5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Neuroblastoma

Elevated expression of TLX (also called as NR2E1) in neuroblastoma (NB) correlates with unfavorable prognosis, and TLX is required for self-renewal of NB cells. Knockdown of TLX has been shown to reduce the NB sphere-forming ability. ASK1 (MAP3K5) and TLX expression are both enhanced in SP (side population) NB and patient-derived primary NB sphere cell lines, but the majority of non-SP NB lines express lower ASK1 expression. We found that ASK1 phosphorylated and stabilized TLX, which led induction of HIF-1α, and its downstream VEGF-A in an Akt dependent manner. In depleting ASK1 upon hypoxia, TLX decreased and the apoptosis ratio of NB cells was enhanced, while low-ASK1-expressing NB cell lines were refractory in TUNEL assay by using flow cytometry. Interestingly, primary NB spheres cell lines express only high levels of active pASK1Thr-838 but the established cell lines expressed inhibitory pASK1Ser-966, and both could be targeted by ASK1 depletion. We report a novel pro-survival role of ASK1 in the tumorigenic NB cell populations, which may be applied as a therapeutic target, inducing apoptosis specifically in cancer stem cells.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Cell Survival; Gene Silencing; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MAP Kinase Kinase Kinase 5; Membrane Potential, Mitochondrial; Mice, SCID; Neuroblastoma; Orphan Nuclear Receptors; Phosphorylation; Protein Binding; Protein Domains; Protein Kinase Inhibitors; Protein Stability; Proto-Oncogene Proteins c-akt; Receptors, Cytoplasmic and Nuclear; Up-Regulation; Xenograft Model Antitumor Assays

Hydrogen sulfide (H(2)S) has been proposed as a novel neuromodulator, which plays critical roles in the central nervous system affecting both neurons and glial cells. However, its relationship with neurodegenerative diseases is unexplored. The present study was undertaken to investigate the effects of H(2)S on cell injury induced by rotenone, a commonly used toxin in establishing in vivo and in vitro Parkinson's disease (PD) models, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report here that sodium hydrosulfide (NaHS), an H(2)S donor, concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death. NaHS also prevented rotenone-induced p38- and c-Jun NH(2)-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) phosphorylation and rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (DeltaPsi(m)) dissipation, cytochrome c release, caspase-9/3 activation and poly(ADP-ribose) polymerase cleavage. Furthermore, 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Thus, we demonstrated for the first time that H(2)S inhibited rotenone-induced cell apoptosis via regulation of mitoK(ATP) channel/p38- and JNK-MAPK pathway. Our data suggest that H(2)S may have potential therapeutic value for neurodegenerative diseases, such as PD.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Formazans; Humans; Hydrogen Sulfide; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Rotenone; Tetrazolium Salts; Uncoupling Agents

Expression of CCAAT/enhancer-binding protein beta (C/EBP beta) and growth-arrest DNA damage-inducible 153/C/EBP beta homology protein (GADD153/CHOP) increased after incubation of human neuroblastoma SH-SY5Y cells with a range of dopamine concentrations. Dopamine (100 microM) caused an increase in C/EBP beta expression between 2 and 12 h of treatment, with no evident intracellular morphological changes. Dopamine (500 microM) led to the appearance of autophagic-like vacuoles and a marked increase in GADD153/CHOP between 6 and 24 h of treatment. The expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease and other neurological disorders, increased with a profile similar to C/EBP beta. In addition, overexpression of C/EBP beta caused a concomitant increase in the expression of alpha-synuclein but not of GADD153. In contrast, the overexpression of GADD153 did not alter the expression of alpha-synuclein. Inhibition of JNK by SP600125 reduced increases in C/EBP beta and alpha-synuclein expression, whereas inhibition of both JNK and p38MAPK (with SB203580) blocked the increase in GADD153 expression. We conclude that dopamine, through a mechanism driven by stress-activated MAPKs, triggers C/EBP beta and GADD153 expression in a dose-dependent way. Given that the promoter region of the alpha-synuclein gene contains distinct zones that are susceptible to regulation by C/EBP beta, this factor could be involved in the increased expression of alpha-synuclein after dopamine-induced cell stress. GADD153 increase seems to be related with the endoplasmic reticulum stress, autophagy and cell death observed at high dopamine concentrations.

Topics: alpha-Synuclein; Amines; Benzimidazoles; Blotting, Western; Carbocyanines; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Cell Count; Cell Death; Cell Line, Tumor; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Molecular Chaperones; Nerve Tissue Proteins; Neuroblastoma; Proteomics; Synucleins; Time Factors; Transcription Factor CHOP; Transcription Factors; Transfection

The pathology of Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. However, the pathogenesis of PD remains unclear. Heat shock proteins (HSPs) have many functions, including inhibition of apoptosis and necrosis, protection from oxidative stress, and maintenance of the mitochondrial membrane potential, that are related to neurodegenerative diseases. 1-Methyl-4-phenylpyridinium ion (MPP(+)) is a neurotoxin that selectively inhibits the mitochondrial functions of DA neurons in the substantia nigra. MPP(+) administration is accepted as a model for PD. In the present study, we found that MPP(+) induced a concentration- and time-dependent decrease in cell viability. Lower concentrations of MPP(+) induced mainly early apoptosis, and, as the concentration increased, the number of late apoptotic and necrotic cells significantly increased. However, treated by heat shock preconditioning or transfection with HDJ-1, a homologue of human Hsp40, cells showed marked improvement in viability after exposure to the same concentrations of MPP(+). Compared with heat shock, HDJ-1 appeared to improve cell viability obviously. Similarly, HDJ-1 elicited significantly stronger protective effects against apoptosis and necrosis. In addition, HDJ-1 transfection maintained more injured cells in early apoptotic stages and inhibited the occurrence of late apoptotic/necrotic events. Heat shock and HDJ-1 both ameliorated MPP(+)-induced cytotoxicity by maintaining the mitochondrial membrane potential and reducing reactive oxygen species (ROS). Therefore, the effects of HSPs, such as reducing apoptosis and necrosis, preserving mitochondrial functions and decreasing oxidative stress, may bring a novel approach for PD therapy.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Benzimidazoles; Blotting, Western; Carbocyanines; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Hot Temperature; Humans; Membrane Potentials; Mitochondria; Necrosis; Neuroblastoma; Reactive Oxygen Species; Time Factors; Transfection

Decreased proteasome activity is an important pathology in Parkinson's disease (PD), which is related to cell death and Lewy body formation. In this study, we show that p53-activity may correlate with neuronal death via the mitochondrial pathway in PD model. The proteasome inhibitor, MG132, induced the accumulation of p53 in human dopaminergic neuroblastoma SH-SY5Y cells. The increased stabilization of p53 upregulated the level of Bax and mitochondrial depolarization. These events were inhibited by the p53 inhibitor, pifithrin-alpha (PFT). Cell viability analyzes demonstrated that PFT partially prevented MG132-induced cell death. These results suggest that p53 is a candidate as an intermediary between the proteasome system and mitochondria-related neuronal death in PD.

Topics: bcl-2-Associated X Protein; Benzimidazoles; Benzothiazoles; Blotting, Western; Carbocyanines; Cell Death; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Fluorescent Antibody Technique; Humans; Leupeptins; Membrane Potentials; Mitochondria; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Time Factors; Toluene; Tumor Suppressor Protein p53

Defects in mitochondrial energy metabolism due to respiratory chain disorders lead to a decrease in mitochondrial membrane potential (DeltaPsim) and induce apoptosis. Since coenzyme Q10 (CoQ10) plays a dual role as an antioxidant and bioenergetic agent in the respiratory chain, it has attracted increasing attention concerning the prevention of apoptosis in mitochondrial diseases. In this study the potential of CoQ10 to antagonize the apoptosis-inducing effects of the respiratory chain inhibitor rotenone was explored by video-enhanced microscopy in SH-SY5Y neuroblastoma cells. The cationic fluorescent dye JC-1 which exhibits potential-dependent accumulation in mitochondria was used as an indicator to monitor changes in DeltaPsim. The relative changes in fluorescence intensity after incubation with rotenone for 15 minutes were calculated. Pre-treatment with CoQ10 (10 or 100 microM) for 48 h led to a significant reduction of rotenone-induced loss of DeltaPsim. These results suggest, that cytoprotection by CoQ10 may be mediated by raising cellular resistance against the initiating steps of apoptosis, namely the decrease of DeltaPsim. Whether these data may provide new directions for the development of neuroprotective strategies has to be investigated in future studies.

Topics: Antioxidants; Apoptosis; Benzimidazoles; Carbocyanines; Coenzymes; Cytoprotection; Electron Transport; Electron Transport Complex I; Energy Metabolism; Enzyme Inhibitors; Fluorescent Dyes; Humans; Membrane Potentials; Mitochondria; Neuroblastoma; Neurons; Rotenone; Tumor Cells, Cultured; Ubiquinone

The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of approximately 100,000 compounds screened at 8 microM, and the calcein assay identifies permeability transition pore-specific inhibitors.

Topics: Benzimidazoles; Carbocyanines; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; Microscopy, Confocal; Mitochondria; Neuroblastoma; Permeability; Tumor Cells, Cultured

Diseases linked to defective mitochondrial function are characterized by morphologically abnormal, swollen mitochondria with distorted cristae. Several lines of evidence now suggest that sporadic forms of Parkinson's disease (PD) and Alzheimer's disease (AD) are linked to mitochondrial dysfunction arising from defects in mitochondrial DNA (mtDNA). Human neuroblastoma (SH-SY5Y) cells that are deficient in mtDNA (Rho(0)) were repopulated with mitochondria from AD or PD patients or age-matched controls. These cytoplasmic hybrid (cybrid) cell lines differ only in the source of their mtDNA. Differences between cybrid cell lines therefore arise from differences in mtDNA and provide a model for the study of how impaired mitochondrial function alters the mitochondria themselves and how these changes adversely affect the neuronal cells they occupy. Cybrid cell mitochondria were labeled with the mitochondrial membrane potential-sensitive dye, JC-1. Analysis of these JC-1 labeled mitochondria by confocal microscopy revealed that mitochondrial membrane potential was significantly reduced in both PD and AD cybrid cells when compared with controls. Ultrastructural examination showed that control cybrid cells contained small, morphologically normal, round or oval mitochondria with a dark matrix and regular distribution of cristae. PD cybrid cells contained a significant and increased percentage of mitochondria that were enlarged or swollen and had a pale matrix with few remaining cristae (0.26-0.65 microm(2)). AD cybrid cells also contained a significantly increased percentage of enlarged or swollen mitochondria (0.25-5.0 microm(2)) that had a pale matrix and few remaining cristae. Other pathological features such as crystal-like intramitochondrial inclusions and cytoplasmic inclusion bodies were also found in PD and AD cybrids. These observations suggest that transfer of PD or AD mtDNA into Rho(0) cells was sufficient to produce pathological changes in mitochondrial ultrastructure that are similar to those seen in other mitochondrial disorders. These data were reported in abstract form (Trimmer et al., 1998, Soc. Neurosci. Abstr. 24: 476).

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Benzimidazoles; Carbocyanines; Electron Transport Complex I; Female; Fluorescent Dyes; Humans; Hybrid Cells; Inclusion Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Mitochondria; NADH, NADPH Oxidoreductases; Neuroblastoma; Organic Chemicals; Parkinson Disease