Stress In Neuroblastoma Cells Research Articles

Ginsenosides Rg1 and Rg2 Activate Autophagy and Attenuate Oxidative Stress in Neuroblastoma Cells Overexpressing Aβ(1-42).

Alzheimer's disease is a neurodegeneration with protein deposits, altered proteolysis, and inflammatory and oxidative processes as major hallmarks. Despite the continuous search for potential therapeutic treatments, no cure is available to date. The use of natural molecules as adjuvants in the treatment of Alzheimer's disease is a very promising strategy. In this regard, ginsenosides from ginseng root show a variety of biological effects. Here, we dissected the role of ginsenosides Rg1 and Rg2 in modulating autophagy and oxidative stress in neuroblastoma cells overexpressing Aβ(1-42). Key hallmarks of these cellular processes were detected through immunomethods and fluorometric assays. Our findings indicate that ginsenosides are able to upregulate autophagy in neuronal cells as demonstrated by increased levels of LC3II and Beclin-1 proteins and decreased amounts of p62. Simultaneously, an activation of lysosomal hydrolases was observed. Furthermore, autophagy activation promoted the clearance of Aβ(1-42). Rg1 and Rg2 also reduced oxidative stress sources and macromolecule oxidation, promoting NRF2 nuclear translocation and the expression of antioxidant enzymes. Our data further clarify the mechanisms of action of Rg1 and Rg2, indicating new insights into their role in the management of disorders like Alzheimer's disease.

Studies regarding influences of ethanol on hypoxemic stress in neuroblastoma cells

: Introduction In the modern world a pathology with increasing frequency is that of spinal cord injury (SCI), with the risk of dysfunction on multiple levels. Following our clinical experience, we have conducted studies on the effect of hypoxic stress (also present in spinal cord injury) on neuronal cell cultures treated for a long time with ethanol (trying to reproduce chronic alcoholism). In this article we present the behavior of neural cells subacutely exposed to ethanol after hypoxic stress, in order to perform a comparative analysis with chronic exposure to alcohol. Materials and methods We performed subacute treatments with ethanol in neural cell cultures. We evaluated gene expression and protein synthesis in the case of experimentally induced hypoxic stress. Disscusions The complexity of the human body is superior to experimental models. This experiment creates a model of extremely complex changes after spinal cord injury. The results cannot undoubtedly overlap the conditions of the physiopathological reality. Results and conclusions The model of neural hypoxic suffering in cell cultures is similar in the case of cell cultures treated subacutely with ethanol, except: the risk of neurodegeneration, the phenomenon of axonal die-back, proapoptotic tendencies, proinflammatory tendencies. The effect of chronic (more than acute/subacute) ethanolic consumption seems to determine geno-molecular neural changes with a potentially favorable effect regarding the response (immediate and long-term) to spinal cord injury

Probiotic Lactiplantibacillus plantarum KU210152 and its fermented soy milk attenuates oxidative stress in neuroblastoma cells

We evaluated the probiotic properties and neuroprotective effects of Lactiplantibacillus plantarum KU210152 and its application in soy milk. L. plantarum KU210152 exhibited high tolerance to artificial gastrointestinal conditions, high adhesion to intestinal cells (HT-29), and safe enzyme production. Conditioned medium acquired from HT-29 cells treated with heat-killed lactic acid bacteria (LAB-CM) was used to evaluate the neuroprotective effects. The CM exhibited neuroprotective effects via cell viability assay, morphological observations, and suppression of ROS production. Heat-killed L. plantarum KU210152 increased brain-derived neurotrophic factor (BDNF) and tyrosine hydroxylase (TH) expression in HT-29 cells. In SH-SY5Y cells, pretreatment with L. plantarum KU210152 CM decreased Bax/Bcl-2 ratio and upregulated BDNF and TH expression. The CM inhibited caspase-9 and caspase-3 activities. The neuroprotective effects of L. plantarum KU210152 were also confirmed in fermented soy milk. Therefore, both L. plantarum KU210152 and the fermented soy milk can be used as functional ingredients with neuroprotective effects against oxidative stress.

Xanthine-Dopamine Hybrid Molecules as Multitarget Drugs with Potential for the Treatment of Neurodegenerative Diseases.

Multitarget drugs based on a hybrid dopamine-xanthine core were designed as potential drug candidates for the treatment of neurodegenerative diseases. Monoamine oxidase B (MAO-B) inhibitors with significant ancillary A2A adenosine receptor (A2AAR) antagonistic properties were further developed to exhibit additional phosphodiesterase-4 and -10 (PDE4/10) inhibition and/or dopamine D2 receptor (D2R) agonistic activity. While all of the designed compounds showed MAO-B inhibition in the nanomolar range mostly combined with submicromolar A2AAR affinity, significant enhancement of PDE-inhibitory and D2R-agonistic activity was additionally reached for some compounds through various structural modifications. The final multitarget drugs also showed promising antioxidant properties in vitro. In order to evaluate their potential neuroprotective effect, representative ligands were tested in a cellular model of toxin-induced neurotoxicity. As a result, protective effects against oxidative stress in neuroblastoma cells were observed, confirming the utility of the applied strategy. Further evaluation of the newly developed multitarget ligands in preclinical models of Alzheimer's and Parkinson's diseases is warranted.

Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Protection of Iron-Induced Oxidative Damage in Neuroblastoma (SH-SY5Y) Cells by Combination of 1-(N-Acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and Green Tea Extract.

Iron is a crucial trace element and essential for many cellular processes; however, excessive iron accumulation can induce oxidative stress and cell damage. Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, have been associated with altered iron homoeostasis causing altered iron distribution and accumulation in brain tissue. This study aims to investigate the protective effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in combination with green tea extract (GTE) on iron-induced oxidative stress in neuroblastoma (SH-SY5Y) cells. Cells were cultured in medium with or without ferric chloride loading. Their viability and mitochondrial activity were assessed using MTT and JC-1 staining methods. Levels of the cellular labile iron pool (LIP), reactive oxygen species (ROS), and lipid-peroxidation products were determined using calcein acetoxymethyl ester, 2′,7′-dichlorohydrofluorescein diacetate, and TBARS-based assays, respectively. The viability of iron-loaded cells was found to be significantly increased after treatment with CM1 (10 µM) for 24 h. CM1 co-treatment with GTE resulted in a greater protective effect than their monotherapy. Combination of CM1 and GTE also reduced mitochondrial disruption and LIP content and ROS and TBARS production. In conclusion, the combination of CM1 and GTE exhibits protection against iron-induced oxidative stress in neuroblastoma cells.

Sea Cucumber-Derived Peptides Alleviate Oxidative Stress in Neuroblastoma Cells and Improve Survival in C. elegans Exposed to Neurotoxic Paraquat.

Oxidative stress results when the production of oxidants outweighs the capacity of the antioxidant defence mechanisms. This can lead to pathological conditions including cancer and neurodegeneration. Consequently, there is considerable interest in compounds with antioxidant activity, including those from natural sources. Here, we characterise the antioxidant activity of three novel peptides identified in protein hydrolysates from the sea cucumber Apostichopus japonicus. Under oxidative stress conditions, synthetic versions of the sea cucumber peptides significantly compensate for glutathione depletion, decrease mitochondrial superoxide levels, and alleviate mitophagy in human neuroblastoma cells. Moreover, orally supplied peptides improve survival of the Caenorhabditis elegans after treatment with paraquat, the latter of which leads to the production of excessive oxidative stress. Thus, the sea cucumber peptides exhibit antioxidant activity at both the cellular and organism levels and might prove attractive as nutritional supplements for healthy ageing.

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

Acetyl Mimicking K274Q Mutation Enhances TAU Aggregation, Increases the Affinity of TAU for Metal Ions and Reduces its Ability to Protect DNA

Tau is primarily acetylated at K174, K274, K280, and K281 residues in Alzheimer's disease. The acetylation of K274-tau is associated with dementia. In this study, we have examined the mechanism of the toxicity of acetylated K274-tau. The surface plasmon resonance experiment indicated that an acetylation mimicking mutation (K274Q) in tau strongly reduced the ability of tau to bind to the tubulin. Further, light scattering, sedimentation and electron microscopic analysis showed that K274Q mutation decreased the ability of tau to polymerize tubulin while K274R mutation did not reduce the ability of tau to bind or polymerize tubulin. Further, Zn2+ and Al3+ increased the liquid-liquid phase separation of wild type tau, an initial stage of tau aggregation. In addition to this K274Q mutation also strongly decreased the critical concentration for the liquid-liquid phase separation of tau. Using far-UV circular dichroism and fluorescence spectroscopy, Zn2+ and Al3+ were found to induce conformational changes in tau. The K274Q mutation also enhanced the binding affinity of tau for Zn2+ and Al3+. In addition, Zn2+ and Al3+ significantly enhanced the aggregation propensity of K274Q tau in comparison to wild type tau. Interestingly, wild type tau binds to DNA with a higher affinity than K274Q tau and protects DNA from the DNase treatment in vitro and heat stress in neuroblastoma cells more efficiently than K274Q tau. The results indicated that the acetylation of K274 residue of tau may increase metal ions induced toxicity and diminish the ability of tau to protect DNA against stress.

The Acetyl Mimicking Mutation, K274Q in Tau, Enhances the Metal Binding Affinity of Tau and Reduces the Ability of Tau to Protect DNA.

The aggregation of tau, a microtubule-associated protein, is known to play an important role in several neurological disorders including Alzheimer's disease. Alzheimer's disease is considered to be associated with the dyshomeostasis of metal ions such as aluminum, zinc, copper, and ferric ions. Tau is predominately acetylated at the K274 residue in Alzheimer's disease, and the acetylation of the K274 residue is thought to be linked with dementia. Using acetyl mimicking K274Q mutation in tau, we have examined the effects of the acetylation at K274 residue of tau on the interactions of tau with metal ions and also on the ability of tau to protect DNA from the heat and other stressors. We found that Zn2+ and Al3+ increased the liquid-liquid phase separation of tau, an initial stage of tau aggregation. Further, Zn2+ and Al3+ considerably reduced the critical concentration for the phase separation of K274Q tau. Using far-UV circular dichroism and fluorescence spectroscopy, we provide evidence suggesting that the binding of Zn2+ and Al3+ induces conformational changes in tau. The K274Q mutation enhanced the binding affinity of tau for Zn2+, Al3+, Cu2+, and Fe3+ ions. In addition, Zn2+, Al3+, Cu2+, and Fe3+ significantly enhanced the aggregation propensity of K274Q tau in comparison to tau. Interestingly, tau binds to DNA with a higher affinity than K274Q tau. Tau protects DNA from the DNase treatment in vitro as well as from the heat stress in neuroblastoma cells more efficiently than K274Q tau. The results indicated that the acetylation of K274 residue of tau may increase metal ion-induced toxicity and diminish the ability of tau to protect DNA.

P 183 - Differentiation modifies Bach1 dependent regulation of HO-1 expression and increases sensitivity to oxidative stress in neuroblastoma cells

Neuronal adaptation to oxidative stress is crucial to prevent degenerative diseases. The role of the Nrf2/HO-1 system in cell response to hydrogen peroxide (H2O2) has been investigated using SH-SY5Y neuroblastoma cells as undifferentiated or after differentiation with all-trans retinoic acid (ATRA). We showed that undifferentiated cells resisted to oxidative stimulus and up-regulated HO-1 which was responsible for their survival, since its silencing decreased viability in cells exposed to H2O2. On the contrary, in ATRA-treated cells the viability decreased in response to increasing concentration of H2O2 and HO-1 was not induced. However, bilirubin supplementation restores cell viability, underlining the role of HO-1-derived bilirubin in cell resistance to oxidative stress. Investigating the mechanisms of HO-1 induction, we showed that in undifferentiated cells the nuclear level of Bach1, repressor of HO-1 transcription, decreased as well as its binding to the promoter of HO-1. In the same condition, Nrf2 binding to the same DNA region increased. Yet, in differentiated cells Bach1 nuclear level was not modified by the exposure to H2O2 as well as its binding to HO-1 promoter and, as a consequence, the binding of Nrf2 to HO-1 promoter was not modified by H2O2. In conclusion, our findings highlight the role of Bach1/HO-1/bilirubin in neuronal response to oxidative stress.

Subcellular Parkinson's Disease-Specific Alpha-Synuclein Species Show Altered Behavior in Neurodegeneration.

Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein α-synuclein. α-synuclein is considered an intrinsically disordered 14kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that α-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different α-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa α-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific α-synuclein features in neurodegeneration will allow for the identification of neurotoxic α-synuclein species, which represent prime targets to reduce α-synuclein pathogenicity.

Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain.

Neuronal ELAV-like (nELAVL) RNA binding proteins have been linked to numerous neurological disorders. We performed crosslinking-immunoprecipitation and RNAseq on human brain, and identified nELAVL binding sites on 8681 transcripts. Using knockout mice and RNAi in human neuroblastoma cells, we showed that nELAVL intronic and 3' UTR binding regulates human RNA splicing and abundance. We validated hundreds of nELAVL targets among which were important neuronal and disease-associated transcripts, including Alzheimer's disease (AD) transcripts. We therefore investigated RNA regulation in AD brain, and observed differential splicing of 150 transcripts, which in some cases correlated with differential nELAVL binding. Unexpectedly, the most significant change of nELAVL binding was evident on non-coding Y RNAs. nELAVL/Y RNA complexes were specifically remodeled in AD and after acute UV stress in neuroblastoma cells. We propose that the increased nELAVL/Y RNA association during stress may lead to nELAVL sequestration, redistribution of nELAVL target binding, and altered neuronal RNA splicing.

Protective Effects of Acetyl-l-Carnitine on Cisplatin Cytotoxicity and Oxidative Stress in Neuroblastoma

The most widely used platinum-derived drug is cisplatin in neuroblastoma (NB) chemotherapy, which is severely neurotoxic. Acetyl-L-Carnitine (ALC) is a natural occurring compound with a neuroprotective activity in several experimental paradigms. The aim of this study was to determine the effects of ALC on cisplatin induced cytotoxicity and oxidative stress in NB cells. SH-SY5Y (N-Myc negative) and KELLY (N-Myc positive) human NB cell lines were used. Cisplatin induced apoptosis was assessed by using a Cell Death Detection ELISA(PLUS) kit. Lipid peroxidation levels were determined by HPLC analysis. Glutathione levels were determined spectrophotometrically. ALC was used prophylactic or after cisplatin application. The level of cisplatin doses were determined in both type of NB cells at which 50% cell death occurred along with synchronized apoptosis induced. Prophylactic 10 and 50 micromol of ALC concentrations were decreased cisplatin induced lipid peroxidation compared to controls that normally exhibited apoptosis especially in SH-SY5Y cells. Cisplatin caused oxidative stress through decreasing glutathione levels in both cell types. ALC were effectively inhibited the increase in cisplatin induced oxidized glutathione and lipid peroxidation formation in NB cells. We suggested that prophylactic ALC would be a useful agent for cisplatin induced toxicity in NB cells.

ERdj5 Sensitizes Neuroblastoma Cells to Endoplasmic Reticulum Stress-induced Apoptosis

Down-regulation of the unfolded protein response (UPR) can be therapeutically valuable in cancer treatment, and endoplasmic reticulum (ER)-resident chaperone proteins may thus be targets for developing novel chemotherapeutic strategies. ERdj5 is a novel ER chaperone that regulates the ER-associated degradation of misfolded proteins through its associations with EDEM and the ER stress sensor BiP. To investigate whether ERdj5 can regulate ER stress signaling pathways, we exposed neuroblastoma cells overexpressing ERdj5 to ER stress inducers. ERdj5 promoted apoptosis in tunicamycin, thapsigargin, and bortezomib-treated cells. To provide further evidence that ERdj5 induces ER stress-regulated apoptosis, we targeted Bcl-2 to ER of ERdj5-overexpressing cells. Targeting the Bcl-2 to ER prevented the apoptosis induced by ER stress inducers but not by non-ER stress apoptotic stimuli, suggesting induction of ER stress-regulated apoptosis by ERdj5. ERdj5 enhanced apoptosis by abolishing the ER stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) and the subsequent translational repression. ERdj5 was found to inhibit the eIF2alpha phosphorylation under ER stress through inactivating the pancreatic endoplasmic reticulum kinase. The compromised integrated stress response observed in ERdj5-overexpressing ER-stressed cells due to repressed eIF2alpha phosphorylation correlated with impaired neuroblastoma cell resistance under ER stress. These results demonstrate that ERdj5 decreases neuroblastoma cell survival by down-regulating the UPR, raising the possibility that this protein could be a target for anti-tumor approaches.

An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15–25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction.

A caspase for stress

Stress in the endoplasmic reticulum (ER)—caused by anything from ischemia to expression of mutant proteins—triggers several responses. Cells slow translation, produce more chaperones, degrade more unfolded proteins, and as a last resort, induce apoptosis. Hitomi et al. (page 347) identify caspase-4 as a critical component of the pathway leading from ER stress to apoptosis, specifically after the expression of cytotoxic Aβ from Alzheimer's patients. Figure siRNA of caspase-4 (right) prevents most cell death after ER stress. Caspase-12 has already been identified as mediating such a function in mice, but the caspase-12 gene in humans has frame-shifts and a premature stop codon. Hitomi et al. therefore screened cDNA libraries with mouse caspase-12 and came up with human caspase-4. Although some caspase-4 is found on mitochondria, the protein is also found on the ER, and its cleavage after ER stress or incubation with Aβ is not prevented by overexpression of mitochondrial Bcl-2. Removal of caspase-4 with siRNA halved cell death after ER stress in neuroblastoma cells and reduced cell death by two thirds after Aβ treatment. But the incomplete suppression, especially in other cell lines, suggests that other caspases may share this responsibility. Sorting out possible contributions by caspase-2 and caspase-5 will be important before caspase-based therapy is attempted for Alzheimer's. ▪