. Propranolol and ICI 118551, but not atenolol, demonstrated a concentration-dependent inhibition of caspase 3-like activity. Propranolol and ICI 118551 directly inhibited the enzymatic activity of recombinant caspase 9 while atenolol did not; however, none of the beta-adrenoceptor blockers that were examined directly blocked caspases 2 or 3 activity. In isolated mitochondria, propranolol and ICI 118551 inhibited staurosporine-induced cytochrome c release while atenolol did not. We conclude that propranolol and ICI 118551 protect SH-SY5Y cells against staurosporine-induced apoptosis through a dual action on the mitochondria and on caspase 9 in a cell type and an apoptotic paradigm where the conventional inhibitors of mitochondrial permeability transition such as cyclosporin A and bongkrekic acid demonstrate no protection.

Topics: Adrenergic beta-Antagonists; Apoptosis; Atenolol; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytochromes c; Cytoprotection; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Humans; In Situ Nick-End Labeling; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; Propanolamines; Propranolol; Protein Kinase C; Protein Kinase Inhibitors; Staurosporine; Time Factors

Modification of catalytic residue His-47 with p-bromophenacyl bromide (BPB) abolished the enzymatic activity of Naja naja atra phospholipase A2 (PLA2). Additionally, alterations in the global structure and the spatial positions of Trp residues were noted in His-modified PLA2. The cell viability of human neuroblastoma SK-N-SH cells was decreased by approximately 40% and 20% after treatment with 10 microM PLA2 and BPB-PLA2, respectively. Native and His-modified PLA2 induced a necrotic cell death accompanied with an activation of p38 MAPK, the loss of mitochondrial membrane potential (DeltaPsim) and cytochrome c release. Pretreatment with SB202190 (p38 MAPK inhibitor) and cyclosporine A (inhibitor of mitochondria permeability transition pore) rescued cell viability, DeltaPsim and cytochrome c release of PLA2-treated cells. Taken together, our data indicate that PLA2 activity does not play an indispensable role on the cytotoxicity of N. naja atra PLA2, and suggest a novel function of secretory PLA2 in inducing cell death of neuroblastoma. Moreover, the reduced cytotoxicity noted with BPB-PLA2 may be partly attributed to conformational distortion after modification of His-47.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cytochromes c; Elapid Venoms; Elapidae; Enzyme Inhibitors; Humans; Imidazoles; Membrane Potential, Mitochondrial; Necrosis; Neuroblastoma; p38 Mitogen-Activated Protein Kinases; Phospholipase A2 Inhibitors; Phospholipases A2; Pyridines

Among the new microtubule-targeted agents, the epothilone family of molecules has shown promising anticancer potential, and clinical trials are currently underway for patupilone (epothilone B) in various cancer indications. In this study, we characterized novel aspects of patupilone's cellular action that may underlie its potent cytotoxicity in human neuroblastoma cells. Patupilone induced mitochondrial membrane potential collapse, mitochondrial morphological changes, and cytochrome c release, leading to apoptosis. Within the first 2 h, patupilone increased the generation of reactive oxygen species (ROS; i.e., superoxides and hydrogen peroxide, 33+/-6 and 51+/-3% increase, respectively), specifically from mitochondria. ROS scavengers and mitochondrial DNA depletion [rho(-) cells] significantly protected cells against patupilone cytotoxicity, indicating that ROS generation is a key event in the initial phase of apoptosis. Although the Bim expression level was not modified by patupilone, this proapoptotic protein accumulated in the mitochondrial compartment (2.4-fold increase at IC70) after only a 6-h treatment. In contrast, Bax and Bcl-2 mitochondrial levels were not changed during treatment. It is noteworthy that ROS inhibition prevented Bim relocalization to mitochondria and mitochondrial membrane changes induced by patupilone. Altogether, our data reveal that patupilone-mediated ROS production by mitochondria initiates the intrinsic signaling cascade by inducing Bim accumulation in mitochondria. These results might explain the superior activity of patupilone in tumor cells compared with paclitaxel that is, until now, the clinical reference among microtubule-stabilizing agents. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator and integrator of apoptotic signals triggered by patupilone.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Epothilones; Humans; Indicators and Reagents; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Time Factors; Tubulin Modulators

Naja naja atra cardiotoxin 3 (CTX3) induced apoptotic death on human neuroblastoma SK-N-SH cells. The apoptosis signals of CTX3 included reactive oxygen species (ROS) generation, disruption of mitochondrial membrane potential (DeltaPsim), cytochrome c release to the cytosol and activation of caspase-9 and -3. However, CTX3-induced increase in mitochondrial permeability transition was not initiated by proteins of the Bcl-2 family. The collapse of DeltaPsim, release of cytosolic cytochrome c, production of ROS and subsequent apoptotic cell death in CTX-treated cells could not be completely abolished by either N-acetylcysteine (ROS scavenger) or cyclosporin A (an inhibitor of mitochondrial permeability transition). Co-incubation with rotenone, an inhibitor of mitochondrial electron transport chain complexes I, resulted in partial inhibition of CTX3-induced ROS generation but not the loss of DeltaPsim. Obviously, the dissipation of DeltaPsim was not an upstream event for ROS generation or vice versa. Given that CTX3 was able to induce the leakage of isolated mitochondria, our data indicate that CTX3-induced apoptotic death of SK-N-SH cells is mediated through mitochondrial alteration and ROS generation.

Topics: Acetylcysteine; Apoptosis; Caspases; Cell Line, Tumor; Cobra Cardiotoxin Proteins; Cyclosporine; Cytochromes c; Elapid Venoms; Enzyme Activation; Free Radical Scavengers; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Reactive Oxygen Species; Rotenone; Uncoupling Agents

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although understanding of the pathogenesis of PD remains incomplete, increasing evidence from human and animal studies has suggested that oxidative stress is an important mediator in its pathogenesis. Astaxanthin (Asx), a potent antioxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress-related diseases. This study examined the protective effects of Asx on 6-hydroxydopamine (6-OHDA)-induced apoptosis in the human neuroblastoma cell line SH-SY5Y. Pre-treatment of SH-SY5Y cells with Asx suppressed 6-OHDA-induced apoptosis in a dose-dependent manner. In addition, Asx strikingly inhibited 6-OHDA-induced mitochondrial dysfunctions, including lowered membrane potential and the cleavage of caspase 9, caspase 3, and poly(ADP-ribose) polymerase. In western blot analysis, 6-OHDA activated p38 MAPK, c-jun NH(2)-terminal kinase 1/2, and extracellular signal-regulated kinase 1/2, while Asx blocked the phosphorylation of p38 MAPK but not c-jun NH(2)-terminal kinase 1/2 and extracellular signal-regulated kinase 1/2. Pharmacological approaches showed that the activation of p38 MAPK has a critical role in 6-OHDA-induced mitochondrial dysfunctions and apoptosis. Furthermore, Asx markedly abolished 6-OHDA-induced reactive oxygen species generation, which resulted in the blockade of p38 MAPK activation and apoptosis induced by 6-OHDA treatment. Taken together, the present results indicated that the protective effects of Asx on apoptosis in SH-SY5Y cells may be, at least in part, attributable to the its potent antioxidative ability.

Topics: Adrenergic Agents; Annexin A5; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Oxidopamine; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Xanthophylls

Arsenic is an environmental toxicant that recently has been shown to have anticancer activity against a number of types of cancer cells by inducing apoptosis. Glycogen synthase kinase-3 (GSK3), a serine/threonine kinase, is an important pro-apoptotic signaling enzyme. Although GSK3 has been shown to promote apoptosis caused by a wide variety of insults, a role for GSK3 in arsenic-induced apoptosis has not yet been identified. Investigation of the involvement of GSK3 in arsenite-induced apoptosis demonstrated that arsenite induced apoptosis in SH-SY5Y human neuroblastoma cells, activating the executioner caspase-3 which caused cleavage of poly-ADP ribose-polymerase (PARP). Two selective GSK3 inhibitors, lithium and SB216763, attenuated caspase-3 activation and PARP cleavage induced by arsenite treatment indicating that GSK3 contributed to arsenite-induced apoptosis. Apoptotic signaling following exposure to arsenite involved cytochrome C release from mitochondria, and this was reduced by inhibition of GSK3 indicating that GSK3 promotes arsenite-induced apoptotic signaling upstream of mitochondrial disruption. Moreover, arsenite induced the translocation of Bax and p53 to the mitochondria and the activation-associated oligomerization of Bax, and these crucial events were reduced by inhibition of GSK3, indicating that GSK3 promotes arsenite-induced apoptosis by facilitating signals leading to mitochondrial apoptotic events. Taken together, the findings from this study reveal that GSK3 promotes arsenite-induced apoptosis by facilitating signaling leading to disruption of mitochondria.

Topics: Apoptosis; Arsenites; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Glycogen Synthase Kinase 3; Humans; Indoles; Lithium Compounds; Maleimides; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Sodium Compounds; Tumor Suppressor Protein p53

Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.

Topics: Adrenergic Agents; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Benzofurans; Calcium; Caspase 3; Cell Line, Tumor; Cytochromes c; Drugs, Chinese Herbal; Humans; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Oxidopamine; Protein Kinase C; Reactive Oxygen Species; Salvia miltiorrhiza; Sympatholytics

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.

Topics: Apoptosis; Blotting, Western; Calcium; Catechols; Cell Line, Tumor; Cysteinyldopa; Cytochromes c; Humans; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; Signal Transduction

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; Dynamins; Embryo, Mammalian; Fibroblasts; Fluorescent Antibody Technique; GTP Phosphohydrolases; Humans; Hydroxydopamines; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Tumor Suppressor Protein p53

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca(2+)](i)) levels using flow cytometry and observed an increase in the intracellular Ca(2+) concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 microM) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of [Ca(2+)](i) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Dizocilpine Maleate; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Enzyme Induction; Flame Retardants; Halogenated Diphenyl Ethers; Humans; Mitochondria; Neuroblastoma; Phenyl Ethers; Polybrominated Biphenyls; Reactive Oxygen Species

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspases; Cell Nucleolus; Chromatography, Affinity; Cytochromes c; Cytosol; Humans; Immunoprecipitation; Male; Mice; Mitochondria; Molecular Chaperones; Neuroblastoma; Nuclear Proteins; Nucleophosmin; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

In response to different stress signals, the c-Jun NH(2)-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (DeltaPsi(M)) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the DeltaPsi(M), an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation.

Topics: Animals; Cell Death; Culture Media, Serum-Free; Cytochromes c; Enzyme Inhibitors; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitogen-Activated Protein Kinases; Necrosis; Neurilemmoma; Neuroblastoma; Oxidative Stress; Rats; Reactive Oxygen Species; Tumor Cells, Cultured

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 microM DAS and 50 microM DADS but slightly affected with 100 microM DADS. Treatment with 50 and 100 microM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 microM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca(2+)](i), increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFkappaB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.

Topics: Allyl Compounds; Anticarcinogenic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Calcium; Calpain; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cytochromes c; Disulfides; Enzyme Activation; Enzyme Inhibitors; Garlic; Humans; I-kappa B Proteins; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neuroblastoma; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Plant Oils; Proto-Oncogene Proteins c-bcl-2; Spectrin; Sulfides

Neuroblastoma is the most common solid tumour in infants characterized by a high resistance to apoptosis. Recently, the cyclo-oxygenase pathway has been considered a potential target in the treatment of different kinds of tumours. The aim of the present work was to investigate a possible relationship between cyclo-oxygenase pathway and stauroporine-induced apoptosis in the neuroblastoma cell line SH-SY5Y.. Cellular viability was measured by release of LDH. DNA fragmentation was visualized by electrophoresis on agarose gel containing ethidium bromide. Cyclo-oxygenase activity was measured in microsomal fractions obtained from cells by quantification of its final product PGE2 by RIA. Caspase-3 activity was measured fluorimetrically and Western blot analysis was performed to assess cytochrome c expression.. We have found that staurosporine (500 nM) induced cellular death in a time-dependent manner in SH-SY5Y human neuroblastoma cells. Cyclo-oxygenase enzymatic activity was present in SH-SY5Y human neuroblastoma cells under basal conditions and pharmacological experiments using COX inhibitors indicate that cyclo-oxygenase-1 and cyclo-oxygenase-3 are the active isoforms in these cells. Co-incubation of SH-SY5Y cells with staurosporine (500 nM) and acetaminophen for 24 h potentiated staurosporine-mediated cellular death in a concentration-dependent manner. This process is mediated by an increase in cytochrome c release and caspase 3 activation and is prevented by N-acetylcysteine or the superoxide dismutase mimetic, MnTBAP.. Acetaminophen potentiates staurosporine-mediated neuroblastoma cell death. The mechanism of action of acetaminophen seems to be related to production of reactive oxygen species and decreased intracellular glutathione levels.

Topics: Acetaminophen; Acetylcysteine; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochromes c; Dinoprostone; Disulfiram; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Free Radical Scavengers; Glutathione; Humans; Metalloporphyrins; Neuroblastoma; Prostaglandin-Endoperoxide Synthases; Staurosporine; Time Factors

1-Methyl-4-phenylpyridinium ion (MPP(+)), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with elevation of intracellular reactive oxygen species (ROS) level and apoptotic death. Rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, has been known to show various non-hypoglycemic effects, including anti-inflammatory, anti-atherogenic, and anti-apoptotic. In the present study, we investigated the protective effects of rosiglitazone on MPP(+) induced cytotoxicity in human neuroblastoma SH-SY5Y cells, as well as underlying mechanism. Our results suggested that the protective effects of rosiglitazone on MPP(+) induced apoptosis may be ascribed to its anti-oxidative properties, anti-apoptotic activity via inducing expression of SOD and catalase and regulating the expression of Bcl-2 and Bax. These data indicated that rosiglitazone might provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; Herbicides; Humans; Hypoglycemic Agents; Mitochondria; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Rosiglitazone; Thiazolidinediones

Salsolinol, an endogenous neurotoxin, is known to be involved in the pathogenesis of Parkinson's disease (PD). In the present study, we have investigated the effects of salsolinol on the activation of two different signaling pathways that involve c-Jun N-terminal kinase (JNK), and nuclear factor-kappaB, (NF-kappaB) in human dopaminergic neuroblastoma SH-SY5Y cells. Salsolinol treatment caused upregulation in the levels of c-Jun and phosphorylated c-Jun. It also caused degradation of IkappaBalpha and translocated the active NF-kappaB into the nucleus. The binding activity of NF-kappaB to DNA was enhanced by salsolinol in a concentration dependent manner. Furthermore, salsolinol decreased the levels of the anti-apoptotic protein Bcl-2, and increased pro-apoptotic protein Bax, while enhancing the release of cytochrome-c from mitochondria. Mitochondrial complex-I activity was significantly decreased and reactive oxygen species (ROS) were increased in salsolinol treated cells. These results partly suggest that salsolinol-induced JNK and NF-kappaB signaling pathways may be involved in induction of apoptosis in human dopaminergic neurons, as seen in Parkinson's disease.

Topics: Cell Line, Tumor; Cytochromes c; Electron Transport Complex I; Humans; Isoquinolines; JNK Mitogen-Activated Protein Kinases; Mitochondria; Neuroblastoma; NF-kappa B; Phosphorylation; Reactive Oxygen Species; Signal Transduction

Akt is a serine/threonine protein kinase that plays a vital role in promoting cellular survival. Predominantly cytosolic, upon stimulation with growth-factors or stress, active Akt translocates into mitochondria, but the functions of Akt in mitochondria are not yet fully understood. Mitochondria play a central role in apoptotic pathways and given Akt's functions in the cytoplasm, Akt in mitochondria may help preserve mitochondrial integrity during cellular stress. To test if the translocation of Akt into mitochondria is neuroprotective, adenoviral vectors expressing a constitutively active Akt, Ad-HA-Akt (DD), and a constitutively active Akt with a mitochondrial targeting signal, Ad-Mito-HA-Akt (DD), were generated. Human SH-SY5Y neuroblastoma cells expressing the adenoviral constructs were treated with staurosporine to initiate intrinsic apoptotic cell death and several aspects of the mitochondrial apoptotic pathway were evaluated. Expression of active Akt targeted to mitochondria was found to be sufficient to significantly reduce staurosporine-induced activation of caspase-3 and caspase-9, the release of cytochrome c from mitochondria, and Bax oligomerization at mitochondria. These findings demonstrate that intramitochondrial active Akt results in efficient protection against apoptotic signaling.

Topics: Amino Acid Sequence; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Molecular Sequence Data; Neuroblastoma; Poly(ADP-ribose) Polymerases; Protein Sorting Signals; Proto-Oncogene Proteins c-akt; Staurosporine

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).

Topics: Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Cytosol; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurites; Neuroblastoma; Ouabain; Phosphorylation; Protein Isoforms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Tretinoin; Tumor Suppressor Protein p53

Morphine is recommended as a first-line opioid analgesic in the pain management of cancer patients. Accumulating evidence shows that morphine has anti-apoptotic activity, but its impact on the therapeutic applications of antineoplastic drugs is not well known. The present study was undertaken to test the hypothesis that morphine might antagonize the pro-apoptotic activity of DOX (doxorubicin), a commonly used antitumour drug for the treatment of neuroblastoma, in cultured SH-SY5Y cells. In the present study we demonstrated that morphine suppressed DOX-induced inhibition of cell proliferation and programmed cell death in a concentration-dependent, and naloxone as well as pertussis toxin-irreversible, manner. Further studies showed that morphine inhibited ROS (reactive oxygen species) generation, and prevented DOX-mediated caspase-3 activation, cytochrome c release and changes of Bax and Bcl-2 protein expression. The antioxidant NAC (N-acetylcysteine) also showed the same effects as morphine on DOX-induced ROS generation, caspase-3 activation and cytochrome c release and changes in Bax (Bcl-2-associated X protein) and Bcl-2 protein expression. Additionally, morphine was found to suppress DOX-induced NF-kappaB (nuclear factor kappaB) transcriptional activation via a reduction of IkappaBalpha (inhibitor of nuclear factor kappaB) degradation. These present findings support the hypothesis that morphine can inhibit DOX-induced neuroblastoma cell apoptosis by the inhibition of ROS generation and mitochondrial cytochrome c release, as well as by blockade of NF-kappaB transcriptional activation, and suggests that morphine might have an impact on the antitumour efficiency of DOX.

Topics: Acetylcysteine; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Cytoprotection; Doxorubicin; Enzyme Activation; Humans; I-kappa B Kinase; Morphine; Neuroblastoma; NF-kappa B; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Transcriptional Activation

The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody-positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro.. SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods.. SH-Sy5Y cells exposed to antineuronal antibody-positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody-negative sera. Neuroblasts exposed to antineuronal antibody-negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG-depleted celiac disease sera had an apoptotic effect similar to controls. Anti-caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera.. Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis.

Topics: Adult; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Bisbenzimidazole; Caspase 3; Caspase 9; Celiac Disease; Cell Line, Tumor; Citrate (si)-Synthase; Cytochromes c; Electron Transport Complex I; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Dyes; Humans; Immunoglobulin G; In Situ Nick-End Labeling; In Vitro Techniques; Male; Middle Aged; Mitochondria; Nerve Degeneration; Neuroblastoma

Dopamine (DA), as a neurotoxin, can elicit severe Parkinson's disease-like syndrome by elevating intracellular reactive oxygen species (ROS) levels and apoptotic activity. In this study, we examined the effect of esculin, which was extracted from Fraxinus sielboldiana blume, on DA-induced cytotoxicity and the underlying mechanism in human neuroblastoma SH-SY5Y cells. Our results suggest that the protective effects of esculin (10(-7), 10(-6) and 10(-5) M) on DA-induced cytotoxicity may be ascribed to its anti-oxidative properties by reducing ROS level, and its anti-apoptotic effect via protecting mitochondrion membrane potential (DeltaPsim), enhancing superoxide dismutaese (SOD) activity and reduced glutathione (GSH) levels, and regulating P53, Bax and Bcl-2 expression. In addition, esculin inhibited the release of cytochrome c and apoptosis-inducing factor (AIF), and the protein expression of activated caspase 3. These data indicate that esculin may provide a useful therapeutic strategy for the treatment of progressive neurodegenerative diseases such as Parkinson's disease (PD).

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Dopamine; Dopamine Antagonists; Dose-Response Relationship, Drug; Esculin; Excitatory Amino Acids; Glutathione; Humans; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Tumor Suppressor Protein p53; Up-Regulation

Several studies have indicated that apoptotic pathways are responsible for the loss of motor neurons that constitute the hallmark of amyotrophic lateral sclerosis (ALS). In this study, we demonstrate that apoptosis induced by the expression of several mutant Cu,Zn superoxide dismutases (SOD1) typical of familial ALS is mediated by Apaf1, a scaffold protein involved in neural development. Using different cell lines of neuronal origin and modulating the expression of both mutant SOD1s and Apaf1, we show that the removal of Apaf1 prevents cells death. Interestingly, intercepting activation of the caspases cascade is also effective in preventing both the mitochondrial damage and the increase in the production of reactive oxygen species induced by fALS-SOD1, even in the presence of cytochrome c release. This death pathway may be crucial also for the pathogenesis of the sporadic form of the disease, where markers of increased oxidative stress and mitochondria damage have been found.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Cell Line, Tumor; Cytochromes c; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mitochondria; Nerve Degeneration; Neuroblastoma; Oxidative Stress; Proteins; Superoxide Dismutase; Superoxide Dismutase-1

Despite much evidence that lithium and valproate, two commonly used mood stabilizers, exhibit neuroprotective properties against an array of insults, the pharmacological relevance of such effects is not clear because most of these studies examined the acute effect of these drugs in supratherapeutic doses against insults which were of limited disease relevance to bipolar disorder. In the present study, we investigated whether lithium and valproate, at clinically relevant doses, protects human neuroblastoma (SH-SY5Y) and glioma (SVG and U87) cells against oxidative stress and endoplasmic reticulum stress in a time-dependent manner. Pretreatment of SH-SY5Y cells for 7 days, but not 1 day, with 1 mM of lithium or 0.6 mM of valproate significantly reduced rotenone and H2O2-induced cytotoxicity, cytochrome c release and caspase-3 activation, and increased Bcl-2 levels. Conversely, neither acute nor chronic treatment of SH-SY5Y cells with lithium or valproate elicited cytoprotective responses against thapsigargin-evoked cell death and caspase-3 activation. Moreover, inhibitors of glycogen synthase kinase-3 (GSK-3), kenpaullone and SB216763, abrogated rotenone-induced, but not H2O2-induced, cytotoxicity. Thus the cytoprotective effects of lithium and valproate against H2O2-induced cell death is likely independent of GSK-3 inhibition. On the other hand, chronic lithium or valproate treatment did not ameliorate cytotoxicity induced by rotenone, H2O2, and thapsigargin in SVG astroglial and U87 MG glioma cell lines. Our results suggest that lithium and valproate may decrease vulnerability of human neural, but not glial, cells to cellular injury evoked by oxidative stress possibly arising from putative mitochondrial disturbances implicated in bipolar disorder.

Topics: Antimanic Agents; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Cytoprotection; Electron Transport Complex I; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hydrogen Peroxide; Lithium Compounds; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rotenone; Thapsigargin; Up-Regulation; Valproic Acid

A preferential loss of brain cholinergic neurons in the course of Alzheimer's disease and other encephalopathies is accompanied by a proportional impairment of acetyl-CoA synthesizing capacity in affected brains. Particular susceptibility of cholinergic neurons to neurodegeneration might results from insufficient supply of acetyl-CoA for energy production and acetylcholine synthesis in these conditions. Exposure of SN56 cholinergic neuroblastoma cells to dibutyryl cAMP and retinoic acid for 3 days caused their morphologic differentiation along with the increase in choline acetyltransferase activity, acetylcholine content and release, calcium content, and the expression of p75 neurotrophin receptors. Acetyl-CoA content correlated inversely with choline acetyltransferase activity in different lines of SN56 cells. In differentiated cells, aluminum (1 mM), amyloid beta(25-35) (0.001 mM), and sodium nitroprusside (1 mM), caused much greater decrease of pyruvate dehydrogenase and choline acetyltransferase activities and cell viability than in nondifferentiated ones. Aluminum (1 mM) aggravated suppressory effects of amyloid beta on choline acetyltransferase and pyruvate dehydrogenase activities and viability of differentiated cells. Similar additive inhibitory effects were observed upon combined exposure of differentiated cells to sodium nitroprusside and amyloid beta(25-35). None or much smaller suppressory effects of these neurotoxins were observed in nondifferentiated cells. Increase in the fraction of nonviable differentiated cells positively correlated with losses of choline acetyltransferase, pyruvate dehydrogenase activities, and cytoplasmic cytochrome c content in different neurotoxic conditions. These data indicate that highly differentiated cholinergic neurons may be more susceptible to aluminum and other neurotoxins than the nondifferentiated ones due to relative shortage of acetyl-CoA, increased content of Ca(2+), and expression of p75 receptors, yielding increase in cytoplasmic cytochrome c and subsequently grater rate of death of the former ones.

Topics: Acetyl Coenzyme A; Acetylcholine; Aluminum; Amyloid beta-Peptides; Animals; Autonomic Nervous System Diseases; Brain Neoplasms; Calcium; Cell Differentiation; Cell Line, Tumor; Choline O-Acetyltransferase; Cytochromes c; DNA, Complementary; Energy Metabolism; Immunohistochemistry; Mice; Neuroblastoma; Neurotoxins; Nitroprusside; Peptide Fragments; Phenotype; Rats; Receptor, trkA; Trypan Blue

Acetaldehyde, an inhibitor of mitochondrial function, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with elevation of the intracellular reactive oxygen species (ROS) level and apoptosis. Adiponectin, secreted from adipose tissue, mediates systemic insulin sensitivity with liver and muscle as target organs. In this study, we investigated the protective effects of adiponectin on acetaldehyde-induced apoptosis in human neuroblastoma SH-SY5Y cells and attempted to examine its mechanism. Acetaldehyde-induced apoptosis was moderately reversed by adiponectin treatment. Our results suggest that the protective effects of adiponectin on acetaldehyde-induced apoptosis may be ascribed to ability to induce the expression of anti-oxidant enzymes and to regulate Bcl-2 and Bax expression. These data indicate that adiponectin may provide a useful therapeutic strategy for the prevention of progressive neurodegenerative disease such as Parkinson's disease.

Topics: Acetaldehyde; Adiponectin; Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species; Receptors, Adiponectin; Receptors, Cell Surface; RNA, Messenger

Germanium (Ge) is commonly used in the semiconductor industry as well as health-promoting and medical field. Biologically, germanium possesses erythropoietic, anti-microbial, anti-tumor, anti-amyloidosis, and immunomodulative effects. However, toxic effects of Ge-containing compounds on kidney, muscle, neuronal cells, and nerves have been reported. Mitochondrial dysfunction was found to be involved in the pathogenesis of GeO(2)-induced nephropathy and myopathy. Since it is well known that mitochondria play a major role in apoptosis triggered by many stimuli, an effort was made to examine whether the Ge-induced neurotoxicity occurs through mitochondria-mediated apoptosis. A mouse neuroblastoma cell line, Neuro-2A, was used in the present study. After incubating with 0.1-800microM of GeO(2) for 0-72h, the cell viability of Neuro-2A cells was inhibited in a dose- and time-dependent manner. Further analysis showed that aside from the changes in the nuclear morphology responsible for apoptosis, the release of cytochrome c, the loss of mitochondrial membrane potential, the translocation of Bax, and the reduction of Bcl-2 expression were also observed in Neuro-2A cells after GeO(2) treatment. These results indicate that the mitochondria-mediated apoptosis is involved in this in vitro model of GeO(2)-induced neurotoxicity.

Topics: Acridine Orange; Analysis of Variance; Animals; Antimutagenic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Ethidium; Flow Cytometry; Germanium; Immunohistochemistry; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Time Factors

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carcinoma, Hepatocellular; Caspases; Chick Embryo; Chickens; Cytochromes c; Fibroblasts; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Newcastle disease virus; Oncolytic Viruses; Polymerase Chain Reaction; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Bax is a proapoptotic protein implicated in cell death involved in several neurodegenerative diseases. Intracellularly expressed antibody (Ab) fragments (intrabodies) inhibiting Bax function would have potential for developing therapeutics for the aforementioned diseases and can serve as research tools. We report identification, cloning, and functional characterization of several Bax-specific single-domain antibodies (sdAbs). These minimal size Ab fragments, which were isolated from a llama V(H)H phage display library by panning, inhibited Bax function in in vitro assays. Importantly, as intrabodies, these sdAbs, which were stably expressed in mammalian cells, were nontoxic to their host cells and rendered them highly resistant to oxidative-stress-induced apoptosis. The intrabodies prevented mitochondrial membrane potential collapse and apoptosis after oxidative stress in the host cells. These anti-Bax V(H)Hs could be used as tools for studying the role of Bax in oxidative-stress-induced apoptosis and for developing novel therapeutics for the degenerative diseases involving oxidative stress.

Topics: Amino Acid Sequence; Antibodies; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cloning, Molecular; Cytochromes c; Gene Expression Regulation; Humans; Membrane Potential, Mitochondrial; Molecular Sequence Data; Neuroblastoma; Oxidative Stress

A pathogenic truncation of an amber mutation at codon 145 (Y145STOP) in Gerstmann-Straussler-Scheinker disease (GSS) was investigated through the real-time imaging in living cells, by utilizing GFP-PrP constructs. GFP-PrP(1-144) exhibited an aberrant localization to mitochondria in mouse neuroblastoma neuro2a (N2a) and HpL3-4 cells, a hippocampal cell line established from prnp gene-ablated mice, whereas full-length GFP-PrP did not. The aberrant mitochondrial localization was also confirmed by Western blot analysis. Since GFP-PrP(1-121), as previously reported, and full-length GFP-PrP do not exhibit such mitochondrial localization, the mitochondrial localization of GFP-PrP(1-144) requires not only PrP residues 121-144 (in human sequence) but also COOH-terminal truncation in the current experimental condition. Subsequently, the GFP-PrP(1-144) induced a change in the mitochondrial innermembrane potential (DeltaPsi(m)), release of cytochrome c from the intermembrane space into the cytosol, and DNA fragmentation in these cells. Non-fluorescent PrP(1-144) also induced the DNA fragmentation in N2a and HpL3-4 cells after the proteasomal inhibition. These data may provide clues as to the molecular mechanism of the neurotoxic property of Y145STOP mutation. Furthermore, immunoelectron microscopy revealed numerous electron-dense deposits in mitochondria clusters of GFP-PrP(1-144)-transfected N2a cells, whereas no deposit was detected in the cells transfected with full-length GFP-PrP. Co-localization of GFP/PrP-immunogold particles with porin-immunogold particles as a mitochondrial marker was observed in such electron-dense vesicular foci, resembling those found in autophagic vacuoles forming secondary lysosomes. Whether such electron-dense deposits may serve as a seed for the growth of amyloid plaques, a characteristic feature of GSS with Y145STOP, awaits further investigations.

Topics: Animals; Apoptosis; Blotting, Western; Codon; Cytochromes c; Cytosol; DNA; Gerstmann-Straussler-Scheinker Disease; Lysosomes; Mice; Mitochondria; Mutation; Neuroblastoma; Neurons; Neuroprotective Agents; Prions

1-methyl-4-phenylpyridinium ion (MPP(+)), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with elevation of intracellular reactive oxygen species (ROS) level and apoptotic death. Salvianic acid A (SA), isolated from the Chinese herbal medicine Salvia miltiorrhiza, is capable of protecting diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the protective effects of SA on MPP(+)-induced cytotoxicity in human neuroblastoma SH-SY5Y cells, as well as the underlying mechanism. Treatment of SH-SY5Y cells with MPP(+) caused the loss of cell viability, and condensation and fragmentation of nuclei, which was associated with the elevation of ROS level, the increase in Bax/Bcl-2 ratio, and the activation of caspase-3. MPP(+) induced mitochondria dysfunction characterized by mitochondrial membrane potential loss and cytochrome c release. These phenotypes induced by MPP(+) were reversed by SA. Our results suggested that the protective effects of SA on MPP(+)-induced cytotoxicity may be ascribed to its antioxidative properties and anti-apoptotic activity via regulating the expression of Bcl-2 and Bax. These data indicated that SA might provide a useful therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; Apoptosis; bcl-2-Associated X Protein; Bisbenzimidazole; Blotting, Western; Caffeic Acids; Caspase 3; Caspases; Catalase; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cytochromes c; Dithioerythritol; Dose-Response Relationship, Drug; Drug Interactions; Flow Cytometry; Humans; Lactates; Membrane Potentials; Mitochondria; Necrosis; Neuroblastoma; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfhydryl Reagents; Superoxide Dismutase

Histone deacetylase inhibitors (HDACIs) are therapeutic drugs that inhibit deacetylase activity, thereby increasing acetylation of many proteins, including histones. HDACIs have antineoplastic effects in preclinical and clinical trials and are being considered for cancers with unmet therapeutic need, including neuroblastoma (NB). Uncertainty of how HDACI-induced protein acetylation leads to cell death, however, makes it difficult to determine which tumors are likely to be responsive to these agents. Here, we show that NB cells are sensitive to HDACIs, and that the mechanism by which HDACIs induce apoptosis involves Bax. In these cells, Bax associates with cytoplasmic Ku70, a protein that typically increases chemotherapy resistance. Our data show that in NB cells Ku70 binds to Bax in an acetylation-sensitive manner. Upon HDACI treatment, acetylated Ku70 releases Bax, allowing it to translocate to mitochondria and trigger cytochrome c release, leading to caspase-dependent death. This study shows that Ku70 is an important Bax-binding protein, and that this interaction can be therapeutically regulated in NB cells. Whereas the Bax-binding ability of Ku70 allows it to block apoptosis in response to certain agents, it is also a molecular target for the action of HDACIs, and in this context, a mediator of NB cell death.

Topics: Acetylation; Animals; Antigens, Nuclear; Apoptosis; bcl-2-Associated X Protein; Butyrates; Cytochromes c; DNA-Binding Proteins; Flow Cytometry; Green Fluorescent Proteins; Histone Deacetylase Inhibitors; Hydroxamic Acids; Immunoblotting; Immunoprecipitation; Ku Autoantigen; Mice; Mitochondria; Neuroblastoma; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Transfection; Tumor Cells, Cultured

Myristicin, 1-allyl-3,4-methylenedioxy-5-methoxybenzene, is a naturally occurring alkenylbenzene compound found in the nutmeg. The present study was conducted to assess the cytotoxic and apoptotic effects of myristicin on the human neuroblastoma SK-N-SH cells. We found that a dose-dependent reduction in cell viability occurs at myristicin concentration > or =0.5 mM in SK-N-SH cells. Apoptotic cell death was confirmed using DNA fragmentation, terminal deoxyribonucelotidyl transferase-mediated dUTP nick end labeling and by 4,6-diamidino-2-phenylindole staining. Microscopy was used to observe apoptotic cell morphology. Western blotting was used to investigate the protein expression of known apoptotic mediators including cytochrome c, caspase-3, and PARP. The apoptosis triggered by myristicin was accompanied by an accumulation of cytochrome c and by the activation of caspase-3. The results obtained suggest that myristicin induces cytotoxicity in human neuroblastoma SK-N-SH cells by an apoptotic mechanism. This myristicin-induced apoptosis provides further insight of the molecular mechanisms of myristicin toxicity.

Topics: Allylbenzene Derivatives; Apoptosis; Benzyl Compounds; Caspase 3; Caspases; Cytochromes c; Dioxolanes; DNA Damage; Dose-Response Relationship, Drug; Humans; In Situ Nick-End Labeling; Neuroblastoma; Pyrogallol; Tumor Cells, Cultured

Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

Topics: Antifungal Agents; Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Humans; L-Lactate Dehydrogenase; Microscopy, Fluorescence; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Subcellular Fractions; Tetrazolium Salts; Thiazoles; Thimerosal

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 3; Caspases; Cell Fusion; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; Glutamic Acid; Humans; Ionomycin; Ionophores; Mice; Mitochondria; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Spectrin; Spinal Cord

Fraxetin belongs to an extensive group of natural phenolic anti-oxidants. In the present study, using a human neuroblastoma SH-SY5Y cells, we have investigated the protective effects of this compound on modifications in endogenous reduced glutathione (GSH), intracellular oxygen species (ROS) and apoptotic death on rotenone-mediated cytoxicity. Incubation of cells with the fraxetin led to a significant elevation dose-dependent of cellular GSH and this was accompanied by a marked protection against rotenone-mediated toxicity, which was also significantly reversed in the cells with buthionine sulfoximine (BSO) co-treatment. Taken together, this study suggested that intracellular GSH appeared to be an important factor in fraxetin-mediated cytoprotection against rotenone-toxicity in SH-SY5Y cells. Fraxetin at 10-100 muM inhibited the formation of ROS, cytochrome c release, activation of caspase-3 and 9, and suppressed the up-regulation of Bax, whereas no significant change occurred in Bcl-2 levels. Our results indicated that the anti-oxidative and anti-apoptotic properties render this natural compound potentially protective against rotenone-induced cytotoxicity.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Count; Cell Line, Tumor; Cell Survival; Coumarins; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glutathione; Humans; Insecticides; Mitochondria; Neuroblastoma; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Rotenone

Point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase-3 (GSK-3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimer's disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol-3-kinase (PI3-K)/Akt and GSK-3 pathway as well as the caspase-3 pathway, VSC4.1 motoneuron cells were transfected with G93A- or A4V-mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24 h after neuronal differentiation, their viability and intracellular signals, including PI3-K/Akt, GSK-3, heat shock transcription factor-1 (HSTF-1), cytochrome c, caspase-3 and poly(ADP-ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK-3beta-mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK-3beta inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK-3beta inhibitor increased their viability by activating HSTF-1 and by reducing cytochrome c release, caspase-3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3-K/Akt and GSK-3beta. These results suggest that the G93A or A4V mutations inhibit PI3-K/Akt and activate GSK-3beta and caspase-3, thus becoming vulnerable to oxidative stress, and that the GSK-3beta-mediated cell death mechanism is important in G93A and A4V cell death.

Topics: Alanine; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Survival; Collagen Type XI; Cytochromes c; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heat-Shock Proteins; Humans; Hybrid Cells; Indoles; Motor Neurons; Mutagenesis; Neuroblastoma; Phosphatidylinositol 3-Kinases; Point Mutation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Trypan Blue; Valine

Several antidepressants, mainly selective serotonin-reuptake inhibitors (SSRIs) and some tricyclic antidepressants (TCAs), have been shown to possess potent apoptotic activity in different cell lines. Our aim was to screen and select those agents with significant activity and elucidate the molecular pathway underlying this process in rat glioma and human neuroblastoma cell lines. We studied the effect of different antidepressants on apoptotic markers, including: cell viability, DNA fragmentation, cytochrome c (Cyt c) release from mitochondria, and caspase-3- like activity. In addition, the involvement of MAPK genes, c-Jun, and ERK was determined. Paroxetine and fluoxetine, SSRIs, clomipramine, a TCA, but not imipramine or mianserin (an atypical antidepressant), caused apoptosis in both cell lines, as assessed by flow cytometry of propidium iodide-stained C6 cells and typical fluorescence microscopy in glioma cells. These apoptotic changes were preceded by rapid increase in p-c-Jun levels, Cyt c release from mitochondria, and increased caspase-3-like activity. Assessment of paroxetine cytotoxicity in primary mouse brain and neuronal cultures showed significantly lower sensitivity to the drug's proapoptotic activity. These results strongly suggest that selected antidepressants induce apoptosis in neuronal and glial cell lines. Activation of p-c-Jun and subsequent increased Cyt c mitochondrial release participate in the apoptotic mechanism of the antidepressant. The high sensitivity to these drugs of the cancer cell, compared with primary brain tissue, suggests the potential use of these agents in the treatment of brain-derived tumors.

Topics: Animals; Antidepressive Agents; Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Mianserin; Mice; Neuroblastoma; Neurons; Paroxetine; Rats

There is a worldwide increasing concern over the neurological risks of thimerosal (ethylmercury thiosalicylate) which is an organic mercury compound that is commonly used as an antimicrobial preservative. In this study, we show that thimerosal, at nanomolar concentrations, induces neuronal cell death through the mitochondrial pathway. Thimerosal, in a concentration- and time-dependent manner, decreased cell viability as assessed by calcein-ethidium staining and caused apoptosis detected by Hoechst 33258 dye. Thimerosal-induced apoptosis was associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. Although thimerosal did not affect cellular expression of Bax at the protein level, we observed translocation of Bax from cytosol to mitochondria. Finally, caspase-9 and caspase-3 were activated in the absence of caspase-8 activation. Our data suggest that thimerosal causes apoptosis in neuroblastoma cells by changing the mitochondrial microenvironment.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Enzyme Activation; Membrane Potentials; Mitochondria; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Thimerosal; Time Factors

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.

Topics: Actins; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Cytoskeleton; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Humans; Membrane Potentials; Mitochondria; Neuroblastoma; Okadaic Acid; Pharmaceutical Preparations; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Topics: Aged; alpha-Synuclein; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Line; Cysteine Endopeptidases; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Immunohistochemistry; Lewy Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Staining and Labeling; Synucleins; Transgenes; Tubulin; Tyrosine; Ubiquitin; Ubiquitin-Protein Ligases

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.

Topics: Amyotrophic Lateral Sclerosis; Animals; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Cytosol; DNA-Binding Proteins; Enzyme Activation; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mice; Mitochondria; Mutation; Neuroblastoma; Neurons; Recombinant Fusion Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Ubiquitin-Protein Ligases

Apoptosis is an active process that is regulated by different signalling pathways. One of the more important organelles involved in apoptosis regulation is the mitochondrion. Electron chain transport disruption increases free radical production leading to multiple conductance channel opening, release of cytochrome c and caspase activation. This death pathway can be blocked by anti-apoptotic members of the Bcl-2 protein family that might shift redox potential to a more reduced state, preventing free radical-mediated damage. 6-Hydroxydopamine (6-OHDA) has been widely used to generate Parkinson's disease-like models. It is able to generate free radicals and to induce catecholaminergic cell death. In this paper we have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-x(L) as a model to gain insights into the mechanisms through which Bcl-x(L) blocks 6-OHDA-induced cell death and to identify the molecular targets for this action. Herein, we present evidence supporting that the Bcl-x(L)-anti-apoptotic signal pathway seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase-3 like activity following 6-OHDA treatment in the human neuroblastoma cell line SH-SY5Y.

Topics: Antioxidants; Apoptosis; bcl-X Protein; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Cytoprotection; Enzyme Activation; Humans; Mitochondria; Neuroblastoma; Oxidopamine; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

On the basis of clinical studies showing that arsenic trioxide (As(2)O(3)), via an apoptotic mechanism, and with minimal toxicity induces complete remission in patients with refractory acute promyelocytic leukemia and that multidrug-resistant and p53-mutated neuroblastoma cells are sensitive to As(2)O(3) both in vitro and in vivo, we searched for molecular mechanisms involved in the As(2)O(3)-induced neuroblastoma cell death.. We have studied the effect of As(2)O(3) on the expression and cellular localization of proteins involved in drug-induced death in two neuroblastoma cell lines with intact p53 and two with mutated p53, the latter two displaying multidrug resistance.. As(2)O(3) provoked Bax expression in all tested neuroblastoma cell lines, including SK-N-BE(2) cells with mutated p53 and LA-N-1 cells, which have a deleted p53. In all cell lines exposed to As(2)O(3), p21 Bax was proteolytically cleaved in a calpain-dependent way into the more proapoptotic p18 Bax, which was detected exclusively in a mitochondria-enriched subcellular fraction. As(2)O(3) also caused an increase of cytoplasmic cytochrome c, translocation of antiapoptosis-inducing factor to the nuclei, and a slight activation of caspase 3. However, inhibition of caspase 3 did not prevent cell death, whereas inhibition of Bax cleavage was associated with a decreased As(2)O(3)-induced cell death.. We show that multidrug-resistant neuroblastoma cells die after exposure to As(2)O(3), independent of functional p53, suggesting activation of a cytotoxic pathway different from that induced by conventional chemotherapeutic agents. We further propose that proteolytic activation of Bax is an important event in As(2)O(3)-induced cell death.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; Blotting, Western; Calpain; Carboplatin; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Humans; Mutation; Neuroblastoma; Oxides; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Suppressor Protein p53; Vincristine

This study investigated whether nicastrin can induce apoptotic cell death in SK-N-MC cells. MTT assays revealed the transfected cells expressing mutant nicastrin, compared with those expressing wild nicastrin or the control vector, showing significantly increased cell death. The mutant nicastrin transfectants were also observed to induce cytosolic cytochrome c release from the mitochondria, and Bax protein expression in response, to increased cell death. These observations suggested that nicastrin, as well as the APP and PS proteins, were also involved in the upregulated Bax mediated neuroblastoma cell death and the release of cytochrome c in the neuroblastoma.

Topics: Amyloid Precursor Protein Secretases; bcl-2-Associated X Protein; Blotting, Western; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Mutational Analysis; Gene Expression Regulation; Humans; Membrane Glycoproteins; Mitochondria; Mutagenesis, Site-Directed; Mutation; Neuroblastoma; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles; Transfection

Activation of the c-Jun N-terminal kinase (JNK) pathway is suggested to be required for neuronal apoptosis. We investigated the role of JNK on phosphorylation of c-Jun, Bcl-2, and apoptotic translocation of cytochrome c (cyt c) in UV-induced apoptosis in human neuroblastoma SH-SY5Y cells. We confirm that UV irradiation induces both apoptosis and necrosis in SH-SY5Y cells and that phosphorylation of JNK at Thr183/Tyr185 in SH-SY5Y cells treated with UV is an early event preceding apoptosis. We also demonstrate that phosphorylation of c-Jun at Ser63 is an early event coinciding with JNK activation, and that the phosphorylation of c-Jun is partially prevented by the JNK inhibitor SP600125. Despite the use of SP600125, the amount of cyt c released into the cytoplasm is not diminished and SP600125 is also unable to decrease the extent of UV-induced apoptosis. These data support the hypothesis that in this system, UV-induced apoptosis is not dependent exclusively on JNK activation. Possible involvement of cyclin-dependent kinases (CDKs) in c-Jun phosphorylation at Ser63 was excluded by pretreating UV-irradiated SH-SY5Y cells with the CDK1/2/5 inhibitor roscovitine.

Topics: Anthracenes; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-jun; Time Factors; Ultraviolet Rays

Trichloroethylene, a common industrial solvent and a metabolic precursor of chloral hydrate, occurs widely in the environment. Chloral hydrate, which is also used as a hypnotic, has been found to condense spontaneously with tryptamine, in vivo, to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) that has a structural analogy to the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Earlier studies have revealed the relative permeability of the molecule through the blood-brain barrier and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo induces an apoptotic pathway in the human neuroblastoma cell line, SK-N-SH, involving the translocation of mitochondrial cytochrome c to the cytosol and activation of caspase 3. TaClo-induced apoptosis shows considerable differences from that mediated by other Parkinson-inducing agents such as MPTP, rotenone and manganese. Although it is not clear if the clinically administered dosage of chloral hydrate or the relatively high environmental levels of trichloroethylene could lead to an onset of Parkinson's disease, the spontaneous in vivo formation of TaClo and its pro-apoptotic properties, as shown in this report, should be considered.

Topics: Apoptosis; Carbolines; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Induction; Gene Expression; Humans; Mitochondria; Neuroblastoma; Neurons; Parkinson Disease; Protein Kinase C

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.

Topics: Amino Acid Sequence; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Binding Sites; Carrier Proteins; Cell Membrane Permeability; Cyclosporine; Cytochromes c; GTP-Binding Proteins; Humans; Membrane Proteins; Mitochondria; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis; Neuroblastoma; Protein Conformation; Protein Glutamine gamma Glutamyltransferase 2; Proto-Oncogene Proteins c-bcl-2; Sequence Alignment; Transfection; Transglutaminases; Tumor Cells, Cultured

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. An unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD and apoptosis was previously shown to be responsible for the neuronal cell death of the disease. In this study, we utilized human neuronal SK-N-SH cells stably transfected with HA-tagged full-length MJD with 78 polyglutamine repeats to examine the effects of polyglutamine expansion on neuronal cell survival in the early stage of disease. Various pro-apoptotic agents were used to assess the tolerance of the mutant cells and to compare the differences between cells with and without mutant ataxin-3. Concentration- and time-dependent experiments showed that the increase in staurosporine-induced cell death was more pronounced and accelerated in cells containing expanded ataxin-3 via MTS assays. Interestingly, under basal conditions, Western blot and immunocytochemical analyses showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, exogenous expression of Bcl-2 desensitized SK-N-SH-MJD78 cells to poly-Q toxicity. These results indicated that mitochondrial-mediated cell death plays a role in the pathogenesis of MJD. In our cellular model, full-length expanded ataxin-3 that leads to neurodegenerative disorders significantly impaired the expression of Bcl-2 protein, which may be, at least in part, responsible for the weak tolerance to polyglutamine toxicity at the early stage of disease and ultimately resulted in an increase of stress-induced cell death upon apoptotic stress.

Topics: Apoptosis; Ataxin-3; Cell Line, Tumor; Cytochromes c; Fibroblasts; Humans; Machado-Joseph Disease; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Neurons; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Staurosporine; Trinucleotide Repeat Expansion

We have investigated the ability of pramipexole, a dopamine agonist used in the symptomatic treatment of Parkinson's disease (PD), to protect against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and rotenone in dopaminergic and non-dopaminergic cells. Pre-incubation with either the active (-)- or inactive (+)-enantiomer forms of pramipexole (10 microm) decreased cell death in response to MPP+ and rotenone in dopaminergic SHSY-5Y cells and in non-dopaminergic JK cells. The protective effect was not prevented by dopamine receptor blockade using sulpiride or clozapine. Protection occurred at concentrations at which pramipexole did not demonstrate antioxidant activity, as shown by the failure to maintain aconitase activity. However, pramipexole reduced caspase-3 activation, decreased the release of cytochrome c and prevented the fall in the mitochondrial membrane potential induced by MPP+ and rotenone. This suggests that pramipexole has anti-apoptotic actions. The results extend the evidence for the neuroprotective effects of pramipexole and indicate that this is not dependent on dopamine receptor occupation or antioxidant activity. Further evaluation is required to determine whether the neuroprotective action of pramipexole is translated to a disease-modifying effect in PD patients.

Topics: 1-Methyl-4-phenylpyridinium; Aconitate Hydratase; Antioxidants; Benzothiazoles; Carbocyanines; Caspase 3; Caspases; Cell Count; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Flow Cytometry; Humans; Jurkat Cells; L-Lactate Dehydrogenase; Mitochondria; Necrosis; Neuroblastoma; Pramipexole; Rotenone; Thiazoles

Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy.. Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting.. Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN.. These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.

Topics: Apoptosis; Bromodeoxyuridine; Caspases; Cell Hypoxia; Cell Survival; Cyclin-Dependent Kinases; Cytochromes c; DNA; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; G1 Phase; G2 Phase; Genes, myc; Growth Inhibitors; Humans; Hypoxia; In Situ Nick-End Labeling; Neuroblastoma; Piperidines; Proto-Oncogene Mas; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (deltapsim) were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVD-CHO, couldn't completely rescue N2a cells from apoptosis. Administration of cyclosporine A, an inhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could completely reverse DNA fragmentation, but can't completely inhibit caspase3 activity. It was concluded that there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cytochromes c; Mice; Mitochondria; Neuroblastoma; Neurons; Reperfusion Injury; Tumor Cells, Cultured

Current evidence suggests that amyloid beta peptides (Abeta) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by Abeta(1-40) and Abeta(1-42) peptides as well as the relationships between apoptosis and oxidative stress. Abeta peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by alpha-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38(MAPK), involved early elevation of p53 protein levels. Pretreatment of neurons with alpha-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of Abeta induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid beta operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.

Topics: Acetylcysteine; alpha-Tocopherol; Amyloid beta-Peptides; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Caspase 3; Caspases; Cell Differentiation; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neuroblastoma; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Peptide Fragments; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Toluene; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria.

Topics: Androstadienes; Brain Neoplasms; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Cell Line; Cell Membrane; Cell Nucleus; Cytochromes c; Cytosol; Drug Interactions; Electron Transport Complex IV; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoblotting; Insulin-Like Growth Factor I; Ionophores; Kidney; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Porins; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Superoxide Dismutase; Threonine; Time Factors; Voltage-Dependent Anion Channel 1; Wortmannin

To observe the effect of Jiunaoyizhi capsul on the signal transduction pathway of SY5Y cell lines and explore the mechanism of the function of the capsul's enhancing neuronal growth.. Human neuroblastoma was used as cell models and they were divided into control group and experimental group. Supernatant of cell lysate was taken and immunoprecipitation was done with antibodies to proteins related to signal transduction pathway, and the immunoprecipitates were analyzed by Western blotting.. After treatment with Jiunaoyizhi capsul, expression of Akt/PKB, CREB, P-CREB was clearly increased and expression of cytochrome C decreased more than the control group.. Jiunaoyizhi capsul can promote expression of some proteins related with signal transduction pathway in SY5Y cell lines. Mechanism of Jiunaoyizhi capsul's enhancing neuronal growth is relevant to expression of some proteins in signal transduction pathway.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cytochromes c; Drug Combinations; Drugs, Chinese Herbal; Humans; Neuroblastoma; Neurons; Plants, Medicinal; Signal Transduction