M17 Human Neuroblastoma Cells Research Articles

Retraction Note: Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Retraction Note: Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Crystalline H-aggregate nanoparticles for detecting dopamine release from M17 human neuroblastoma cells.

Dopamine (DA) is an important neurotransmitter, which is essential for transmitting signals in neuronal communications. The deficiency of DA release from neurons is implicated in neurological disorders. There has been great interest in developing new optical probes for monitoring the release behavior of DA from neurons. H-aggregates of organic dyes represent an ordered supramolecular structure with delocalized excitons. In this paper, we use the self-assembly of 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)) in ammonia solution to develop crystalline H-aggregate nanoparticles, in which DiSC2(5) molecules show long-range π-π stacking. The crystalline H-aggregate nanoparticles are stable in cell culture medium and can serve as an efficient photo-induced electron transfer (PET) probe for the detection of DA with the concentration as low as 0.1 nM in cell culture medium. Furthermore, the crystalline H-aggregate nanoparticle-based PET probe is used to detect the release behavior of DA from the M17 human neuroblastoma cells. We find that the DA release from the cells is enhanced by nicotine stimulations. Our results highlight the potential of crystalline H-aggregate nanoparticle-based PET probes for diagnosing nervous system diseases and verifying therapies.

Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.

Amyloid β peptide promotes lysosomal degradation of clusterin via sortilin in hippocampal primary neurons

Progressive accumulation of amyloid-β peptide (Aβ) in the brain is implicated as the central event in the development of Alzheimer's disease (AD). It is thought that extracellular Aβ triggers toxic signals leading to neurodegeneration. The events downstream of Aβ however are not entirely clear. Clusterin (Apo J) is one of the major risk factors for sporadic form of AD. Clusterin binds to Aβ and prevents Aβ aggregation. In addition, clusterin promotes Aβ degradation and accelerates Aβ clearance from the brain. Clusterin thus protects neurons from Aβ and loss of clusterin level in the brain is implicated as promoting AD pathology. In this study, we found that the level of clusterin protein but not mRNA is reduced in the brains of 3xTg-AD mice. When rat hippocampal primary neurons were treated with Aβ1-42, level of clusterin protein but not mRNA was downregulated. Aβ1-42-induced downregulation of clusterin was blocked by lysosome inhibitors bafilomycin A1 and ammonium chloride. In neurons, Aβ1-42 induced expression of sortilin, a lysosomal sorting protein that targets proteins to lysosome for degradation. In BE(2) M17 human neuroblastoma cells, clusterin bound to sortilin and when sortilin expression was silenced, Aβ1-42-induced clusterin downregulation was almost completely blocked. Our data demonstrate that in neurons, Aβ1-42 promotes lysosomal degradation of clusterin by inducing expression of sortilin and provide a novel mechanism by which Aβ promotes AD pathogenesis.

Type-1 interferons contribute to oxygen glucose deprivation induced neuro-inflammation in BE(2)M17 human neuroblastoma cells

BackgroundHypoxic-ischaemic injuries such as stroke and traumatic brain injury exhibit features of a distinct neuro-inflammatory response in the hours and days post-injury. Microglial activation, elevated pro-inflammatory cytokines and macrophage infiltration contribute to core tissue damage and contribute to secondary injury within a region termed the penumbra. Type-1 interferons (IFNs) are a super-family of pleiotropic cytokines that regulate pro-inflammatory gene transcription via the classical Jak/Stat pathway; however their role in hypoxia-ischaemia and central nervous system neuro-inflammation remains unknown. Using an in vitro approach, this study investigated the role of type-1 IFN signalling in an inflammatory setting induced by oxygen glucose deprivation (OGD).MethodsHuman BE(2)M17 neuroblastoma cells or cells expressing a type-1 interferon-α receptor 1 (IFNAR1) shRNA or negative control shRNA knockdown construct were subjected to 4.5 h OGD and a time-course reperfusion period (0 to 24 h). Q-PCR was used to evaluate IFNα, IFNβ, IL-1β, IL-6 and TNF-α cytokine expression levels. Phosphorylation of signal transducers and activators of transcription (STAT)-1, STAT-3 and cleavage of caspase-3 was detected by western blot analysis. Post-OGD cellular viability was measured using a MTT assay.ResultsElevated IFNα and IFNβ expression was detected during reperfusion post-OGD in parental M17 cells. This correlated with enhanced phosphorylation of STAT-1, a downstream type-1 IFN signalling mediator. Significantly, ablation of type-1 IFN signalling, through IFNAR1 knockdown, reduced IFNα, IFNβ, IL-6 and TNF-α expression in response to OGD. In addition, MTT assay confirmed the IFNAR1 knockdown cells were protected against OGD compared to negative control cells with reduced pro-apoptotic cleaved caspase-3 levels.ConclusionsThis study confirms a role for type-1 IFN signalling in the neuro-inflammatory response following OGD in vitro and suggests its modulation through therapeutic blockade of IFNAR1 may be beneficial in reducing hypoxia-induced neuro-inflammation.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

BackgroundImmortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.ResultsWe studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG.ConclusionTaken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Effects of intravenous immunoglobulin on alpha synuclein aggregation and neurotoxicity

α-Synuclein is thought to contribute to the pathogenesis of Parkinson's disease (PD). It is the main protein in Lewy bodies, the pathognomonic inclusion bodies in the PD substantia nigra, and mutations which increase its aggregation and/or expression are associated with familial early-onset parkinsonism. Soluble oligomers are considered to be α-synuclein's most neurotoxic conformation. We previously reported that intravenous immunoglobulin (IVIG) products contain specific antibodies to α-synuclein which do not prevent development of four-day α-synuclein oligomers. The objective of this study was to further examine IVIG's effects on α-synuclein's aggregation and neurotoxicity. The IVIG product Gammagard (Baxter Healthcare) did not prevent the development of nine-day α-synuclein oligomers, nor did it degrade preformed oligomers, as shown by western blots performed on gels run under reducing/denaturing conditions and native gels. In western blots of native gels, an additional low molecular weight band (~22kDa) was detected in α-synuclein incubated for four days in Gammagard, but not in Gammagard alone. No significant differences were found for Thioflavin-T reactivity between α-synuclein amorphous aggregates grown in Gammagard vs. those grown in phosphate-buffered saline. Gammagard partially protected SK-N-BE(2)M17 human neuroblastoma cells against α-synuclein oligomer toxicity (p=0.007 vs. protective effects of normal human IgG). These findings suggest that although IVIG does not prevent α-synuclein aggregation, it still may reduce α-synuclein neurotoxicity through an unknown mechanism.

Parkinson’s disease‐associated DJ‐1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.

Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.

The Hsp90 Kinase Co-chaperone Cdc37 Regulates Tau Stability and Phosphorylation Dynamics

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

Parkin Attenuates Wild-Type τ Modification in the Presence of β-Amyloid and α-Synuclein

Changes in tau (τ) metabolism comprise important pathological landmarks in the tauopathies with parkinsonism as well as Parkinson’s disease and Alzheimer’s disease. Mutations in the parkin gene are associated with Parkinson’s disease. Deposits of amyloid proteins, including Aβ and α-synuclein coexist in the brains of patients with dementia with Lewy bodies; however, it is not known how either of them interacts with τ to provoke neurofibrillary tangle formation across the tauopathies. Here, we show a role for parkin against τ pathology in the presence of intracellular Aβ or α-synuclein. Parkin attenuates four-repeat human τ, but not mutant P301L, hyperphosphorylation in the presence of intracellular Aβ1–42, or α-synuclein and decreases GSK-3β activity in amyloid-stressed M17 human neuroblastoma cells. These data suggest that parkin may counteract the alteration of τ metabolism in certain neurodegenerative diseases with τ cytopathy and parkinsonism.

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.

Parkin Attenuates Wild-Type τ Modification in the Presence of β-Amyloid and α-Synuclein

Changes in tau (tau) metabolism comprise important pathological landmarks in the tauopathies with parkinsonism as well as Parkinson's disease and Alzheimer's disease. Mutations in the parkin gene are associated with Parkinson's disease. Deposits of amyloid proteins, including Abeta and alpha-synuclein coexist in the brains of patients with dementia with Lewy bodies; however, it is not known how either of them interacts with tau to provoke neurofibrillary tangle formation across the tauopathies. Here, we show a role for parkin against tau pathology in the presence of intracellular Abeta or alpha-synuclein. Parkin attenuates four-repeat human tau, but not mutant P301L, hyperphosphorylation in the presence of intracellular Abeta(1-42), or alpha-synuclein and decreases GSK-3beta activity in amyloid-stressed M17 human neuroblastoma cells. These data suggest that parkin may counteract the alteration of tau metabolism in certain neurodegenerative diseases with tau cytopathy and parkinsonism.

Clioquinol inhibits peroxide-mediated toxicity through up-regulation of phosphoinositol-3-kinase and inhibition of p53 activity

A growing body of evidence supports a central role for biometals in neurodegenerative disorders. Biometals induce oxidative stress through the generation of reactive oxygen species and contribute to neuronal cell dysfunction in Alzheimer's disease (AD), prion disorders and Parkinson's disease (PD). Therapies based on modulation of biometal metabolism are currently being developed and the metal ligand, 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol or CQ) has been investigated for the treatment of AD. CQ has also shown therapeutic benefits in an animal model of PD. However, little is known about the neuroprotective processes of CQ in vivo. In this study, we examined the effect of CQ in BE(2)-M17 human neuroblastoma cells exposed to increased oxidative stress (hydrogen peroxide (H 2O 2) treatment). Although CQ alone induced a moderate toxic effect on cells, when added to H 2O 2-treated M17 cells, CQ induced a significant inhibition of H 2O 2 toxicity. This correlated with up-regulation of phosphoinositol-3-kinase (PI3K) activity in CQ-treated cells. The protective action of CQ was not observed in murine N2a neuroblastoma cells treated with H 2O 2 and this cell-line did not reveal CQ-mediated increases in PI3K activation. The protective effect was specific for CQ and was not induced by a number of different metal ligands. Inhibition of PI3K activity with LY294002 prevented CQ protection against H 2O 2 toxicity, demonstrating a crucial role for CQ activation of PI3K in protection against oxidative stress. Furthermore, CQ inhibited H 2O 2-mediated up-regulation of p53 activity in the M17 cells and this was dependent on PI3K activation. Our studies demonstrate that in human M17 cells, CQ can protect against oxidative stress by activating the PI3K-dependent survival pathway and blocking p53-mediated cell death. These findings have important implications for the development of protective metal ligand-based therapies for treatment of disorders involving oxidative stress.

SU-FF-T-275: Invitro Dose and Dose Rate Feasibility Study for Effective Tomotherapy TMI Treatment

Purpose: We reported the successful use of Tomotherapy for the total marrow irradiation (TMI) treatment. However, Tomotherapy delivers treatment at a much higher dose rate (400–900 cGy/min) than current TBI treatment (10 – 25 cGy/min) delivered using a standard medical accelerator. Even with the more accurate dosimetry possible with Tomotherapy, the effect of high dose rates even at limited doses is unclear at best. We aim to study the effect of high dose and dose rate on cell survival. Method and Materials: A549 human lungcells, BE(2)‐M17 human neuroblastoma cells and N2A mouse neuroblastoma cells were used. Cells were plated in T‐25 flasks with appropriate medium. After 8 hours attachment period, each flask of cells were irradiated with a different dose: 2, 4, 6, 9 and 12 Gy. As the dose rate, 112 cGy/min was used in A549 and BE(2)‐M17 cells and 400 cGy/min was used in N2A cells.Cells were cultured for 10–15 days and survived cells were making colonies. Colonies were fixed with a mixture of methanol and acetic acid (10:1 v/v) and stained with 1% crystal violet. Colonies containing more than 30 cells were scored. Results: BE(2)‐M17 cells were more sensitive to radiation than A549 and N2A cells. Mean lethal dose, D0, were 89, 136 and 188 rad in BE(2)‐M17, A549 and N2A cells in each. Plating efficiency at 6Gy were 0.50, 6.23 and 6.97 % in BE(2)‐M17, A549 and N2A cells in each. Extrapolation number were 8.17, 5.20 and 2.51 in BE(2)‐M17, A549 and N2A cells in each. Conclusions: We show that different cell lines have different dose response invitro. Currently we are continuing the survival study for dose rate from 10 cGy/min to 800 cGy/min which will be reported in the conference.

P38 Activation Mediates Amyloid-β Cytotoxicity

Amyloid-beta is a leading candidate factor in the development of Alzheimer disease (AD), however the mechanisms involved are unclear. As such, there has been considerable interest in evidence showing that the neuronal damage caused by amyloid-beta is mediated by oxidative stress. Notably, oxidative stress leads to activation of stress-activated protein kinases, which we and others have shown are also involved in AD pathogenesis. One SAPK in particular, p38, appears to be crucial in AD and therefore, in the current study, we investigated the role of p38 activation in amyloid-beta cytotoxicity. Our data showed p38 activation was induced by amyloid-beta in a concentration-dependent manner in M17 human neuroblastoma cells. Notably, amyloid-beta toxicity was significantly decreased by inhibition of p38 activity by overexpressing dominant negative p38. Consistent with this, in primary cortical neurons amyloid-beta also induced p38 activation and amyloid-beta toxicity was significantly diminished when p38 was inhibited by its specific inhibitor, SB203580. Taken together, these data suggest that p38 is a key downstream effector of amyloid-beta-induced neuronal death and blocking this pathway may be of therapeutic value.

Neuroprotective properties of Bcl-w in Alzheimer disease.

While there is a host of pro-apoptotic stimuli that target neurons in Alzheimer disease (AD), given the chronicity of the disease and the survival of many neurons, those neurons must either avoid or, at minimum, delay apoptotic death signaling. In this study, we investigated Bcl-w, a novel member of the Bcl-2 family that promotes cell survival. In AD, we found increased levels of Bcl-w associated with neurofibrillary pathology and punctate intracytoplasmic structures whereas, in marked contrast, there are only low diffuse levels of Bcl-w in the neuronal cytoplasm of age-matched control cases. Immunoblot analysis confirmed that Bcl-w levels were significantly increased in AD. By electron microscopy, we determined that the increased Bcl-w expression in AD was ultrastructurally localized to mitochondria and neurofibrillary pathology. To investigate the cause and consequence of Bcl-w up-regulation in neurons, we found that fibrillized amyloid-beta led to increased Bcl-w protein levels in M17 human neuroblastoma cells, and that overexpression of Bcl-w significantly protected neurons against staurosporine- and amyloid-beta-induced apoptosis. Taken together, these series of results suggest that Bcl-w may play an important protective role in neurons in the diseased brain and that this aspect could be therapeutically harnessed to afford neuroprotection.