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Sample records for lines sy-5y neuroblastoma

  1. Cytotoxicity induced by cypermethrin in Human Neuroblastoma Cell Line SH-SY5Y.

    PubMed

    Raszewski, Grzegorz; Lemieszek, Marta Kinga; Łukawski, Krzysztof

    2016-01-01

    The purpose of this study was to evaluate the cytotoxic potential of Cypermethrin (CM) on cultured human Neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with CM at 0-200µM for 24, 48, and 72 h, in vitro. It was found that CM induced the cell death of Neuroblastoma cells in a dose- and time-dependent manner, as shown by LDH assays. Next, some aspects of the process of cell death triggered by CM in the human SH-SY5Y cell line were investigated. It was revealed that the pan-caspase inhibitor Q-VD-OPh, sensitizes SH-SY5Y cells to necroptosis caused by CM. Furthermore, signal transduction inhibitors PD98059, SL-327, SB202190, SP600125 failed to attenuate the effect of the pesticide. Finally, it was shown that inhibition of TNF-a by Pomalidomide (PLD) caused statistically significant reduction in CM-induced cytotoxicity. Overall, the data obtained suggest that CM induces neurotoxicity in SH-SY5Y cells by necroptosis.

  2. Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

    PubMed

    Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

    2016-02-17

    Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

  3. Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

    PubMed Central

    Shipley, Mackenzie M.; Mangold, Colleen A.; Szpara, Moriah L.

    2016-01-01

    Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1–4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease. PMID:26967710

  4. Silicon as neuroprotector or neurotoxic in the human neuroblastoma SH-SY5Y cell line.

    PubMed

    Garcimartín, Alba; Merino, José Joaquín; Santos-López, Jorge Arturo; López-Oliva, María Elvira; González, María Pilar; Sánchez-Muniz, Francisco José; Benedí, Juana

    2015-09-01

    Silicon (Si) is a trace element that has been considered to be an environmental contaminant for many years, although different studies have recently reported it is an essential element for living cells. The present study tested the ability of different concentrations of Si G57™ to induce neuroprotection or neurotoxicity over 24 h in the SH-SY5Y human neuroblastoma cell line. Cell viability, cellular proliferation, LDH release, ROS, antioxidant capacity, TBARS, caspase-3, -8 and -9, DNA fragmentation, and TNF-α levels were evaluated. Low Si doses (50-250 ng mL(-1)) increased the cell viability and reduced caspase-3 and -8 activities and TNF-α level. The increase in cell viability was independent of any proliferative effect as there was no variation in cyclin E and PCNA levels. At higher concentrations, Si increased caspase-3, as well as TBARS, LDH, DNA fragmentation, and TNF-α releases. Altogether, these results suggest that Si could act either as a neuroprotector or a neurotoxic agent depending on the concentration tested. This study emphasizes the importance of developing new neuroprotective therapies based on low Si doses.

  5. Autophagy inhibition by caffeine increases toxicity of methamphetamine in SH-SY5Y neuroblastoma cell line.

    PubMed

    Pitaksalee, Rujiraporn; Sanvarinda, Yupin; Sinchai, Theerin; Sanvarinda, Pantip; Thampithak, Anusorn; Jantaratnotai, Nattinee; Jariyawat, Surawat; Tuchinda, Patoomratana; Govitrapong, Piyarat; Sanvarinda, Pimtip

    2015-05-01

    Methamphetamine (METH) is a highly addictive CNS stimulant that its long-term use is associated with the loss of neurons in substantia nigra and development of Parkinson's disease later in life. Common form of METH is Ya-Ba tablet, in which, large portion of caffeine is added to the mass to enhance the stimulatory effect. Previous study demonstrated that caffeine potentiates the toxic effect of METH in association with the production of reactive oxygen species and the induction of apoptosis. Since METH causes induction of autophagy, the question was raised whether this pathway participates in the potentiating effect of caffeine on METH neurotoxicity. We used SH-SY5Y, a neuroblastoma cell line, as an in vitro model to study the effect of METH and caffeine. Co-treatment of non-toxic concentrations of METH, at 0.5 mM, and caffeine, at 1 mM, caused reduction of the cell viability. Reduction of the cell viability was associated with attenuation of autophagy, demonstrated by reduction of LC3-II levels and the number of autophagosome puncta, together with increase of caspase-3 activation. Similar effect was produced by treatment with autophagy inhibitors, 3-MA and wortmanin. Our results suggested that caffeine potentiates METH toxicity through inhibition of autophagy and that autophagy serves as a protective mechanism. In conclusion, we proposed the augmented hazard associated with caffeine and METH combination in Ya-Ba abusers.

  6. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Neuroprotective, antiapoptotic and antioxidant effects of l-carnitine against caffeine-induced neurotoxicity in SH-SY5Y neuroblastoma cell line.

    PubMed

    Bavari, Marzieh; Tabandeh, Mohammad Reza; Najafzadeh Varzi, Hosein; Bahramzadeh, Somayeh

    2016-01-01

    Caffeine is the most widely consumed nervous stimulant that induces oxidative-mediated apoptosis and cell cycle arrest in neural cells. Due to low toxicity, high accumulation in neural cells, reduction of the free fatty acids and antioxidant property, l-carnitine (LC) is an interesting compound to be used in vivo against several neuropathies. This study was conducted to evaluate the protective effect of LC against caffeine-induced cytotoxicity in SH-SY5Y neuroblastoma cell line. SH-SY5Y neuroblastoma cells were incubated with cytotoxic doses of caffeine (5 and 10 mM) in the presence or absence of LC (1 and 5 mM) for duration of 18-24 h. The antioxidant factors, DNA fragmentation and cytotoxic markers were assessed in treated cells. Our results showed that 5 mM LC for 18 h protected SH-SY5Y cells against cytotoxicity induced by both doses of caffeine. This protection was related to the inhibition of reactive oxygen species generation, the increase in the superoxide dismutase and catalase activities and glutathione content and the prevention of lipid peroxidation in cultured SH-SY5Y cells. Apoptosis markers such as DNA fragmentation and caspase-3 activity were also inhibited by 5 mM LC in caffeine-treated cells. Our results suggest that LC could protect SH-SY5Y cells from caffeine-induced injury through the inhibition of oxidative damage, mitochondria dysfunction and inhibition of cell apoptosis. Our results indicate that LC therapy may be a valuable approach for the suppression of oxidative stress-related apoptosis in various neural diseases.

  8. Bovine herpesvirus 1 can efficiently infect the human (SH-SY5Y) but not the mouse neuroblastoma cell line (Neuro-2A).

    PubMed

    Thunuguntla, Prasanth; El-Mayet, Fouad S; Jones, Clinton

    2017-03-15

    Bovine herpesvirus 1 (BoHV-1) is a significant bovine pathogen that establishes a life-long latent infection in sensory neurons. Previous attempts to develop immortalized bovine neuronal cells were unsuccessful. Consequently, our understanding of the BoHV-1 latency-reactivation cycle has relied on studying complex virus-host interactions in calves. In this study, we tested whether BoHV-1 can infect human (SH-SY5Y) or mouse (Neuro-2A) neuroblastoma cells. We provide new evidence that BoHV-1 efficiently infects SH-SY5Y cells and yields virus titers approximately 100 fold less than bovine kidney cells. Conversely, virus titers from productively infected Neuro-2A cells were approximately 10,000 fold less than bovine kidney cells. Using a β-Gal expressing virus (gC-Blue), we demonstrate that infection of Neuro-2A cells (actively dividing or differentiated) does not result in efficient virus spread, unlike bovine kidney or SH-SY5Y cells. Additional studies demonstrated that lytic cycle viral gene expression (bICP4 and gE) was readily detected in SH-SY5Y cells: conversely bICP4 was not readily detected in productively infected Neuro-2A cells. Finally, infection of SH-SY5Y and bovine kidney cells, but not Neuro-2A cells, led to rapid activation of the Akt protein kinase. These studies suggest that the Neuro-2A cell line may be a novel cell culture model to identify factors that regulate BoHV-1 productive infection in neuronal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Diclofenac-Induced Apoptosis in the Neuroblastoma Cell Line SH-SY5Y: Possible Involvement of the Mitochondrial Superoxide Dismutase

    PubMed Central

    Cecere, Francesca; Iuliano, Annarita; Albano, Francesco; Zappelli, Claudia; Castellano, Immacolata; Grimaldi, Pasquale; Masullo, Mariorosario; De Vendittis, Emmanuele; Ruocco, Maria Rosaria

    2010-01-01

    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. PMID:20625417

  10. Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

    PubMed

    Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

    2013-06-01

    Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

  11. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents

    PubMed Central

    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-01-01

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil. To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. PMID:27036033

  12. Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y

    PubMed Central

    Catino, Stefania; Paciello, Fabiola; Miceli, Fiorella; Rolesi, Rolando; Troiani, Diana; Calabrese, Vittorio; Santangelo, Rosaria; Mancuso, Cesare

    2016-01-01

    Over the past years, several lines of evidence have pointed out the efficacy of ferulic acid (FA) in counteracting oxidative stress elicited by β-amyloid or free radical initiators, based on the ability of this natural antioxidant to up-regulate the heme oxygenase-1 (HO-1) and biliverdin reductase (BVR) system. However, scarce results can be found in literature regarding the cytoprotective effects of FA in case of damage caused by neurotoxicants. The aim of this work is to investigate the mechanisms through which FA exerts neuroprotection in SH-SY5Y neuroblastoma cells exposed to the neurotoxin trimethyltin (TMT). FA (1–10 μM for 6 h) dose-dependently increased both basal and TMT (10 μM for 24 h)-induced HO-1 expression in SH-SY5Y cells by fostering the nuclear translocation of the transcriptional activator Nrf2. In particular, the co-treatment of FA (10 μM) with TMT was also responsible for the nuclear translocation of HO-1 in an attempt to further increase cell stress response in SH-SY5Y cells. In addition to HO-1, FA (1–10 μM for 6 h) dose-dependently increased the basal expression of BVR. The antioxidant and neuroprotective features of FA, through the increase of HO activity, were supported by the evidence that FA inhibited TMT (10 μM)-induced lipid peroxidation (evaluated by detecting 4-hydroxy-nonenal) and DNA fragmentation in SH-SY5Y cells and that this antioxidant effect was reversed by the HO inhibitor Zinc-protoporphyrin-IX (5 μM). Among the by-products of the HO/BVR system, carbon monoxide (CORM-2, 50 nM) and bilirubin (BR, 50 nM) significantly inhibited TMT-induced superoxide anion formation in SH-SY5Y cells. All together, these results corroborate the neuroprotective effect of FA through the up-regulation of the HO-1/BVR system, via carbon monoxide and BR formation, and provide the first evidence on the role of HO-1/Nrf2 axis in FA-related enhancement of cell stress response in human neurons. PMID:26779023

  13. Paullinia cupana Mart. var. Sorbilis protects human dopaminergic neuroblastoma SH-SY5Y cell line against rotenone-induced cytotoxicity.

    PubMed

    de Oliveira, Diêgo Madureira; Barreto, George; Galeano, Pablo; Romero, Juan Ignacio; Holubiec, Mariana Inés; Badorrey, Maria Sol; Capani, Francisco; Alvarez, Lisandro Diego Giraldez

    2011-09-01

    Paullinia cupana Mart. var. Sorbilis, commonly known as Guaraná, is a Brazilian plant frequently cited for its antioxidant properties and different pharmacological activities on the central nervous system. The potential beneficial uses of Guaraná in neurodegenerative disorders, such as in Parkinson's disease (PD), the pathogenesis of which is associated with mitochondrial dysfunction and oxidative stress, has not yet been assessed. Therefore, the main aim of the present study was to evaluate if an extract of commercial powdered seeds of Guaraná could protect human dopaminergic neuroblastoma SH-SY5Y cell line against rotenone-induced cytotoxicity. Two concentration of Guaraná dimethylsulfoxide extract (0.312 and 0.625 mg/mL) were added to SH-SY5Y cells treated with 300 nM rotenone for 48 h, and the cytoprotective effects were assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, measuring lactate dehydrogenase (LDH) levels, and analyzing nuclear integrity with Hoechst33258 stain. Results showed that the addition of Guaraná extract significantly increased the cell viability of SH-SY5Y cells treated with rotenone, in a dose-dependent manner. On the other hand, LDH levels were significantly reduced by addition of 0.312 mg/mL of Guaraná, but unexpectedly, no changes were observed with the higher concentration. Moreover, chromatin condensation and nuclear fragmentation were significantly reduced by addition of any of both concentrations of the extract. The results obtained in this work could provide relevant information about the mechanisms underlying the degeneration of dopaminergic neurons in PD and precede in vivo experiments. Further studies are needed to investigate which active constituent is responsible for the cytoprotective effect produced by Paullinia cupana.

  14. Melatonin inhibits angiogenesis in SH-SY5Y human neuroblastoma cells by downregulation of VEGF.

    PubMed

    González, Alicia; González-González, Alicia; Alonso-González, Carolina; Menéndez-Menéndez, Javier; Martínez-Campa, Carlos; Cos, Samuel

    2017-04-01

    Vascular endothelial growth factor (VEGF) produced from tumor cells plays a crucial role in the pathogenesis and neovascularization of neuroblastoma. Inhibition of VEGF secretion by tumor cells, as well as VEGF-regulated signaling in endothelial cells, are important to reduce the angiogenesis and growth of neuroblastoma. Since melatonin has anti-angiogenic effects in tumor cell lines, the aim of the present study was to study melatonin modulation of the pro-angiogenic effects of VEGF in neuroblastoma cells (SH-SY5Y). We used co-cultures of SH-SY5Y and endothelial cells. VEGF expression and protein levels were analyzed by quantitative RT-PCR and ELISA, respectively. Endothelial cell migration was assessed by wound-healing assay and endothelial angiogenesis by a tube formation assay. Melatonin inhibited the pro-angiogenic effects of SH-SY5Y cells. The conditioned medium collected from the neuroblastoma cells was angiogenically active and stimulated proliferation, migration and tube formation in endothelial cells. This effect was significantly counteracted by the addition of either anti-VEGF or melatonin. Melatonin inhibited VEGF expression and secretion in SH-SY5Y cells, decreasing the levels of VEGF available for endothelial cells. Melatonin has anti-angiogenic effects at different steps of the angiogenic process in SH-SY5Y neuroblastoma cells, through the downregulation of VEGF.

  15. The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

    PubMed

    Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

    2016-05-01

    Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

    PubMed

    Wang, Chao; Zeng, Zhenyu; Liu, Qiong; Zhang, Renli; Ni, Jiazuan

    2014-11-18

    Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

  17. Se-Methylselenocysteine Inhibits Apoptosis Induced by Clusterin Knockdown in Neuroblastoma N2a and SH-SY5Y Cell Lines

    PubMed Central

    Wang, Chao; Zeng, Zhenyu; Liu, Qiong; Zhang, Renli; Ni, Jiazuan

    2014-01-01

    Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection. PMID:25411798

  18. Expression of CD38 in human neuroblastoma SH-SY5Y cells.

    PubMed

    Orciani, M; Trubiani, O; Cavaletti, G; Guarnieri, S; Salvolini, E; Tredici, G; Di Primio, R

    2008-01-01

    Human CD38 antigen is a 42-45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+ -releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of approximately 42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release.

  19. Royal jelly lipophilic fraction induces antiproliferative effects on SH-SY5Y human neuroblastoma cells.

    PubMed

    Gismondi, Angelo; Trionfera, Eleonora; Canuti, Lorena; Di Marco, Gabriele; Canini, Antonella

    2017-09-01

    Royal jelly (RJ) is one the most important bee product because it strongly influences the larval development in the hive, including the queen bee. In literature, RJ is known for its antioxidant, immunoregulatory, antifungal, antibiotical, erythropoietic, hypoglycemic, anticholesteremic, antithyroidic, anti-osteoporotic and estrogenic properties. However, it is surprising how rare the scientific evidence about RJ antineoplastic capacity are. That being said, we investigated, for the first time, the in vitro bioactivity of six different RJs on the growth of three different mammalian cell lines: immortalized murine myoblasts (C2C12), human prostate cancer (PC3) and human neuroblastoma (SH-SY5Y). These studies were performed treating the cells with the only lipophilic, or hydrophilic, fraction of the RJs, a scientific approach never performed before. Moreover, chemical and protein profiles of all RJs were finely characterized, in qualitative and quantitative terms, by GC-MS and 1D-SDS-PAGE, respectively, in order to give a complete framework to the research. Despite the deep differences we found in the composition of each sample, unexpectedly, RJs showed comparable or very similar biological effects. In particular, our attention was captured by the extraordinary antiproliferative activity of the lipophilic extract of all RJs against SH-SY5Y cells, suggesting a potential medical application of this bee product to prevent the onset and slow down the growth of human neuroblastoma.

  20. Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-dependent, but not nerve growth factor-independent, differentiation and cell cycle arrest in the human neuroblastoma cell line, SY5Y.

    PubMed

    Gryz, Ela A; Meakin, Susan O

    2003-11-27

    TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons and also differentiation of neuroblastomas and apoptosis of medulloblastomas. We have previously shown that aspartic acid and glutamic acid substitution (AspGlu and GluAsp) of the activation loop tyrosines in TrkA (Tyr(683) and Tyr(684)) supports NGF-independent neuritogenesis and cell survival in PC12 cell-derived nnr5 cells. In this study, the AspGlu and GluAsp mutant Trks have been analysed for their ability to support NGF-independent and NGF-dependent neuritogenesis, proliferation and cell signalling in the human neuroblastoma cell line, SY5Y. We find that the AspGlu and GluAsp mutant Trks support NGF-dependent, but not NGF-independent, autophosphorylation, neuritogenic responses and/or inhibit cell cycle progression. The NGF-dependent neuritogenic responses are lower for the mutant Trks (approximately 30-60% for AspGlu and 50-60% for GluAsp), relative to wild-type TrkA. While both the AspGlu and GluAsp mutant Trks support NGF-dependent transient phosphorylation of Shc, PLCgamma-1, AKT, FRS2, SH2B as well as prolonged MAP kinase activation, the GluAsp mutant induces stronger NGF-dependent tyrosine phosphorylation of FRS2 and SH2B, as well as a stronger reduction in bromodeoxyuridine (BrdU) incorporation. Collectively, these data suggest that neither absolute levels of receptor autophosphorylation, high levels of TrkA expression nor the activation of a specific signalling pathway is dominant and absolutely essential for neuritogenesis and cell cycle arrest of SY5Y cells.

  1. Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells

    PubMed Central

    Kim, Han Bit; Yoo, Byung Sun

    2016-01-01

    Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of 3 μg/mL, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis (0.3~3 μg/mL) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to 3 μg/mL propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells. PMID:27437091

  2. Comparative proteomic analysis of human SH-SY5Y neuroblastoma cells under simulated microgravity.

    PubMed

    Zhang, Yongqian; Wang, Hongbin; Lai, Chengjun; Wang, Lu; Deng, Yulin

    2013-02-01

    Microgravity is one of the most important features in spaceflight. Previous evidence has shown that neurophysiological impairment signs occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human SH-SY5Y neuroblastoma cells that were grown in a microgravity environment. The comparative proteomic method based on the (18)O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity. Twenty-two differentially abundant proteins were quantified in human SH-SY5Y neuroblastoma cells. The cell microfilament network was disrupted under simulated microgravity, which was determined by the immunocytochemistry. The concentration of reactive oxygen species, malondialdehyde, and free Ca2+ ion significantly increased, and the level of ATP significantly decreased under simulated microgravity. However, there was no obvious cell apoptosis observed under simulated microgravity. These results provide new molecular evidence for the change in protein abundance in SH-SY5Y cells under simulated microgravity, which might unfold biological mechanisms and the development of effective countermeasures to deal with microgravity-related neurological problems. We believe that the state-of-the-art proteomic assay may be a means by which aerospace scientists will begin to understand the underlying mechanisms of space life activities at the protein level.

  3. The effects of okra (Abelmoschus esculentus Linn.) on the cellular events associated with Alzheimer's disease in a stably expressed HFE neuroblastoma SH-SY5Y cell line.

    PubMed

    Mairuae, Nootchanat; Connor, James R; Lee, Sang Y; Cheepsunthorn, Poonlarp; Tongjaroenbuangam, Walaiporn

    2015-08-31

    It has been reported that persons carrying the H63D variant in their hemochromatosis (HFE) gene are at increased risk of Alzheimer's disease (AD). We investigated the possibility that okra (Abelmoschus esculentus) and quercetin could mitigate this risk factor by examining its effect on AD-associated cellular events in HFE stably expressing SH-SY5Y cells. Treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2), and protein oxidation compared to untreated cells. The levels of tau phosphorylation at serine-199, serine-202, and serine-396 sites were also significantly decreased when cells were treated with okra. Exposure of the H63D and wild type (WT) cells to iron increased tau phosphorylation, but this response was decreased significantly when cells were treated with okra. The mechanism responsible for these changes appears to be related to decreased glycogen synthase kinase (GSK)-3β activity, an upstream signaling kinase of tau phosphorylation. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. Our results provide important in vitro data on the effects of okra on various AD-associated cellular processes in H63D variant HFE cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of AD and other neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this.

  4. Weightlessness influences the cytoskeleton and ROS level in SH-SY5Y neuroblastoma cells

    NASA Astrophysics Data System (ADS)

    Bo, Wang; Lina, Qu; Yingxian, Li; Qi, Li; Lei, Bi; Yinghui, Li

    During Spaceflight the nerve system of astronauts was obviously influenced To investigate how gravity effects nerve system the SH-SY5Y neuroblastoma cells were taken as research object By utilizing clinostat and parabolic flight for the model of gravity changing the level of reactive oxygen species was assayed in different time under simulated microgravity the cytomorphology and cytoskeleton of SH-SY5Y neuroblastoma cells were also observed after parabolic flight and clinostat by the conventional and the confocal laser scanning microscope The data showed that ROS level was enhanced and the cytoskeleton was damaged which microfilaments and microtubules were highly disorganized the cell shape was deteriorated under simulated microgravity indicating the relativity between the ROS level fluctuating and cytoskeleton changing It illuminates signal transduction disturbed by oxidative stress also regulates the cytoskeleton changing in SH-SY5Y cells The results suggest the cytoskeleton which is the receptor for sensing gravity was also regulated by cellular redox state which clues on the complexity of cell for self-adjusting to gravity changing

  5. Aluminium Induced Endoplasmic Reticulum Stress Mediated Cell Death in SH-SY5Y Neuroblastoma Cell Line Is Independent of p53

    PubMed Central

    Mustafa Rizvi, Syed Husain; Parveen, Arshiya; Verma, Anoop K.; Ahmad, Iqbal; Arshad, Md; Mahdi, Abbas Ali

    2014-01-01

    Aluminium (Al) is the third most abundant element in the earth’s crust and its compounds are used in the form of house hold utensils, medicines and in antiperspirant etc. Increasing number of evidences suggest the involvement of Al+3 ions in a variety of neurodegenerative disorders including Alzheimer’s disease. Here, we have attempted to investigate the role of Al in endoplasmic reticulum stress and the regulation of p53 during neuronal apoptosis using neuroblastoma cell line. We observed that Al caused oxidative stress by increasing ROS production and intracellular calcium levels together with depletion of intracellular GSH levels. We also studied modulation of key pro- and anti-apoptotic proteins and found significant alterations in the levels of Nrf2, NQO1, pAKT, p21, Bax, Bcl2, Aβ1-40 and Cyt c together with increase in endoplasmic reticulum (ER) stress related proteins like CHOP and caspase 12. However, with respect to the role of p53, we observed downregulation of its transcript as well as protein levels while analysis of its ubiquitination status revealed no significant changes. Not only did Al increase the activities of caspase 9, caspase 12 and caspase 3, but, by the use of peptide inhibitors of specific and pan-caspases, we observed significant protection against neuronal cell death upon inhibition of caspase 12, demonstrating the prominent role of endoplasmic reticulum stress generated responses in Al toxicity. Overall our findings suggest that Al induces ER stress and ROS generation which compromises the antioxidant defenses of neuronal cells thereby promoting neuronal apoptosis in p53 independent pathway. PMID:24878590

  6. Characterisation of SH-SY5Y Human Neuroblastoma cell growth over glass and SU-8 substrates.

    PubMed

    Ajetunmobi, A; McAllister, D; Jain, Namrata; Brazil, Owen; Corvin, A; Volkov, Y; Tropea, D; Prina-Mello, A

    2017-03-28

    The physical properties of substrates can have profound effects on the structure and function of cultured cells. In this study we aimed to examine the viability, adherence and morphological and functional variations between SH-SY5Y human neuroblastoma cells cultured on SU-8 surfaces compared to control surfaces composed of borosilicate glass, which are routinely used for cell culture. The SU-8 polymer has been extensively studied for its biocompatibility but there has been little investigation into the characteristic differences between cells cultured on SU-8 when compared to glass. SH-SY5Y cells were cultured within Polydimethylsiloxane wells on both SU-8 and glass substrates for up to 72 hrs after which flow cytometry and ELISA analysis was performed to examine cell viability and neurotoxicity. Immunocytochemistry was also performed in order to analyse the morphological and functional characteristics of the cells. Atomic force microscopy was performed to measure surface roughness and to map cell-substrate interactions, Nanoindentation testing was used to characterise the mechanical properties of polymer surface. Results showed that SH-SY5Y cells grown on SU-8 have significantly improved viability and increased morphological and functional characteristics of neurodevelopment. The results from this study suggest that the mechanical properties of the polymer are optimal for the study of cultured cell lines, which could account for the increased viability, adherence and morphological and functional characteristics of neurodevelopment. This article is protected by copyright. All rights reserved.

  7. Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

    SciTech Connect

    Kazmi, S.M.; Mishra, R.K.

    1989-02-15

    The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. (/sup 3/H)Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, (/sup 3/H)Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.

  8. Acrylonitrile induced apoptosis via oxidative stress in neuroblastoma SH-SY5Y cell.

    PubMed

    Watcharasit, Piyajit; Suntararuks, Sumitra; Visitnonthachai, Daranee; Thiantanawat, Apinya; Satayavivad, Jutamaad

    2010-10-01

    Acrylonitrile (ACN) is a chemical that is widely used in the production of plastics, acrylic fibers, synthetic rubbers and resins. It has been reported that ACN can cause oxidative stress, a condition which is well recognized as an apoptotic initiator; however, information regarding ACN-induced apoptosis is limited. This present study investigated whether ACN induces apoptosis in human neuroblastoma SH-SY5Y cells, and whether its apoptotic induction involves oxidative stress. The results showed that ACN caused activation of caspase-3, a key enzyme involved in apoptosis, in a dose- and time-dependent manner. Detection of sub-G1 apoptotic cell death and apoptotic nuclear condensation revealed that ACN caused an increase in the number of apoptotic cells indicating ACN induces apoptosis in SH-SY5Y cells. ACN dose- and time-dependently increased the level of proapoptotic protein, Bax. Pretreatment with N-acetylcysteine (NAC), an antioxidant, attenuated caspase-3 activation by ACN, as evidenced by a reduction in proteolysis of PARP, a known caspase-3 substrate, as well as in the number of sub-G1 apoptotic cells. Moreover, induction of Bax by ACN was abolished by NAC. Taken together, the results indicate that ACN induces apoptosis in SH-SY5Y cells via a mechanism involving generation of oxidative stress-mediated Bax induction.

  9. Ginsenoside Re protects methamphetamine-induced mitochondrial burdens and proapoptosis via genetic inhibition of protein kinase C δ in human neuroblastoma dopaminergic SH-SY5Y cell lines.

    PubMed

    Nam, Yunsung; Wie, Myung Bok; Shin, Eun-Joo; Nguyen, Thuy-Ty Lan; Nah, Seung-Yeol; Ko, Sung Kwon; Jeong, Ji Hoon; Jang, Choon-Gon; Kim, Hyoung-Chun

    2015-08-01

    Recently, we have demonstrated that ginsenoside Re protects methamphetamine (MA)-induced dopaminergic toxicity in mice via genetic inhibition of PKCδ and attenuation of mitochondrial stress. In addition, we have reported that induction of mitochondrial glutathione peroxidase (GPx) is also important for neuroprotection mediated by ginsenoside Re. To extend our knowledge, we examined the effects of ginsenoside Re against MA toxicity in vitro condition using SH-SY5Y neuroblastoma cells. Treatment with ginsenoside Re resulted in significant attenuations against a decrease in the activity of GPx and an increase in the activity of superoxide dismutase (SOD) in the cytosolic and mitochondrial fraction. The changes in glutathione (GSH) paralleled those in GPx in the same experimental condition. Consistently, ginsenoside Re treatment exhibited significant protections against cytosolic and mitochondrial oxidative damage (i.e. lipid peroxidation and protein oxidation), mitochondrial translocation of PKCδ, mitochondrial dysfunction (mitochondrial transmembrane potential and intra-mitochondrial Ca(2+)), apoptotic events [i.e., cytochrome c release from mitochondria, cleavage of caspase-3 and poly(ADP-ribose)polymerase-1, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells], and a reduction in the tyrosine hydroxylase (TH) expression and TH activity induced by MA in SH-SY5Y neuroblastoma cells. These protective effects of ginsenoside Re were comparable to those of PKCδ antisense oligonucleotide (ASO). However, ginsenoside Re did not significantly provide additional protective effects mediated by genetic inhibition of PKCδ. Our results suggest that PKCδ is a specific target for ginsenoside Re-mediated protective activity against MA toxicity in SH-SY5Y neuroblastoma cells. Copyright © 2014 John Wiley & Sons, Ltd.

  10. Calcium Signaling of Lysophosphatidylethanolamine through LPA1 in Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Lee, Jung-Min; Park, Soo-Jin; Im, Dong-Soon

    2017-01-01

    Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca2+ through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA1 antagonists showed LPE induced intracellular Ca2+ increases in an LPA1 GPCR-dependent manner. Furthermore, LPE increased intracellular Ca2+ through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP3 receptors, Ca2+ release from intracellular Ca2+ stores, and subsequent Ca2+ influx across plasma membranes, and LPA acted on LPA1 and LPA2 receptors to induce Ca2+ response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells. PMID:27302965

  11. Effects of dichlorobenzene on acetylcholine receptors in human neuroblastoma SH-SY5Y cells.

    PubMed

    Yan, Ren-Ming; Chiung, Yin-Mei; Pan, Chien-Yuan; Liu, Jenn-Hwa; Liu, Pei-Shan

    2008-11-20

    para-Dichlorobenzene (DCB), a deodorant and an industrial chemical, is a highly volatile compound and is known to be an indoor air contaminant. Because of its widespread use and volatility, the toxicity of DCB presents a concern to industrial workers and public. Some toxic aspects of DCB have already been focused but its effects on neuronal signal transduction have been hitherto unknown. The effects of DCB on the cytosolic calcium homeostasis are investigated in human neuroblastoma SH-SY5Y cells in this study. DCB, above 200 microM, was found to induce a rise in cytosolic calcium concentration that could not be counteracted by nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR) antagonists but was partially inhibited by thapsigargin. To understand the actions of DCB on the acetylcholine receptors, we investigated its effects on the changes of cytosolic calcium concentration following nicotinic AChR stimulation with epibatidine and muscarinic AChR stimulation with methacholine in human neuroblastoma SH-SY5Y cells. DCB inhibited the cytosolic calcium concentration rise induced by epibatidine and methacholine with respective IC(50)s of 34 and 294 microM. The inhibitions of DCB were not the same as thapsigargin's inhibition. In the electrophysiological observations, DCB blocked the influx currents induced by epibatidine. Our findings suggest that DCB interferes with the functional activities of AChR, including its coupling influx currents and cytosolic calcium elevations.

  12. Calcium Signaling of Lysophosphatidylethanolamine through LPA1 in Human SH-SY5Y Neuroblastoma Cells.

    PubMed

    Lee, Jung-Min; Park, Soo-Jin; Im, Dong-Soon

    2017-03-01

    Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca(2+) through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA1 antagonists showed LPE induced intracellular Ca(2+) increases in an LPA1 GPCR-dependent manner. Furthermore, LPE increased intracellular Ca(2+) through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP3 receptors, Ca(2+) release from intracellular Ca(2+) stores, and subsequent Ca(2+) influx across plasma membranes, and LPA acted on LPA1 and LPA2 receptors to induce Ca(2+) response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells.

  13. Recognition and identification of active components from Radix Bupleuri using human neuroblastoma SH-SY5Y cells.

    PubMed

    Zhang, Yan; Liu, Feihu; Zhang, Xiaohong; Xu, Tanghui; Quan, Wei; Wang, Hui; Shi, Jianguo; Dai, Zunxiao; Wu, Bin; Wu, Qiangju

    2016-03-01

    The aim of the study was to screen active components of Radix Bupleuri (a traditional Chinese herb) and discover novel anti-schizophrenic candidate drugs using human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were used for preparation of the stationary phase in the cell membrane chromatography model. Retention components by the SH-SY5Y/CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the SH-SY5Y/CMC method using amisulpride and haloperidol as standard compounds, this method was applied to screening active components from the extracts of Radix Bupleuri. Retention components of SH-SY5Y/CMC model were saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C and saikosaponin D, which were identified by the GC/MS method. In vitro pharmacological trials-MTT, saikosaponin B1, saikosaponin B2 and saikosaponin C could protect SY5Y cells. The protective effects of saikosaponin B1 and saikosaponin C were concentration dependent. Saikosaponin A and saikosaponin D inhibited cell viability at concentrations >30 µg/mL (p < 0.05). Via SH-SY5Y/CMC method and SH-SY5Y MTT trial, we rapidly detected target components from Radix Bupleuri, accurately identified them and determined their different effects on SH-SY5Y cells. Saikosaponin B1, saikosaponin B2 and saikosaponin C may be anti-schizophrenic candidate drugs.

  14. Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells.

    PubMed

    Sarkanen, Jertta-Riina; Nykky, Jonna; Siikanen, Jutta; Selinummi, Jyrki; Ylikomi, Timo; Jalonen, Tuula O

    2007-09-01

    Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

  15. TRPC1-mediated Inhibition of 1-Methyl-4-phenylpyridinium Ion Neurotoxicity in Human SH-SY5Y Neuroblastoma Cells*

    PubMed Central

    Bollimuntha, Sunitha; Singh, Brij B.; Shavali, Shaik; Sharma, Sushil K.; Ebadi, Manuchair

    2013-01-01

    Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca2+ channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP+ showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP+. MPP+-induced α-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP+ was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP+ neurotoxicity, which was partially dependent on external Ca2+. Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP+ treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson’s disease-inducing agents. PMID:15542611

  16. Rosiglitazone protects human neuroblastoma SH-SY5Y cells against acetaldehyde-induced cytotoxicity

    SciTech Connect

    Jung, Tae Woo; Lee, Ji Young; Shim, Wan Sub; Kang, Eun Seok; Kim, Soo Kyung; Ahn, Chul Woo; Lee, Hyun Chul; Cha, Bong Soo . E-mail: bscha@yumc.yonsei.ac.kr

    2006-02-03

    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 level and apoptosis. Rosiglitazone, a peroxisome proliferator-activated receptor-{gamma} agonist, has been known to show various non-hypoglycemic effects, including anti-inflammatory, anti-atherogenic, and anti-apoptotic. In this study, we investigated the protective effects of rosiglitazone on acetaldehyde-induced apoptosis in human neuroblastoma SH-SY5Y cells and attempted to examine its mechanism. Acetaldehyde-induced apoptosis was moderately reversed by rosiglitazone treatment. Our results suggest that the protective effects of rosiglitazone 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 rosiglitazone may provide a useful therapeutic strategy for the prevention of progressive neurodegenerative disease such as Parkinson's disease.

  17. JWH-133, a Selective Cannabinoid CB₂ Receptor Agonist, Exerts Toxic Effects on Neuroblastoma SH-SY5Y Cells.

    PubMed

    Wojcieszak, Jakub; Krzemień, Wojciech; Zawilska, Jolanta B

    2016-04-01

    Endocannabinoid system plays an important role in the regulation of diverse physiological functions. Although cannabinoid type 2 receptors (CB2) are involved in the modulation of immune system in peripheral tissues, recent findings demonstrated that they are also expressed in the central nervous system and could constitute a new target for the treatment of neurodegenerative disorders. At present, very little is known about the potential effects of CB2-mimetic drugs on neuronal cells. This study aimed to examine whether JWH-133, a selective CB2 receptor agonist, affects the survival of SH-SY5Y neuroblastoma cell line, a widely used experimental in vitro model to study mechanisms of toxicity and protection in nigral dopaminergic neurons. Cell viability was assessed using two complementary methods: MTT test measuring mitochondrial activity and LDHe test indicating disruption of cell membrane integrity. In addition, cell proliferation was measured using BrdU incorporation assay. JWH-133 (10-40 μM) induced a concentration-dependent decrease of SH-SY5Y cell viability and proliferation rate. Using AM-630, a reverse agonist of CB2 receptors, as well as Z-VAD-FMK, a pan-caspase inhibitor, we demonstrated that the cytotoxic effect of JWH-133 presumably was not mediated by activation of CB2 receptors or by caspase pathway. Results of this work suggest that agonists of CB2 receptors when administered in multiple/high doses may induce neuronal damage.

  18. The biologic role of ganglioside in neuronal differentiation--effects of GM1 ganglioside on human neuroblastoma SH-SY5Y cells.

    PubMed Central

    Lee, M. C.; Lee, W. S.; Park, C. S.; Juhng, S. W.

    1994-01-01

    Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal growth factor) and PDGF receptors. The experiment was designed to study the effects of GM1 ganglioside on growth of human neuroblastoma SH-SY5Y cells stimulated with trophic factor in vitro. The cells were plated in Eagle's minimum essential medium without serum. The number and morphologic change of SH-SY5Y cells were evaluated in the serum free medium added GM1 ganglioside with insulin or PDGF. SH-SY5Y cells were maintained for six days in serum-free medium, and then cultured for over two weeks in serum-free medium containing either insulin or PDGF. The effect of insulin on cell proliferation developed earlier and was more potent than that of PDGF. These proliferative effects were inhibited by GM1 ganglioside, and the cells showed prominent neurites outgrowth. These findings suggest that GM1 ganglioside inhibits the cell proliferation mediated by tyrosine kinase receptors and directly induces neuritogenesis as one of the neurotrophic factors. PMID:7986393

  19. Cx43 Mediates Resistance against MPP+-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway

    PubMed Central

    Kim, In-Su; Ganesan, Palanivel; Choi, Dong-Kug

    2016-01-01

    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. PMID:27809287

  20. [Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y].

    PubMed

    Surin, A M; Kriukova, E V; Strukov, A S; Zhmak, M N; Talka, R; Tuominen, R; Salminen, O; Khiroug, L; Kasheverov, I E; Tsetlin, V I

    2012-01-01

    Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.

  1. Transcriptional profile of SH-SY5Y human neuroblastoma cells transfected by Toxoplasma rhoptry protein 16

    PubMed Central

    Fan, Weiwei; Chang, Shuang; Shan, Xiumei; Gao, Dejun; Zhang, Steven Qian; Zhang, Jin; Jiang, Nan; Ma, Duan; Mao, Zuohua

    2016-01-01

    Toxoplasma rhoptry protein 16 (ROP16) is crucial in the host-pathogen interaction by acting as a virulent factor during invasion. To improve understanding of the molecular function underlying the effect of ROP16 on host cells, the present study analyzed the transcriptional profile of genes in the ROP16-transfected SH-SY5Y human neuroblastoma cell line. The transcriptional profile of the SH-SY5Y human neuroblastoma cell line overexpressing ROP16 were determined by microarray analysis in order to determine the host neural cell response to the virulent factor. Functional analysis was performed using the Protein Analysis Through Evolutionary Relationships classification system. The ToppGene Suite was used to select candidate genes from the differentially expressed genes. A protein-protein interaction network was constructed using Cytoscape software according to the interaction associations determined using the Search Tool for the Retrieval of Interacting Genes/Proteins. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of the selected genes confirmed the results of the microarray. The results showed that 383 genes were differentially expressed in response to ROP16 transfection, of which 138 genes were upregulated and 245 genes were downregulated. Functional analysis indicated that the differentially expressed genes (DEGs) were involved in several biological processes, including developmental process, biological regulation and apoptotic process. A total of 15 candidate genes from the DEGs were screened using the ToppGene Suite. No significant differences in expression were observed between the RT-qPCR data and the microarray data. Transfection with ROP16 resulted in alterations of several biological processes, including nervous system development, apoptosis and transcriptional regulation. Several genes, including CXCL12, BAI1, ZIC2, RBMX, RASSF6, MAGE-A6 and HOX, were identified as significant DEGs. Taken together, these results may

  2. Endoplasmic reticulum stress is involved in the lidocaine-induced apoptosis in SH-SY5Y neuroblastoma cells.

    PubMed

    Li, Kehan; Han, Xuechang

    2015-05-01

    Lidocaine has been indicated to promote apoptosis and to promote endoplasmic reticulum (ER) stress. However, the mechanism underlining ER stress-mediated apoptosis is unclear. In the present study, we investigated the promotion to ER stress in the lidocaine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Firstly, we confirmed that lidocaine treatment induced apoptosis in SH-SY5Y cells, time-dependently and dose-dependently, via MTT cell viability assay and annexin V/FITC apoptosis detection with a FACScan flow cytometer. And the anti-apoptosis Bcl-2 and Bcl-xL were downregulated, whereas the apoptosis-executive caspase 3 was promoted through Western blot assay and caspase 3 activity assay. Moreover, the ER stress-associated binding immunoglobulin protein (BiP), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP) were also upregulated at both mRNA and protein levels by lidocaine treatment. On the other hand, downregulation of the ER stress-associated BiP by RNAi method not only blocked the lidocaine-promoted ER stress but also attenuated the lidocaine-induced SH-SY5Y cell apoptosis. In conclusion, the present study confirmed the involvement of ER stress in the lidocaine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Our study provides a better understanding on the mechanism of lidocaine's neurovirulence.

  3. Vasoactive intestinal peptide-induced neuritogenesis in neuroblastoma SH-SY5Y cells involves SNAP-25.

    PubMed

    Héraud, Céline; Chevrier, Lucie; Meunier, Annie Claire; Muller, Jean-Marc; Chadéneau, Corinne

    2008-01-01

    Vasoactive intestinal peptide (VIP) is a neuropeptide known to regulate proliferation and differentiation in normal and tumoral cells. We previously reported that VIP induced neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium. This neuritogenesis was associated with a regulated expression of neuronal cytoskeleton markers. To further characterize the neuroblastic cell differentiation induced by VIP in human SH-SY5Y cells, we investigated expression of synaptosomal-associated protein of 25 kDa (SNAP-25), a protein implicated in exocytosis associated with different processes, including neurite outgrowth. Western immunoblotting and real-time RT-PCR analyses revealed that VIP increased expression of the SNAP-25 protein and the level of both SNAP-25a and SNAP-25b mRNA isoforms. Immunofluorescence experiments indicated that SNAP-25 was mainly located in neurites and at the plasma membrane in SH-SY5Y cells treated with VIP. RNA interference experiments demonstrated that SNAP-25 was involved in VIP-induced neuritogenesis. In conclusion, SNAP-25 is up-regulated and implicated in neuritogenesis in human neuroblastoma SH-SY5Y cells treated with the neuropeptide VIP.

  4. Arsenic trioxide (As{sub 2}O{sub 3}) induced calcium signals and cytotoxicity in two human cell lines: SY-5Y neuroblastoma and 293 embryonic kidney (HEK)

    SciTech Connect

    Florea, Ana-Maria; Splettstoesser, Frank; Buesselberg, Dietrich . E-mail: Dietrich.Buesselberg@uni-due.de

    2007-05-01

    Arsenic trioxide (As{sub 2}O{sub 3}) has anticancer properties; however, its use also leads to neuro-, hepato- or nephro-toxicity, and therefore, it is important to understand the mechanism of As{sub 2}O{sub 3} toxicity. We studied As{sub 2}O{sub 3} influence on intracellular calcium ([Ca{sup 2+}]{sub i}) homeostasis of human neuroblastoma SY-5Y and embryonic kidney cells (HEK 293).We also relate the As{sub 2}O{sub 3} induced [Ca{sup 2+}]{sub i} modifications with cytotoxicity. We used Ca{sup 2+} sensitive dyes (fluo-4 and rhod-2) combined with laser scanning microscopy or fluorescence activated cell sorting to measure Ca{sup 2+} changes during the application of As{sub 2}O{sub 3} and we approach evaluation of cytotoxicity. As{sub 2}O{sub 3} (1 {mu}M) increased [Ca{sup 2+}]{sub i} in SY-5Y and HEK 293 cells. Three forms of [Ca{sup 2+}]{sub i}-elevations were found: (1) steady-state increases (2) transient [Ca{sup 2+}]{sub i}-elevations and (3) Ca{sup 2+}-spikes. [Ca{sup 2+}]{sub i} modifications were independent from extracellular Ca{sup 2+} but dependent on internal calcium stores. The effect was not reversible. Inositol triphosphate (IP{sub 3}) and ryanodine (Ry) receptors are involved in regulation of signals induced by As{sub 2}O{sub 3}. 2-APB and dantrolene significantly reduced the [Ca{sup 2+}]{sub i}-rise (p < 0.001, t-test) but did not completely abolish [Ca{sup 2+}]{sub i}-elevation or spiking. This indicates that other Ca{sup 2+} regulating mechanisms are involved. In cytotoxicity tests As{sub 2}O{sub 3} significantly reduced cell viability in both cell types. Staining with Hoechst 33342 showed occurrence of apoptosis and DNA damage. Our data suggest that [Ca{sup 2+}]{sub i} is an important messenger in As{sub 2}O{sub 3} induced cell death.

  5. Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells.

    PubMed

    Cocco, Stefania; Secondo, Agnese; Del Viscovo, Adelaide; Procaccini, Claudio; Formisano, Luigi; Franco, Cristina; Esposito, Alba; Scorziello, Antonella; Matarese, Giuseppe; Di Renzo, Gianfranco; Canzoniero, Lorella Maria Teresa

    2015-01-01

    Chronic exposure to polychlorinated biphenyls (PCBs), ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254) in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 μg/ml) reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase), measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.

  6. Zinc oxide nanoparticles and SH-SY5Y cell line

    NASA Astrophysics Data System (ADS)

    Zheng, Jinghui

    The Arctic and sub-arctic regions are impacted by the growth of the global nanotechnology industry. Nanomaterials have unique chemical and physical properties that may lead to toxicological effects that interfere with normal cellular metabolism. Zinc oxide nanoparticles (ZnO NPs) are now very common and widely used in daily life. In industry, ZnO NPs are used to protect different materials from damage caused by UV exposure. The scientific literature suggests that ZnO NPs can have negative impacts on both living organisms and plants. However, there is a paucity of research on the mechanisms by which ZnO NPs may affect the neuronal cells. This study investigates how ZnO NPs interact with the neuroblastoma cell line SH-SY5Y. Using transmission electron microscopy, we observed that the ZnO NPs form 36 nm particles on average, and increase the level of vascular endothelial growth factor (VEGF) in extracellular fluid, as measured by an enzyme-linked immunosorbent assay (ELISA). Moreover, ZnO NPs, in presence of tumor necrosis factor-alpha (TNF-alpha), can also decrease the level of extracellular VEGF compared with TNF-alpha treatment alone. These findings suggest the basis for more studies on understanding the mechanism by which ZnO NPs impact cytokine signaling. Another direction is using ELISA technology to observe the interactions of NPs with different cell types such as neuronal stem cells.

  7. Melatonin attenuates methamphetamine-induced disturbances in mitochondrial dynamics and degeneration in neuroblastoma SH-SY5Y cells.

    PubMed

    Parameyong, Arisa; Charngkaew, Komgrid; Govitrapong, Piyarat; Chetsawang, Banthit

    2013-10-01

    Methamphetamine (METH) is a psychostimulant drug that can cause toxicity and degeneration in the brain. The toxicity due to METH involves multiple pathways, including the mitochondrial-dependent death pathway. Several pieces of evidence have emphasized that the fragmentation of mitochondria into smaller structures plays some role in the cell-death process. In this study, we investigated the role of mitochondrial dynamics in METH-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced toxicity was investigated. Our results show that METH significantly decreased cell viability and increased the levels of the mitochondrial fission protein, Fis1 and the Drp1 oligomer. However, the levels of the mitochondrial fusion proteins OPA1 and Mfn1 did not change in METH-treated cells. Melatonin can reverse the toxic effects of the METH-induced reduction in cell viability and the production of the Fis1 protein and the Drp1 oligomer. Moreover, the morphological alteration of mitochondria was investigated in METH-treated cells in the presence of melatonin using transmission electron microscopy (TEM). At 24 hr after METH exposure, typical cell shrinkage was observed in SH-SY5Y cells. Mitochondria were fragmented into small globular structures in a large proportion of METH-treated cells, but tubular networks of mitochondria were present in large proportions of control-untreated cells and METH-treated cells in the presence of melatonin. The results of the present study demonstrate the potential of melatonin to reduce cell death and restore mitochondrial function in neurons affected by METH-induced toxicity.

  8. Protective effects of Cervus nippon Temminck velvet antler polypeptides against MPP+‑induced cytotoxicity in SH‑SY5Y neuroblastoma cells.

    PubMed

    Xin, Ji-Le; Zhang, Yang; Li, Yan; Zhang, Lian-Zhu; Lin, Yong; Zheng, Lian-Wen

    2017-10-01

    The aim of the present study was to examine the protective effects and mechanism of sika deer (Cervus nippon Temminck) velvet antler polypeptides (VAPs) against MPP+ exposure in the SH‑SY5Y human neuroblastoma cell line. MPP+ cytotoxicity and the protective effects of VAPs on the SH‑SY5Y cells were determined using an MTT assay. Cell apoptosis and mitochondrial membrane potential were detected using Hoechst 33342 and Rhodamine123 staining, respectively. Endoplasmic reticulum (ER) stress‑related reactive oxygen species (ROS) production in the SH‑SY5Y cells was detected using 2',7'‑dichlorodihydrofluorescein diacetate fluorescent probes. The expression levels of proteins, including caspase‑12, glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c‑Jun N‑terminal kinase (p‑JNK) were detected using western blot analysis. The results showed that the half inhibitory concentration of MPP+ at 72 h was 120.9 µmol/l, and that 62.5, 125, and 250 µg/ml concentrations of VAPs protected the SH‑SY5Y cells under MPP+ exposure. When exposed to 120.9 µmol/l MPP+, changes in cell nucleus morphology, mitochondrial membrane potential and intracellular ROS were observed. VAPs at concentrations of 62.5, 125, 250 µg/ml reduced this damage. Western blot analysis showed that protein expression levels of caspase‑12, GRP78 and p‑JNK were upregulated in the SH‑SY5Y cells exposed to 120.9 µmol/l MPP+ for 72 h. In addition, 62.5, 125, and 250 µg/ml VAPs downregulated the expression levels of caspase‑12 and p‑JNK in a concentration‑ dependent manner, particularly the p‑JNK pathway. The effects of VAPs on GRP78 and CHOP were weak. In conclusion, MPP+‑induced SH‑SY5Y cell death may be linked to ER stress. VAPs prevented MPP+‑induced SH‑SY5Y cell death by affecting the p‑JNK pathway and caspase‑12‑mediated apoptosis. These findings assist in understanding the mechanism

  9. Different mechanisms of lysophosphatidylcholine-induced Ca(2+) mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells.

    PubMed

    Li, Xiao-Hua; Long, Ding-Xin; Li, Wei; Wu, Yi-Jun

    2007-08-31

    In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca(2+) overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca(2+) level ([Ca(2+)](i)) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca(2+) channel blocker, did not affect the LPC-induced Ca(2+) increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca(2+)-free medium, LPC produced a significant [Ca(2+)](i) decrease in N2a cells, while it caused 64% of total [Ca(2+)](i) increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca(2+)](i) increase was produced in each cell line through different mechanisms. In particular, the [Ca(2+)](i) increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca(2+) channels as well as by Ca(2+) release from intracellular Ca(2+) stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca(2+) overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.

  10. Cearoin Induces Autophagy, ERK Activation and Apoptosis via ROS Generation in SH-SY5Y Neuroblastoma Cells.

    PubMed

    Bastola, Tonking; An, Ren-Bo; Kim, Youn-Chul; Kim, Jaehyo; Seo, Jungwon

    2017-02-06

    Neuroblastomas are the most common solid extracranial tumors in childhood. We investigated the anticancer effect of cearoin isolated from Dalbergia odorifera in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with various doses of cearoin. The viability was measured by MTT assay. DCFDA fluorescence assay and Griess assay were used for the measurement of intracellular reactive oxygen species (ROS) and nitric oxide (NO), respectively. Western blot analysis was performed to clarify the molecular pathway involved. Cearoin induced cell death in a dose-dependent manner. Cearoin increased the phosporylation of ERK, the conversion of LC3B-I to LC3B-II, decrease in Bcl2 expression, the activation of caspase-3, and the cleavage of PARP, indicating the induction of autophagy and apoptosis. Furthermore, cearoin treatment increased the production of ROS and NO. Co-treatment with the antioxidant N-acetylcysteine completely abolished cearoin-mediated autophagy, ERK activation and apoptosis, suggesting the critical role of ROS in cearoin-induced anticancer effects. Moreover, co-treatment with ERK inhibitor PD98059 partially reversed cearoin-induced cell death, indicating the involvement of ERK in cearoin anticancer effects. These data reveal that cearoin induces autophagy, ERK activation and apoptosis in neuroblastoma SH-SY5Y cells, which is mediated primarily by ROS generation, suggesting its therapeutic application for the treatment of neuroblastomas.

  11. Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells.

    PubMed

    Kuhla, Björn; Lüth, Hans-Joachim; Haferburg, Dietrich; Weick, Michael; Reichenbach, Andreas; Arendt, Thomas; Münch, Gerald

    2006-06-01

    In Alzheimer's disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p-bromobenzylglutathione cyclopentyl diester (pBrBzGSCp(2)), to SH-SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 microM pBrBzGSCp(2) leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca(2+)](i), and activation of caspase-3. However, pBrBzGSCp(2) did not lead to tau "hyper"-phosphorylation despite activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser(202)/Thr(205) and Ser(396)/Ser(404) epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP-2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.

  12. Inhibition of WNT signaling reduces differentiation and induces sensitivity to doxorubicin in human malignant neuroblastoma SH-SY5Y cells.

    PubMed

    Suebsoonthron, Junjira; Jaroonwitchawan, Thiranut; Yamabhai, Montarop; Noisa, Parinya

    2017-02-24

    Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

  13. Effect of 8-hydroxyquinoline and derivatives on human neuroblastoma SH-SY5Y cells under high glucose

    PubMed Central

    Suwanjang, Wilasinee; Prachayasittikul, Supaluk

    2016-01-01

    8-Hydroxyquinoline and derivatives exhibit multifunctional properties, including antioxidant, antineurodegenerative, anticancer, anti-inflammatory and antidiabetic activities. In biological systems, elevation of intracellular calcium can cause calpain activation, leading to cell death. Here, the effect of 8-hydroxyquinoline and derivatives (5-chloro-7-iodo-8-hydroxyquinoline or clioquinol and 8-hydroxy-5-nitroquinoline or nitroxoline) on calpain-dependent (calpain-calpastatin) pathways in human neuroblastoma (SH-SY5Y) cells was investigated. 8-Hydroxyquinoline and derivatives ameliorated high glucose toxicity in SH-SY5Y cells. The investigated compounds, particularly clioquinol, attenuated the increased expression of calpain, even under high-glucose conditions. 8-Hydroxyquinoline and derivatives thus adversely affected the promotion of neuronal cell death by high glucose via the calpain-calpastatin signaling pathways. These findings support the beneficial effects of 8-hydroxyquinolines for further therapeutic development. PMID:27635352

  14. Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

    SciTech Connect

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C. . E-mail: hclee2@ym.edu.tw

    2007-05-01

    Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in Brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects.

  15. Profiling transcriptomes of human SH-SY5Y neuroblastoma cells exposed to maleic acid

    PubMed Central

    Wang, Chia-Chi; Lin, Yin-Chi; Cheng, Yin-Hua

    2017-01-01

    Background Maleic acid is a multi-functional chemical widely used in the field of industrial chemistry for producing food additives and food contact materials. As maleic acid may contaminate food by the release from food packages or intentional addition, it raises the concern about the effects of excessive dietary exposure to maleic acid on human health. However, the influence of maleic acid on human health has not been thoroughly studied. In silico toxicogenomics approaches have found the association between maleic acid and nervous system disease in human. The aim of this study is to experimentally explore the effects of maleic acid on human neuronal cells. Methods A microarray-based transcriptome profiling was performed to offer a better understanding of the effects of maleic acid on human health. Gene expression profiles of human neuroblastoma SH-SY5Y cells exposed to three concentrations of maleic acid (10, 50, and 100 μM) for 24 h were analyzed. Genes which were differentially expressed in dose-dependent manners were identified and further analyzed with an enrichment analysis. The expression profile of selected genes related to the inferred functional changes was validated using quantitative polymerase chain reaction (qPCR). Specific fluorescence probes were applied to observe the inferred functional changes in maleic acid-treated neuronal cells. Results A total of 316 differentially expressed genes (141 upregulated and 175 downregulated) were identified in response to the treatment of maleic acid. The enrichment analysis showed that DNA binding and metal ion binding were the significant molecular functions (MFs) of the neuronal cells affected by maleic acid. Maleic acid exposure decreased the expression of genes associated with calcium and thiol levels of the cells in a dose-dependent manner. The levels of intracellular calcium and thiol levels were also affected by maleic acid dose-dependent. Discussion The exposure to maleic acid is found to decrease the

  16. Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy.

    PubMed

    Deng, Yong-Ning; Shi, Jie; Liu, Jie; Qu, Qiu-Min

    2013-07-01

    Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson's disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P<0.001), decreased cell apoptosis (by 54.38%, P<0.001), increased SOD and GSH (by 120.53% and 90.46%, P<0.01), reduced accumulation of α-synuclein (by 35.93%, P<0.001) and ROS generation (by 33.99%, P<0.001), preserved MMP (33.93±3.62%, vs. 15.10±0.71% of JC-1 monomer, P<0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P<0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P<0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.

  17. PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes

    PubMed Central

    Ferguson, Ross; Subramanian, Vasanta

    2016-01-01

    Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease. PMID:27391595

  18. Effect of graphene oxide on undifferentiated and retinoic acid-differentiated SH-SY5Y cells line

    NASA Astrophysics Data System (ADS)

    Lv, Min; Zhang, Yujie; Liang, Le; Wei, Min; Hu, Wenbing; Li, Xiaoming; Huang, Qing

    2012-06-01

    Graphene oxide (GO), has created an unprecedented opportunity for development and application in biology, due to its abundant functional groups and well water solubility. Recently, the potential toxicity of GO in the environment and in humans has garnered more and more attention. In this paper, we systematically studied the cytotoxicity of GO nanosheets via examining the effect of GO on the morphology, viability and differentiation of a human neuroblastoma SH-SY5Y cell line, which was an ideal model used to study neuronal disease in vitro. The results suggested that GO had no obvious cytotoxicity at low concentration (<80 μg mL-1) for 96 h, but the viability of cells exhibited dose- and time-dependent decreases at high concentration (>=80 μg mL-1). Moreover, GO did not induce apoptosis. Very interestingly, GO significantly enhanced the differentiation of SH-SY5Y induced-retinoic acid (RA) by evaluating neurite length and the expression of neuronal marker MAP2. These data provide a promising application for neurodegenerative diseases.

  19. delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Connor, M.; Henderson, G.

    1996-01-01

    1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h

  20. Neuroprotective role of sphingosine-1-phosphate in L-BMAA treated neuroblastoma cells (SH-SY5Y).

    PubMed

    Muñoz-Sáez, Emma; de Munck García, Estefanía; Arahuetes Portero, Rosa María; Vicente, Francisca; Ortiz-López, Francisco Javier; Cantizani, Juan; Gómez Miguel, Begoña

    2015-04-23

    Sphingosine-1-phosphate (S1P) is a bioactive lipid which regulates proliferation, cell migration, survival and differentiation by specific receptors activation. We studied its effects on L-BMAA treated neuroblastoma cells (SH-SY5Y), an amino acid that can trigger neurodegenerative diseases such as amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC). We found that S1P protects from necrosis and prevents the GSK3 increasing as long as the PI3K/AKT pathway is active. Moreover, GSK3 inhibition protects against neuronal death caused by L-BMAA.

  1. Melatonin attenuates methamphetamine-induced neuroinflammation through the melatonin receptor in the SH-SY5Y cell line.

    PubMed

    Wongprayoon, Pawaris; Govitrapong, Piyarat

    2015-09-01

    Methamphetamine is a well-known psychostimulant drug, the abuse of which is a serious worldwide public health issue. In addition to its addictive effect, methamphetamine exposure has been shown to be associated with neuroinflammation in several brain areas. Several lines of evidence indicate that TNFα plays an important role in the methamphetamine-induced neuroinflammatory processes that result in apoptotic cell death. Many investigators have demonstrated the anti-neuroinflammatory effects of melatonin, but the mechanism by which this occurs still needs to be explored. In this study, we investigated the effect of methamphetamine on TNFα expression and NFκB activation in the neuroblastoma cell line SH-SY5Y. We demonstrated the time-dependent effect of methamphetamine on the induction of TNFα expression as well as IκB degradation and NFκB nuclear translocation. Furthermore, we investigated the effect of melatonin on methamphetamine-induced TNFα overexpression and NFκB activation. The results showed that pretreatment with 100nM melatonin could prevent the TNFα overexpression caused by methamphetamine exposure. This attenuating effect was prevented by pre-incubation with luzindole, an antagonist of the melatonin MT1/MT2 receptors. Furthermore, methamphetamine-induced IκB degradation and NFκB nuclear translocation were also suppressed by pretreatment with melatonin, and pretreatment with luzindole diminished these protective effects. MT2 knockdown by siRNA abrogated the anti-inflammatory effect exerted by melatonin. From these findings, we propose that melatonin exerts its protective effects on methamphetamine-induced neuroinflammation through the membrane receptor, at least in part MT2 subtype, in the SH-SY5Y neuroblastoma cell line.

  2. Force spectroscopy of membrane hardness of SH-SY5Y neuroblastoma cells before and after differentiation

    NASA Astrophysics Data System (ADS)

    Kwon, Sangwoo; Yang, Woochul; Choi, Yun Kyong; Park, Jung Keuck

    2014-05-01

    Atomic force microscopy (AFM) is utilized in many studies for measuring the structure and the physical characteristics of soft and bio materials. In particular, the force spectroscopy function in the AFM system allows us to explore the mechanical properties of bio cells. In this study, we probe the variation in the membrane hardness of human neuroblastoma SH-SY5Y cells (SH-cells) before and after differentiation by using force spectroscopy. The SH-cell, which is usually differentiated by using a chemical treatment with retinoic acid (RA), is a neuronal cell line employed widely as an in-vitro model for neuroscience research. In force spectroscopy, the force-distance curves are obtained from both the original and the RA-treated cells while the AFM tip approaches and pushes on the cell membranes. The slope deduced from linear region in the force-distance curve is the spring constant and corresponds to the hardness of the cell membrane. The spring constant of the RA-treated cells (0.597 ± 0.010 nN/nm) was smaller than that of the original cells (0.794 ± 0.010 nN/nm), reflecting a hardness decrease in the cells differentiated with the RA treatments. The results clearly demonstrated that the differentiated cells are softer than the original cells. The change in the elasticity of the differentiated cells might be caused by morphological modification during differentiation process. We suggest that force spectroscopy can be employed as a novel method to determine the degree of differentiation of stem cells into various functional cells.

  3. Methylmercury increases N-methyl-D-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity.

    PubMed

    Ndountse, Leopold Tchapda; Chan, Hing Man

    2008-07-30

    Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl-D-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [(3)H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25-1 microM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25-5 microM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 microM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 microM) partially attenuated the neurotoxicity effect of 1 microM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.

  4. Neuroprotective effects of Hu-Yi-Neng, a diet supplement, on SH-SY5Y human neuroblastoma cells.

    PubMed

    Yang, Y-H; Hsieh, T-J; Tsai, M-L; Chen, C-H; Lin, H-T; Wu, S-J

    2014-01-01

    Oxidative stress is considered the potential risk to the development of dementia. Some medicines, vitamins, and diet supplements have been suggested to have possible benefits via the antioxidative effects to slow the decline of cognitive function in demented and non-demented individuals. However, few studies were conducted to examine their functions, especially in composite diet supplements. Hu-Yi-Neng is a composite diet supplement, including ginkgo biloba, extract of pine bark, phosphatidyl serine, docosahexaenoic acid, and folic acid, used extensively in Taiwan. Therefore, our aim is to investigate the potential protective effects of Hu-Yi-Neng on human neuron cells. MATERALS AND METHODS: H2O2-induced neuronal toxicity was characterized in SH-SY5Y human neuroblastoma cells by the decrease of cell viability using PrestoBlue™ assay and by the increase of intracellular reactive oxygen species (ROS) level using DCFH-DA (2', 7'-dichlorodihydrofluorescin diacetate) assays. HO-1 mRNA expression was detected by real-time PCR. Akt and Erk 1/2 proteins were detected by western blotting. Pretreatment with Hu-Yi-Neng significantly reversed the decrease in cell viability induced by H2O2 in SH-SY5Y cells. Furthermore, Hu-Yi-Neng dose-dependently suppressed the elevation of intracellular reactive oxygen species (ROS) level. Hu-Yi-Neng protected SH-SY5Y cells from oxidative stress may via the increase in mRNA expression of heme oxygenase-1 (HO-1), an antioxidant enzyme. In addition, Hu-Yi-Neng inhibited H2O2-induced phosphorylation of Akt kinase but further increased the phosphorylation of Erk 1/2. Our results suggest that Hu-Yi-Neng has protective effect against oxidative stress-induced neuron cell loss and it could be an ideal composite diet supplement for preventing neurodegenerative diseases.

  5. Manganese-Induced Neurotoxicity and Alterations in Gene Expression in Human Neuroblastoma SH-SY5Y Cells.

    PubMed

    Gandhi, Deepa; Sivanesan, Saravanadevi; Kannan, Krishnamurthi

    2017-09-15

    Manganese (Mn) is an essential trace element required for many physiological functions including proper biochemical and cellular functioning of the central nervous system (CNS). However, exposure to excess level of Mn through occupational settings or from environmental sources has been associated with neurotoxicity. The cellular and molecular mechanism of Mn-induced neurotoxicity remains unclear. In the current study, we investigated the effects of 30-day exposure to a sub-lethal concentration of Mn (100 μM) in human neuroblastoma cells (SH-SY5Y) using transcriptomic approach. Microarray analysis revealed differential expression of 1057 transcripts in Mn-exposed SH-SY5Y cells as compared to control cells. Gene functional annotation cluster analysis exhibited that the differentially expressed genes were associated with several biological pathways. Specifically, genes involved in neuronal pathways including neuron differentiation and development, regulation of neurogenesis, synaptic transmission, and neuronal cell death (apoptosis) were found to be significantly altered. KEGG pathway analysis showed upregulation of p53 signaling pathways and neuroactive ligand-receptor interaction pathways, and downregulation of neurotrophin signaling pathway. On the basis of the gene expression profile, possible molecular mechanisms underlying Mn-induced neuronal toxicity were predicted.

  6. Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

    PubMed Central

    Smart, D.; Wandless, A.; Lambert, D. G.

    1995-01-01

    1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells. PMID:8528562

  7. Neurotoxicity effects of atrazine-induced SH-SY5Y human dopaminergic neuroblastoma cells via microglial activation.

    PubMed

    Ma, Kun; Wu, Hao-Yu; Zhang, Bo; He, Xi; Li, Bai-Xiang

    2015-11-01

    Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR) is a broad-spectrum herbicide with a wide range of applications worldwide. However, ATR is neurotoxic; it reduces dopamine levels in the substantia nigra and corpus striatum in the midbrain, affects the absorption of synaptic vesicles and synaptic bodies, and interferes with dopamine storage and uptake in synaptic vesicles, leading to neurodegenerative disorders. Microglia are resident immunocompetent and phagocytic cells that regulate and participate in the microenvironment in the central nervous system. They demonstrate macrophage characteristics after activation by releasing inflammatory cytokines and neurotoxic substances to increase the inflammatory response, and are thus involved in neurodegeneration. The aim of this study was to investigate the neurotoxic effects of ATR-activated microglia-mediated neuronal damage in terms of human dopaminergic neuroblastoma SH-SY5Y cell death. ATR was administered to BV-2 microglial cells at 12.5, 25, and 50 μM for 1, 6, 12, 24 and 48 h, respectively. ATR increased activated-microglia-induced overexpression of reactive oxygen species, inducible nitric oxide synthase, nitric oxide, gp91(phox), p47(phox), and the inflammatory cytokines tumor necrosis factor α and interleukin-1β, thus reducing SH-SY5Y cell viability. These results suggest that activated microglia may play a critical role in inflammation-mediated dopaminergic neuronal death, and provide the basis for further studies on the mechanisms of ATR-induced dopaminergic system toxicity.

  8. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

    SciTech Connect

    Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

    2012-08-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-{kappa}B), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  9. Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells.

    PubMed

    Horner, Kristen A; Zadina, James E

    2004-12-03

    The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

  10. Flavonoids from Potentilla parvifolia Fisch. and Their Neuroprotective Effects in Human Neuroblastoma SH-SY5Y cells In Vitro.

    PubMed

    Yuan, Zhenzhen; Luan, Guangxiang; Wang, Zhenhua; Hao, Xueyan; Li, Ji; Suo, Yourui; Li, Gang; Wang, Honglun

    2017-03-11

    Potentilla parvifolia Fisch. (Rosaceae) is a traditional medicinal plant in China. In this study, seven flavonoids, ayanin (1), tricin (2), quercetin (3), tiliroside (4), miquelianin (5), isoquercitrin (6), and astragalin (7), were separated and purified from ethanol extractive fractions from ethanol extracts of P. parvifolia using a combination of sevaral chromatographic methods. The human neuroblastoma SH-SY5Y cells were differentiated with all trans-retinoic acid and treated with okadaic acid to induce tau protein phosphorylation and synaptic atrophy, which could establish an Alzheimer's disease cell model. The neuroprotective effects of these flavonoids in cellular were evaluated in vitro by this cell model. Results from the western blot and morphology analysis suggested that compounds 3 and 4 had the better neuroprotective effects. This article is protected by copyright. All rights reserved.

  11. Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway.

    PubMed

    Rahman, Md Ataur; Bishayee, Kausik; Huh, Sung-Oh

    2016-02-01

    Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.

  12. Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

    PubMed Central

    Rahman, Md. Ataur; Bishayee, Kausik; Huh, Sung-Oh

    2016-01-01

    Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy. PMID:26674967

  13. Protective Effects of Bacopa Monnieri on Hydrogen Peroxide and Staurosporine: Induced Damage of Human Neuroblastoma SH-SY5Y Cells.

    PubMed

    Łojewski, Maciej; Pomierny, Bartosz; Muszyńska, Bożena; Krzyżanowska, Weronika; Budziszewska, Bogusława; Szewczyk, Agnieszka

    2016-02-01

    Many herbs, and recently their biomass from in vitro cultures, are essential for the treatment of diseases. The aim of this study was to determine the optimal growth of Bacopa monnieri (water hyssop) in an in vitro culture and to examine if extracts of the B. monnieri biomass from the in vitro culture would affect hydrogen peroxide- and staurosporine-induced injury of the human neuroblastoma SH-SY5Y cell line. It has been found that B. monnieri at concentrations of 25, 50, and 100 µg/mL inhibited both hydrogen peroxide-induced efflux of lactate dehydrogenase from damaged cells to culture medium and increased cell viability determined by an MTT assay. Moreover, B. monnieri at concentrations of 10, 25, and 50 µg/mL decreased staurosporine-induced activity of an executive apoptotic enzyme-caspase-3 and protected mitochondrial membrane potential. The obtained data indicate that the biomass from the in vitro culture of B. monnieri prevented SH-SY5Y cell damage related to oxidative stress and had the ability to inhibit the apoptotic process. Thus, this study supports the traditional use of B. monnieri as a neuroprotective therapy, and further in vivo studies on the effects of this preparation on morphology and function of nerve cells could lead to its wider application.

  14. Involvement of Mu Opioid Receptor Signaling in the Protective Effect of Opioid against 6-Hydroxydopamine-Induced SH-SY5Y Human Neuroblastoma Cells Apoptosis

    PubMed Central

    Eftekhar-Vaghefi, Shahrzad; Esmaeili-Mahani, Saeed; Elyasi, Leila; Abbasnejad, Mehdi

    2015-01-01

    Introduction: The neuroprotective role of opioid morphine against 6-hydroxydopamine-induced cell death has been demonstrated. However, the exact mechanism(s) underlying such neuroprotection, especially the role of subtype receptors, has not yet been fully clarified. Methods: Here, we investigated the effects of different opioid agonists on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular calcium, reactive oxygen species and mitochondrial membrane potential were assessed by fluorescence spectrophotometry method. Immunoblot technique was used to evaluate cytochrome-c and activated caspase-3 as biochemical markers of apoptosis induction. Results: The data showed that 6-OHDA caused significant cell damage, loss of mitochondrial membrane potential and increase in intracellular reactive oxygen species and calcium levels as well as activated caspase-3 and cytochrome-c release. Incubation of SH-SY5Y cells with μ-opioid agonists, morphine and DAMGO, but not with δ-opioid agonist, DADLE, elicited protective effect and reduced biochemical markers of cell damage and death. Discussion: The results suggest that μ-opioid receptors signaling participate in the opioid neuroprotective effects against 6-OHDA-induced neurotoxicity. PMID:26904174

  15. Estradiol and testosterone regulate arginine-vasopressin expression in SH-SY5Y human female neuroblastoma cells through estrogen receptors-α and -β.

    PubMed

    Grassi, Daniela; Bellini, Maria Jose; Acaz-Fonseca, Estefania; Panzica, Giancarlo; Garcia-Segura, Luis M

    2013-06-01

    The expression of arginine-vasopressin (AVP) is regulated by estradiol and testosterone (T) in different neuronal populations by mechanisms that are not yet fully understood. Estrogen receptors (ERs) have been shown to participate in the regulation of AVP neurons by estradiol. In addition, there is evidence of the participation of ERβ in the regulation of AVP expression exerted by T via its metabolite 5α-dihydrotestosterone (5α-DHT) and its further conversion in the androgen metabolite and ERβ ligand 3β-diol. In this study we have explored the role of ERs in the regulation exerted by estradiol and T on AVP expression, using the human neuroblastoma cell line SH-SY5Y. Estradiol treatment increased AVP mRNA levels in SH-SY5Y cells in comparison with cells treated with vehicle. The stimulatory effect of estradiol on AVP expression was imitated by the ERα agonist 4,4',4',-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and blocked by the ER antagonist, ICI 182,780, and the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile reduced AVP expression, whereas the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol enhanced the action of estradiol on AVP expression. T increased AVP expression in SH-SY5Y cells by a mechanism that was dependent on aromatase but not on 5α-reductase activity. The T effect was not affected by blocking the androgen receptor, was not imitated by the T metabolite 5α-DHT, and was blocked by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, 5α-DHT had a similar effect as the ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and 3β-diol, reducing AVP expression. These findings suggest that estradiol and T regulate AVP expression in SH-SY5Y cells through ERs, exerting a stimulatory action via ERα and

  16. Role of D-Limonene in autophagy induced by bergamot essential oil in SH-SY5Y neuroblastoma cells.

    PubMed

    Russo, Rossella; Cassiano, Maria Gilda Valentina; Ciociaro, Antonella; Adornetto, Annagrazia; Varano, Giuseppe Pasquale; Chiappini, Carlotta; Berliocchi, Laura; Tassorelli, Cristina; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2014-01-01

    Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005-0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70(S6K) (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125-750 µM) and linalyl acetate (62.5-375 µM), were individually tested at concentrations comparable to those found in 0.005-0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by D-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by D-limonene.

  17. Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Russo, Rossella; Cassiano, Maria Gilda Valentina; Ciociaro, Antonella; Adornetto, Annagrazia; Varano, Giuseppe Pasquale; Chiappini, Carlotta; Berliocchi, Laura; Tassorelli, Cristina; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2014-01-01

    Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d

  18. Quinolinic acid induces neuritogenesis in SH-SY5Y neuroblastoma cells independently of NMDA receptor activation.

    PubMed

    Hernandez-Martinez, Juan-Manuel; Forrest, Caroline M; Darlington, L Gail; Smith, Robert A; Stone, Trevor W

    2017-03-01

    Glutamate and nicotinamide adenine dinucleotide (NAD(+) ) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N-methyl-D-aspartate (NMDA) receptors and also a precursor for the formation of NAD(+) . The effect of QA on cell survival and differentiation has therefore been examined on SH-SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 μm) induced differentiation of SH-SY5Y cells into a neuronal phenotype showing neurite growth. QA (50-150 nm) also caused a concentration-dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker β3-tubulin in whole-cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA-induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene-iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK-801) did not prevent quinolinate-induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of β3-tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD(+) , independently of NMDA receptors.

  19. Neuroprotective effect of arctigenin via upregulation of P-CREB in mouse primary neurons and human SH-SY5Y neuroblastoma cells.

    PubMed

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-09-10

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  20. Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-01-01

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB. PMID:24025424

  1. 2,2',4,4'-Tetrabromodiphenyl ether promotes human neuroblastoma SH-SY5Y cells migration via the GPER/PI3K/Akt signal pathway.

    PubMed

    Tian, P-C; Wang, H-L; Chen, G-H; Luo, Q; Chen, Z; Wang, Y; Liu, Y-F

    2016-02-01

    Neuroblastoma is the predominant tumor of early childhood. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) has the highest concentration among all polybrominated diphenyl ether (PBDE) congeners in human body, particularly for children. Considering that accumulating evidences showed developmental neurotoxicity of PBDE, there is an urgent need to investigate the effects of BDE-47 on the development of neuroblastoma. This study revealed that BDE-47 had limited effects on the cytotoxicity while significantly increased the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells. This was further confirmed by the results that BDE-47 treatment significantly downregulated the expression of E-cadherin and zona occludin-1 and upregulated the expression of matrix metalloproteinase-9 (MMP-9). Silencing of MMP-9 by specific small interfering RNA significantly abolished the BDE-47-induced migration and invasion of SH-SY5Y cells. Further, the signals G protein-coupled estrogen receptor 1 (GPER)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) mediated the BDE-47-induced upregulation of MMP-9 and in vitro migration of SH-SY5Y cells since G15 (GPER inhibitor) and LY 294002 (PI3K/Akt inhibitor) significantly abolished the effects of BDE-47. Our results revealed that BDE-47 significantly triggered the metastasis of human neuroblastoma SH-SY5Y cells via upregulation of MMP-9 by the GPER/PI3K/Akt signal pathway. This study revealed for the first time that BDE-47 can promote the migration of SH-SY5Y cells. It also provided a better understanding about the metastasis of human neuroblastoma induced by environmental endocrine disruptors.

  2. A Potent (R)-alpha-bis-lipoyl Derivative Containing 8-Hydroxyquinoline Scaffold: Synthesis and Biological Evaluation of Its Neuroprotective Capabilities in SH-SY5Y Human Neuroblastoma Cells

    PubMed Central

    Cacciatore, Ivana; Fornasari, Erika; Baldassarre, Leonardo; Cornacchia, Catia; Fulle, Stefania; Di Filippo, Ester Sara; Pietrangelo, Tiziana; Pinnen, Francesco

    2013-01-01

    A novel bis-lipoyl derivative containing 8-hydroxyquinoline scaffold (LA-HQ-LA, 5) was synthesized as a new multifunctional drug candidate with antioxidant, chelant, and neuroprotective properties for the treatment of neurodegenerative diseases. We have investigated the potential effectiveness of LA-HQ-LA against the cytotoxicity induced by 6-OHDA and H2O2 on human neuroblastoma SH-SY5Y cell line. Our outcomes showed that LA-HQ-LA resulted in significant neuroprotective and antioxidant effects against H2O2- and 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells, as assessed by MTT assay. In particular, it showed potent neuroprotective effects against 6-OHDA in RA/PMA differentiated cells at all the tested concentrations. PMID:24275787

  3. Protection against oxidant-induced apoptosis by mitochondrial thioredoxin in SH-SY5Y neuroblastoma cells

    SciTech Connect

    Chen Yan; Yu Min; Jones, Dean P.; Greenamyre, J. Timothy; Cai Jiyang . E-mail: jiyang.cai@vanderbilt.edu

    2006-10-15

    Mitochondrial oxidative stress plays important roles in aging and age-related degenerative disorders. The newly identified mitochondrial thioredoxin (mtTrx; Trx2) is a key component of the mitochondrial antioxidant system which is responsible for the clearance of reactive intermediates and repairs proteins with oxidative damage. Here, we show that in cultured SH-SY5Y human neuroblastoma 1cells, overexpression of mtTrx inhibited apoptosis and loss of mitochondrial membrane potential induced by a chemical oxidant, tert-butylhydroperoxide (tBH). The effects of calcium ionophore (Br-A23187) were not affected by mtTrx, suggesting the protection was specific against oxidative injury. The mitochondrial glutathione pool was oxidized by tBH, and this oxidation was not inhibited by increased mtTrx. Consequently, the antioxidant function of mtTrx is not redundant, but rather in addition, to that of GSH. Mutations of Cys90 and Cys93 to serines rendered mtTrx ineffective in protection against tBH-induced cytoxicity. These data indicate that mtTrx controls the mitochondrial redox status independently of GSH and is a key component of the defensive mechanism against oxidative stress in cultured neuronal cells.

  4. Toxic profile of bergamot essential oil on survival and proliferation of SH-SY5Y neuroblastoma cells.

    PubMed

    Berliocchi, Laura; Ciociaro, Antonella; Russo, Rossella; Cassiano, Maria Gilda Valentina; Blandini, Fabio; Rotiroti, Domenicantonio; Morrone, Luigi Antonio; Corasaniti, Maria Tiziana

    2011-11-01

    Cosmetic, pharmaceutical, food and confectionary industries make increasing use of plant extracts in their products. Despite the widespread use of products containing plant extracts, the mechanisms of their effects are not fully characterized. Bergamot essential oil (BEO; Citrus bergamia, Risso) is a well-known plant extract used in aromatherapy and it has analgesic, anxiolytic and neuroprotective effects in rodents. To elicit neuroprotection, BEO recruits Akt prosurvival pathways. However, Akt stimulates cell proliferation, which may also pose risks for health in case of prolonged use. To study the potential effects of BEO on survival and proliferation of dividing cells, we selected human SH-SY5Y neuroblastoma cells. BEO triggered concentration-dependent mitochondrial dysfunction, cytoskeletal reorganization, cell shrinkage, DNA fragmentation and both caspase-dependent and independent cell death. Analysis of cleavage products of poly-(ADP-ribose) polymerase (PARP) revealed caspase-3 activation, but also activation of additional protease families. As result of increased proteolytic activity, Akt protein levels decreased in BEO-treated cells. Our data show that BEO can be lethal for dividing cells by activating multiple pathways. While this may reduce the risk of unwanted cell proliferation after prolonged use, it does suggest a cautionary approach to the use of inappropriate dilutions of the oil that may cause cell death.

  5. The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells

    PubMed Central

    Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy

    2017-01-01

    Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa’s nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action. PMID:28832626

  6. The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells.

    PubMed

    Leung, How-Wing; Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy; Mitra-Ganguli, Tora; VanDongen, Antonius M J

    2017-01-01

    Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.

  7. Low arsenite concentrations induce cell proliferation via activation of VEGF signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Watcharasit, Piyajit; Visitnonthachai, Daranee; Suntararuks, Sumitra; Thiantanawat, Apinya; Satayavivad, Jutamaad

    2012-01-01

    Arsenic widely contaminates the environment, especially in drinking water. Although it is a known carcinogen in humans, its carcinogenic mechanism has not yet been clarified. Here, we demonstrated that a low concentration of arsenite treatment induced proliferation of human neuroblastoma SH-SY5Y cells as indicated by increases in cell viability and BrdU incorporation. Additionally, arsenite increased VEGF expression and secretion. Inhibition of VEGF-induced signaling by SU4312, the inhibitor of VEGF receptor 2 kinase, and by treatment with anti-VEGF antibody blocked arsenite-induced increases in cell proliferation. Moreover, arsenite caused activation of ERK, a key signaling molecule involved in cell proliferation, and this activation was attenuated by SU4312, suggesting that ERK activation contributes to VEGF-mediated cell proliferation induced by arsenite. Collectively, the present study reveals that a mechanism underlying arsenic-induced cell proliferation may be through induction and activation of VEGF signaling, and this may subsequently contribute to tumor formation. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Quantitative proteomic analysis of HIV-1 Tat-induced dysregulation in SH-SY5Y neuroblastoma cells.

    PubMed

    Ganief, Tariq; Gqamana, Putuma; Garnett, Shaun; Hoare, Jackie; Stein, Dan J; Joska, John; Soares, Nelson; Blackburn, Jonathan M

    2017-03-01

    Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

  9. Neuroprotective effect of Rosmarinus officinalis extract on human dopaminergic cell line, SH-SY5Y.

    PubMed

    Park, Se-Eun; Kim, Seung; Sapkota, Kumar; Kim, Sung-Jun

    2010-07-01

    Hydrogen peroxide (H2O2) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson's disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H2O2-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H2O2-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H2O2. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H2O2-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

  10. Tris (1,3-dichloro-2-propyl) phosphate-induced apoptotic signaling pathways in SH-SY5Y neuroblastoma cells.

    PubMed

    Li, Ruiwen; Zhou, Peijiang; Guo, Yongyong; Lee, Jae-Seong; Zhou, Bingsheng

    2017-01-01

    Tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP, also known as TDCPP), an extensively used flame retardant, is frequently detected in the environment and biota. Recent studies have shown that TDCIPP has neurotoxic effects. In this study, we determined the mechanisms of TDCIPP-induced neurotoxicity in human neuroblastoma (SH-SY5Y) cells. By using morphological examination, flow cytometry, and mitochondrial membrane potential (ΔYm) measurement, we confirmed that exposure to TDCIPP caused apoptosis accompanied by the activation of apoptosis-related genes (e.g. Bax and Bcl-2) and caspase 3 protein in SH-SY5Y cells. Increased reactive oxygen species (ROS) formation and intracellular calcium ions ([Ca(2+)]i) were also observed in TDCIPP-treated SH-SY5Y cells. Exposure to TDCIPP led to the activation of protein markers of endoplasmic reticulum (ER) stress, including eukaryotic translation initiation factor 2a subunit (p-EIF2a), activation transcription factor (ATF4), glucose-regulated protein (GRP78), and the proapoptotic factor C/EBP homologous protein (CHOP). To determine the role of the ER in apoptosis, phenyl butyric acid (PBA), an ER stress inhibitor, was applied. Treatment with PBA effectively attenuated TDCIPP-induced ER stress and protected against apoptotic death in SH-SY5Y cells by inhibition of Bax expression and promotion of Bcl-2 expression. Furthermore, we found that pretreatment of the cells with the ROS scavenger N-acetyl cysteine (NAC) inhibited the ER stress response and prevented apoptosis. The combination of PBA and NAC pretreatment could further prevent TDCIPP induced ER-stress and apoptotic death compared with PBA or NAC pretreatment alone. Thus, in the present study, we demonstrated that TDCIPP induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathways in SH-SY5Y cells.

  11. Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y Cells

    SciTech Connect

    Liu, P.-S. . E-mail: psliu@mail.scu.edu.tw; Chiung, Y.-M.; Kao, Y.-Y.

    2006-03-01

    The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca{sup 2+}]{sub c}) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca{sup 2+} mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca{sup 2+}]{sub c} by releasing Ca{sup 2+} from the intracellular stores and extracellular Ca{sup 2+} influx. 500 {mu}M TDI induced a net [Ca{sup 2+}]{sub c} increase of 112 {+-} 8 and 78 {+-} 6 nM in the presence and absence of extracellular Ca{sup 2+}, respectively. In Ca{sup 2+}-free buffer, TDI induced Ca{sup 2+} release from internal stores to reduce their Ca{sup 2+} content and this reduction was evidenced by a suppression occurring on the [Ca{sup 2+}]{sub c} rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca{sup 2+}, simultaneous exposure to TDI and methacholine led a higher level of [Ca{sup 2+}]{sub c} compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca{sup 2+}]{sub c} homeostasis including releasing Ca{sup 2+} from internal stores and inducing extracellular Ca{sup 2+} influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.

  12. Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y cells.

    PubMed

    Liu, Pei-Shan; Chiung, Yin-Mei; Kao, Yi-Yun

    2006-03-01

    The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.

  13. Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity

    SciTech Connect

    Gustafsson, Helena; Runesson, Johan; Lundqvist, Jessica; Lindegren, Helene; Axelsson, Viktoria; Forsby, Anna

    2010-06-01

    The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.

  14. Mu and Delta opioid receptors activate the same G proteins in human neuroblastoma SH-SY5Y cells

    PubMed Central

    Alt, A; Clark, M J; Woods, J H; Traynor, J R

    2002-01-01

    There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH-SY5Y cells.The [35S]GTPγS binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC50, 45 nM) and SNC80 (EC50, 32 nM) were found to be completely selective for stimulation of [35S]-GTPγS binding through mu and delta opioid receptors respectively. Maximal stimulation of [35S]-GTPγS binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [35S]-GTPγS binding. This effect was also seen when measured at the level of adenylyl cyclase.Receptor activation increased the dissociation of pre-bound [35S]-GTPγS. In addition, the delta agonist SNC80 promoted the dissociation of [35S]-GTPγS from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [35S]-GTPγS from G proteins initially labelled using SNC80.Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity.The findings support the hypothesis that mu- and delta-opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane. PMID:11786497

  15. Hydrogen peroxide modifies both activity and isoforms of acetylcholinesterase in human neuroblastoma SH-SY5Y cells.

    PubMed

    Garcimartín, Alba; López-Oliva, M Elvira; González, M Pilar; Sánchez-Muniz, Francisco J; Benedí, Juana

    2017-08-01

    The involvement of cholinergic system and the reactive oxygen species (ROS) in the pathogenesis of some degenerative diseases has been widely reported; however, the specific impact of hydrogen peroxide (H2O2) on the acetylcholinesterase (AChE) activity as well as AChE isoform levels has not been clearly established. Hence, the purpose of present study is to clarify whether H2O2 alters these parameters. Human neuroblastoma SH-SY5Y cells were treated with H2O2 (1-1000µM) for 24h and AChE activity and AChE and cytochrome c levels were evaluated. AChE activity was strongly increased from 1µM to 1000µM of H2O2. The results of the kinetic study showed that H2O2 affected Vmax but not Km; and also that H2O2 changed the sigmoid kinetic observed in control samples to hyperbolic kinetic. Thus, results suggest that H2O2 acts as an allosteric activators. In addition, H2O2, (100-1000µM) reduced the total AChE content and modified its isoform profile (mainly 50-, 70-, and 132-kDa)·H2O2 from 100µM to 1000µM induced cytochrome c release confirming cell death by apoptosis. All these results together suggest: a) the involvement of oxidative stress in the imbalance of AChE; and b) treatment with antioxidant agents may be a suitable strategy to protect cholinergic system alterations promoted by oxidative stress. Copyright © 2017. Published by Elsevier B.V.

  16. Carvacrol protects neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis by suppressing activation of MAPK/JNK-NF-κB signaling pathway

    PubMed Central

    Cui, Zhen-wen; Xie, Zheng-xing; Wang, Bao-feng; Zhong, Zhi-hong; Chen, Xiao-yan; Sun, Yu-hao; Sun, Qing-fang; Yang, Guo-yuan; Bian, Liu-guan

    2015-01-01

    Aim: Carvacrol (2-methyl-5-isopropylphenol), a phenolic monoterpene in the essential oils of the genera Origanum and Thymus, has been shown to exert a variety of therapeutic effects. Here we examined whether carvacrol protected neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis and explored the underlying mechanisms. Methods: Neuroblastoma SH-SY5Y cells were incubated with Fe2+ for 24 h, and the cell viability was assessed with CCK-8 assay. TUNEL assay and flow cytometric analysis were performed to evaluate cell apoptosis. The mRNA levels of pro-inflammatory cytokines and NF-κB p65 were determined using qPCR. The expression of relevant proteins was determined using Western blot analysis or immunofluorescence staining. Results: Treatment of SH-SY5Y cells with Fe2+ (50–200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L). Treatment with Fe2+ increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis. Furthermore, treatment with Fe2+ significantly increased the gene expression of IL-1β, IL-6 and TNF-α, and induced the nuclear translocation of NF-κB. Treatment with Fe2+ also significantly increased the phosphorylation of p38, ERK, JNK and IKK in the cells. Pretreatment with carvacrol significantly inhibited Fe2+-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis. Moreover, pretreatment with carvacrol inhibited Fe2+-induced phosphorylation of JNK and IKK, but not p38 and ERK in the cells. Conclusion: Carvacrol protects neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis, which may result from suppressing the MAPK/JNK-NF-κB signaling pathways. PMID:26592517

  17. Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Lantto, Tiina A.; Laakso, Into; Dorman, H. J. Damien; Mauriala, Timo; Hiltunen, Raimo; Kõks, Sulev; Raasmaja, Atso

    2016-01-01

    Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds. PMID:27420050

  18. Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid.

    PubMed

    Hall, Susan; Anoopkumar-Dukie, Shailendra; Grant, Gary D; Desbrow, Ben; Lai, Richard; Arora, Devinder; Hong, Yinna

    2017-06-01

    Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100 nM and 10 μM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10 μM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10 nM and 10 μM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1 μM and 10 μM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.

  19. A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.

    PubMed

    Um, Moonkyoung; Gross, Alec W; Lodish, Harvey F

    2007-03-01

    The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits

  20. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    PubMed Central

    Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  1. U18666A, an Activator of Sterol Regulatory Element Binding Protein (SREBP) Pathway Modulates Presynaptic Dopaminergic Phenotype of SH-SY5Y Neuroblastoma Cells.

    PubMed

    Schmitt, Mathieu; Dehay, Benjamin; Bezard, Erwan; Garcia-Ladona, F Javier

    2017-04-13

    The therapeutic use of statins has been associated to a reduced risk of Parkinson's disease (PD) and may hold neuroprotective potential by counteracting the degeneration of dopaminergic neurons. Transcriptional activation of the sterol regulatory element-binding protein (SREBP) is one of the major downstream signalling pathways triggered by the cholesterol-lowering effect of statins. In a previous study in neuroblastoma cells, we have shown that statins consistently induce the up-regulation of presynaptic dopaminergic proteins as well as changes of their function and these effects were accompanied by downstream activation of SREBP. In current study, we aimed to determine the direct role of SREBP pathway in the modulation of dopaminergic phenotype. We demonstrate that treatment of SH-SY5Y cells with U18666A, a SREBP activator, increases the translocation of SREBPs into the nucleus, increases expression of SREBP-1, SREBP-2 and of the presynaptic dopaminergic markers such as vesicular monoamine transporter 2, synaptic vesicle glycoprotein 2A and 2C, synaptogyrin-3 and tyrosine hydroxylase. The addition of SREBP inhibitor, PF-429242, blocks the increase of U18666A-induced expression of SREBPs and of presynaptic markers. Our results, in line with previously reported effects of statins, demonstrate that direct stimulation of SREBP translocation is associated to differentiation towards a dopaminergic-like phenotype and suggest that SREBP-mediated transcriptional activity may lead to the restoration of the presynaptic dopamine markers and may contribute to neuroprotection of dopaminergic neurons. These findings further support the potential protective role of statin in PD and shed light upon SREBP as a potential new target for developing disease-modifying treatment in PD. This article is protected by copyright. All rights reserved.

  2. Low-dose/dose-rate γ radiation depresses neural differentiation and alters protein expression profiles in neuroblastoma SH-SY5Y cells and C17.2 neural stem cells.

    PubMed

    Bajinskis, Ainars; Lindegren, Heléne; Johansson, Lotta; Harms-Ringdahl, Mats; Forsby, Anna

    2011-02-01

    The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.

  3. Effects of bergamot essential oil and its extractive fractions on SH-SY5Y human neuroblastoma cell growth.

    PubMed

    Navarra, Michele; Ferlazzo, Nadia; Cirmi, Santa; Trapasso, Elena; Bramanti, Placido; Lombardo, Giovanni Enrico; Minciullo, Paola Lucia; Calapai, Gioacchino; Gangemi, Sebastiano

    2015-08-01

    The goals were to investigate the mechanisms underlying the antiproliferative effects of bergamot essential oil (BEO) and to identify the compounds mainly responsible for its SH-SY5Y cells growth rate inhibition. Five BEO extractive fractions (BEOs) differing in their chemical composition were used. Cell proliferation was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell count assays. Trypan blue exclusion test and Annexin V/PI staining were performed to assess their cytotoxic activity. Genotoxicity was detected by comet assay. The cell cycle was checked cytofluorimetrically. Reactive oxygen species (ROS) and Δψm were measured fluorimetrically. Western blotting analyses for some apoptosis-related proteins were carried out. Treatment of SH-SY5Y cells with some types of BEOs decreased cell growth rate by a mechanism correlated to both apoptotic and necrotic cell death. Coloured BEOs act by increasing ROS generation, responsible for the drop in Δψm, and modulate p38 and extracellular signal-regulated kinases (ERK ½) mitogen-activated protein kinases, p53, Bcl-2 and Bax signalling pathways. Finally, we identify bergamottin and 5-geranyloxy-7-methoxycoumarin as the bioactive molecules that could play a pivotal role in the antiproliferative effects exerted by coloured BEOs. Our study provides novel insights into the field of the antiproliferative effects of BEO, which could be exploited in the context of a multitarget pharmacological strategy. © 2015 Royal Pharmaceutical Society.

  4. Effects of antidepressants on DSP4/CPT-induced DNA damage response in neuroblastoma SH-SY5Y cells

    PubMed Central

    Wang, Yan; Hilton, Benjamin A.; Cui, Kui; Zhu, Meng-Yang

    2015-01-01

    DNA damage is a form of cell stress and injury. Increased systemic DNA damage is related to the pathogenic development of neurodegenerative diseases. Depression occurs in a relatively high percentage of patients suffering from degenerative diseases, for whom antidepressants are often used to relieve depressive symptoms. However, few studies have attempted to elucidate why different groups of antidepressants have similar effects on relieving symptoms of depression. Previously, we demonstrated that neurotoxins N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)- and camptothecin (CPT)-induced the DNA damage response in SH-SY5Y cells, and DSP4 caused cell cycle arrest which was predominately in the S-phase. The present study shows that CPT treatment also resulted in similar cell cycle arrest. Some classic antidepressants could reduce the DNA damage response induced by DSP4 or CPT in SH-SY5Y cells. Cell viability examination demonstrated that both DSP4 and CPT caused cell death, which was prevented by spontaneous administration of some tested antidepressants. Flow cytometric analysis demonstrated that a majority of the tested antidepressants protect cells from being arrested in S-phase. These results suggest that blocking the DNA damage response may be an important pharmacologic characteristic of antidepressants. Exploring the underlying mechanisms may allow for advances in the effort to improve therapeutic strategies for depression appearing in degenerative and psychiatric diseases. PMID:26038195

  5. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    SciTech Connect

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  6. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  7. Neuroprotective effect of Demethoxycurcumin, a natural derivative of Curcumin on rotenone induced neurotoxicity in SH-SY 5Y Neuroblastoma cells.

    PubMed

    Ramkumar, Muthu; Rajasankar, Srinivasagam; Gobi, Veerappan Venkatesh; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Kalandar, Ameer; Chidambaram, Ranganathan

    2017-04-18

    Mitochondrial dysfunction and oxidative stress are the main toxic events leading to dopaminergic neuronal death in Parkinson's disease (PD) and identified as vital objective for therapeutic intercession. This study investigated the neuro-protective effects of the demethoxycurcumin (DMC), a derivative of curcumin against rotenone induced neurotoxicity. SH-SY5Y neuroblastoma cells are divided into four experimental groups: untreated cells, cells incubated with rotenone (100 nM), cells treated with DMC (50 nM) + rotenone (100 nM) and DMC alone treated. 24 h after treatment with rotenone and 28 h after treatment with DMC, cell viability was assessed using the MTT assay, and levels of ROS and MMP, plus expression of apoptotic protein were analysed. Rotenone induced cell death in SH-SY5Y cells was significantly reduced by DMC pretreatment in a dose-dependent manner, indicating the potent neuroprotective effects of DMC. Rotenone treatment significantly increases the levels of ROS, loss of MMP, release of Cyt-c and expression of pro-apoptotic markers and decreases the expression of anti-apoptotic markers. Even though the results of the present study indicated that the DMC may serve as a potent therapeutic agent particularly for the treatment of neurodegenerative diseases like PD, further pre-clinical and clinical studies are required.

  8. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal.

    PubMed

    Waly, Mostafa; Power-Charnitsky, Verna-Ann; Hodgson, Nathaniel; Sharma, Alok; Audhya, Tapan; Zhang, Yiting; Deth, Richard

    2016-01-01

    The folate and cobalamin (Cbl-) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S-adenosylmethionine (SAM). OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

  9. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

    PubMed Central

    Power-Charnitsky, Verna-Ann; Sharma, Alok; Audhya, Tapan; Zhang, Yiting

    2016-01-01

    The folate and cobalamin (Cbl-) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S-adenosylmethionine (SAM). OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action. PMID:26989453

  10. Neuroprotective Effects of Bioavailable Polyphenol-Derived Metabolites against Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

    PubMed

    González-Sarrías, Antonio; Núñez-Sánchez, María Ángeles; Tomás-Barberán, Francisco A; Espín, Juan Carlos

    2017-02-01

    Oxidative stress is involved in cell death in neurodegenerative diseases. Dietary polyphenols can exert health benefits, but their direct effects on neuronal cells are debatable because most phenolics are metabolized and do not reach the brain as they occur in the dietary sources. Herein, we evaluate the effects of a panel of bioavailable polyphenols and derived metabolites at physiologically relevant conditions against H2O2-induced apoptosis in human neuroblastoma SH-SY5Y cells. Among the 19 metabolites tested, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, gallic acid, ellagic acid, and urolithins prevented neuronal apoptosis via attenuation of ROS levels, increased REDOX activity, and decreased oxidative stress-induced apoptosis by preventing the caspase-3 activation via the mitochondrial apoptotic pathway in SH-SY5Y cells. This suggests that dietary sources containing the polyphenol precursors of these molecules such as cocoa, berries, walnuts, and tea could be potential functional foods to reduce oxidative stress associated with the onset and progress of neurodegenerative diseases.

  11. Paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Yang, Wonsuk; Tiffany-Castiglioni, Evelyn; Koh, Hyun Chul; Son, Il-Hong

    2009-12-15

    Epidemiologic and laboratory studies suggest that paraquat can be an environmental etiologic factor in Parkinson's disease (PD). One mechanism by which paraquat may mediate cell death of dopaminergic neurons is by inducing endoplasmic reticulum (ER) stress, as suggested in a recent report. In this study, we further investigated this linkage by examining ER stress cascades. To this aim, human neuroblastoma cells (SH-SY5Y cells) were treated with paraquat and the signaling cascades through which ER stress results in apoptosis were examined. Then, it was examined whether ER stress is produced by paraquat. Paraquat increased ER stress biomarker proteins, glucose-regulated protein 78 (GRP78), ER degradation-enhancing alpha-mannosidae-like protein (EDEM), and C/EBP homologous protein (CHOP). Then, it was investigated which ER stress cascades are affected by paraquat. Paraquat activated inositol-requiring enzyme 1 (IRE1), apoptosis signal regulating kinase 1 (ASK1), and c-jun kinase (JNK). Also, paraquat activated calpain and caspase 3, but did not affect the levels of intracellular calcium and the activity of caspase 12. Finally, apoptotic DNA damage by paraquat was investigated and this damage was attenuated by salubrinal (ER stress inhibitor), thioredoxin (ASK1 inhibitor) and SP600125 (JNK inhibitor). Therefore, current data indicate that paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in SY5Y cells.

  12. Iron-induced oxidative stress activates AKT and ERK1/2 and decreases Dyrk1B and PRMT1 in neuroblastoma SH-SY5Y cells.

    PubMed

    Bautista, Elizabeth; Vergara, Paula; Segovia, José

    2016-03-01

    Iron is essential for proper neuronal functioning; however, excessive accumulation of brain iron is reported in Parkinson's, Alzheimer's, Huntington's diseases and amyotrophic lateral sclerosis. This indicates that dysregulated iron homeostasis is involved in the pathogenesis of these diseases. To determinate the effect of iron on oxidative stress and on cell survival pathways, such as AKT, ERK1/2 and DyrK1B, neuroblastoma SH-SY5Y cells were exposed to different concentration of FeCl2 (iron). We found that iron induced cell death in SH-SY5Y cells in a concentration-dependent manner. Detection of iNOS and 3-nitrotyrosine confirms the presence of increased nitrogen species. Furthermore, we found a decrease of catalase and protein arginine methyl-transferase 1 (PRMT1). Interestingly, iron increased the activity of ERK and AKT and reduced DyrK1B. Moreover, after FeCl2 treatment, the transcription factors c-Jun and pSmad1/5 were activated. These results indicate that the presence of high levels of iron increase the vulnerability of neurons to oxidative stress. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

    PubMed Central

    Smart, D; Smith, G; Lambert, D G

    1995-01-01

    We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated. PMID:7832776

  14. Effects of Pyridostigmine bromide on SH-SY5Y cells: An in vitro neuroblastoma neurotoxicity model.

    PubMed

    Azzolin, VerÔnica Farina; Barbisan, Fernanda; Lenz, Luana Suéling; Teixeira, Cibele Ferreira; Fortuna, Milena; Duarte, Thiago; Duarte, Marta Maria Frescura Medeiros; da Cruz, Ivana Beatrice Mânica

    2017-11-01

    Pyridostigmine bromide (PB) is a reversible acetylcholinesterase (AChE) inhibitor and the first-choice for the treatment of symptoms associated with myasthenia gravis and other neuromuscular junction disorders. However, evidence suggested that PB could be associated with the Gulf War Illness characterised by the presence of fatigue, headaches, cognitive dysfunction, and musculoskeletal respiratory and gastrointestinal disturbances. Given that a potential neurotoxic effect of PB has not yet been completely elucidated, the present investigation used neural SH-SY5Y cells to evaluate the effect of PB on the cellular viability, cell apoptosis, modulation of the cell cycle, oxidative stress, and genotoxicity variables, which indicate neurodegeneration. As expected, a PB concentration curve based on the therapeutic dose of the drug showed an inhibition of the AChE activity. However, this effect was transient and did not involve differential AChE gene regulation by PB. These results confirmed that undifferentiated SH-SY5Y cells can be used as a cholinergic in vitro model. In general, PB did not trigger oxidative stress, and at a slightly higher PB concentration (80ng/mL), higher levels of protein carbonylation and DNA damage were detected, as determined by the marker 8-deoxyguanosine. The PB genotoxic effects at 80ng/mL were confirmed by the upregulation of the p53 and DNA methyltransferase 1 (DNMT1) genes, which are associated with cellular DNA repair. PB at 40ng/mL, which is the minimal therapeutic dose, led to higher cell proliferation and mitochondrial activity compared with the control group. The effects of PB were corroborated by the upregulation of the telomerase gene. In summary, despite the methodological constrains related to the in vitro protocols, our results suggested that exposure of neural cells to PB, without other chemical and physical stressors did not cause extensive toxicity or indicate any neurodegeneration patterns. Copyright © 2017 Elsevier B.V. All

  15. Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

    PubMed

    Wang, Hong; Sun, Ruowen; Chi, Zuofei; Li, Shuang; Hao, Liangchun

    2017-04-05

    Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

  16. Agaricus blazei extract attenuates rotenone-induced apoptosis through its mitochondrial protective and antioxidant properties in SH-SY5Y neuroblastoma cells.

    PubMed

    Venkatesh Gobi, Veerappan; Rajasankar, Srinivasagam; Ramkumar, Muthu; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Chidambaram, Ranganathan

    2016-09-20

    The present study was aimed to find out the effect of Agaricus blazei mushroom extract against rotenone-induced cellular model. SH-SY5Y neuroblastoma cells are divided into four experimental groups (control, rotenone (100 nM), A. blazei (5 μg/ml) + rotenone (100 nM), and A. blazei alone treated) based on MTT assay, cells were allowed to measure the ROS, TBARS levels, and antioxidants activities. Finally, mitochondrial transmembrane potential (MMP) and expressions of apoptotic proteins were also analyzed. Pre-treatment with A. blazei significantly enhanced cell viability, attenuated rotenone-induced ROS, MMP, and apoptosis. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone-induced cytotoxicity. Therefore, it may be concluded that A. blazei can be further developed as a promising drug for the treatment of Parkinson's disease (PD).

  17. Spirafolide from bay leaf (Laurus nobilis) prevents dopamine-induced apoptosis by decreasing reactive oxygen species production in human neuroblastoma SH-SY5Y cells.

    PubMed

    Ham, Ahrom; Kim, Bora; Koo, Uk; Nam, Kung-Woo; Lee, Sung-Jin; Kim, Kyeong Ho; Shin, Jongheon; Mar, Woongchon

    2010-12-01

    Reactive oxygen species (ROS) are important mediators in many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. This study tested the neuroprotective effects of spirafolide, a compound purified from the leaves of Laurus nobilis L. (Lauraceae), against dopamine (DA)-induced apoptosis in human neuroblastoma SH-SY5Y cells. Following a 24-h exposure of cells to DA (final conc., 0.6 mM), we observed a marked increase in apoptosis, increased generation of ROS and decreased cell viability. Pretreatment of the cells for 24 h with spirafolide (0.4, 2, and 10 μM) before exposure to DA notably increased cell survival (p < 0.01) and lowered intracellular ROS levels (p < 0.01). These results indicate that spirafolide has neuroprotective effects against DA toxicity. These effects may contribute to the treatment of neurodegenerative diseases.

  18. Morphine induces Beclin 1- and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus.

    PubMed

    Zhao, Lixia; Zhu, Yushan; Wang, Dongmei; Chen, Ming; Gao, Ping; Xiao, Weiming; Rao, Guanhua; Wang, Xiaohui; Jin, Haijing; Xu, Lin; Sui, Nan; Chen, Quan

    2010-04-01

    Chronic exposure to morphine can induce drug addiction and neural injury, but the exact mechanism is not fully understood. Here we show that morphine induces autophagy in neuroblastoma SH-SY5Y cells and in the rat hippocampus. Pharmacological approach shows that this effect appears to be mediated by PTX-sensitive G protein-coupled receptors signaling cascade. Morphine increases Beclin 1 expression and reduces the interaction between Beclin 1 and Bcl-2, thus releasing Beclin 1 for its pro-autophagic activity. Bcl-2 overexpression inhibits morphine-induced autophagy, whereas knockdown of Beclin 1 or knockout of ATG5 prevents morphine-induced autophagy. In addition, chronic treatment with morphine induces cell death, which is increased by autophagy inhibition through Beclin 1 RNAi. Our data are the first to reveal that Beclin 1 and ATG5 play key roles in morphine-induced autophagy, which may contribute to morphine-induced neuronal injury.

  19. Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

    PubMed

    Stio, M; Celli, A; Treves, C

    2001-06-01

    This study examines the effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)(2)D(3) or its derivatives, but significantly decreased in the presence of the two retinoids (0.001--10 microM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060, and 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.

  20. Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells: the impact of cell differentiation state.

    PubMed

    Jantas, D; Greda, A; Golda, S; Korostynski, M; Grygier, B; Roman, A; Pilc, A; Lason, W

    2014-08-01

    Recent studies have documented that metabotropic glutamate receptors from group II and III (mGluR II/III) are a potential target in the symptomatic treatment of Parkinson's disease (PD), however, the neuroprotective effects of particular mGluR II/III subtypes in relation to PD pathology are recognized only partially. In the present study, we investigated the effect of various mGluR II/III activators in the in vitro model of PD using human neuroblastoma SH-SY5Y cell line and mitochondrial neurotoxin MPP(+). We demonstrated that all tested mGluR ligands: mGluR II agonist - LY354740, mGluR III agonist - ACPT-I, mGluR4 PAM - VU0361737, mGluR8 agonist - (S)-3,4-DCPG, mGluR8 PAM - AZ12216052 and mGluR7 allosteric agonist - AMN082 were protective against MPP(+)-evoked cell damage in undifferentiated (UN-) SH-SY5Y cells with the highest neuroprotection mediated by mGluR8-specific agents. However, in retinoic acid- differentiated (RA-) SH-SY5Y cells we found protection mediated only by mGluR8 activators. We also demonstrated the cell proliferation stimulating effect for mGluR4 and mGluR8 PAMs. Next, we showed that the protection mediated by mGluR II/III activators in UN-SH-SY5Y was not accompanied by the modulation of caspase-3 activity, however, a decrease in the number of apoptotic nuclei was found. Finally, we showed that the inhibitor of necroptosis, necrostatin-1 blocked the mGluR III-mediated protection. Altogether our comparative in vitro data add a further proof to neuroprotective effects of mGluR agonists or PAMs and point to mGluR8 as a promising target for neuroprotective interventions in PD. The results also suggest the participation of necroptosis-related molecular pathways in neuroprotective effects of mGluR III activation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

    SciTech Connect

    Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O. )

    1988-11-01

    The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

  2. Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Nataraj, Jagatheesan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed

    2015-01-01

    Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington's disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5–200 nM) for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5–200 nM) and then incubation with rotenone (100 nM). Using effective dose of vanillin (100 nM), mitochondrial membrane potential, levels of reactive oxygen species (ROS), and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD. PMID:26664453

  3. Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells.

    PubMed

    Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Nataraj, Jagatheesan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed

    2015-01-01

    Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington's disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5-200 nM) for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5-200 nM) and then incubation with rotenone (100 nM). Using effective dose of vanillin (100 nM), mitochondrial membrane potential, levels of reactive oxygen species (ROS), and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD.

  4. Aldo-keto reductases mediate constitutive and inducible protection against aldehyde toxicity in human neuroblastoma SH-SY5Y cells.

    PubMed

    Lyon, Robert C; Li, Dan; McGarvie, Gail; Ellis, Elizabeth M

    2013-01-01

    Reactive aldehydes including methyl glyoxal, acrolein and 4-hydroxy-2-nonenal (4-HNE) have been implicated in the progression of neurodegenerative diseases. The reduction of aldehydes to alcohols by the aldo-keto reductase (AKR) family of enzymes may represent an important detoxication route within neuronal cells. In this study, the ability of AKR enzymes to protect human neuroblastoma SH-SY5Y cells against reactive aldehydes was assessed. Using gene-specific RNA interference (RNAi), we report that AKR7A2 makes a significant contribution to the reduction of methyl glyoxal in SH-SY5Y cells, with its knockdown altering the IC(50) from 410 to 25.8μM, and that AKR1C3 contributes to 4-HNE reduction, with its knockdown lowering the IC(50) from 1.25 to 0.58μM. In addition, we have shown that pretreatment of cells with sub-lethal concentrations of 4-HNE or methyl glyoxal leads to a significant increase in IC(50) when cells are exposed to higher concentrations of the toxic aldehyde. The IC(50) for methyl glyoxal increased from 410μM to 1.9mM, and the IC(50) for 4-HNE increased from 120 to 690nM. To investigate this protection, we show that pretreatment of cells with the AKR inhibitor sorbinil lead to decreased resistance to aldehydes. We show that AKR1C can be induced 8-fold in SH-SY5Y cells by treatment with sub-lethal concentrations of methyl glyoxal, and 5-fold by 4-HNE treatment. AKR1B is not induced by methyl glyoxal but is induced 10-fold by 4-HNE treatment. Furthermore, we have shown that this adaptive response can also be induced using the chemoprotective agent tert-butyl hydroquinone (t-BHQ), and that this also evokes an increase in the expression and activity of AKR1B and AKR1C. These findings highlight the potential for the interventional upregulation of AKR via non-toxic derivatives or natural compounds as a novel therapeutic approach towards the detoxication of aldehydes, with the aim of halting the progression of aldehyde-dependent neurodegenerative

  5. The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

    PubMed Central

    Auer, Simon; Rinnerthaler, Mark; Bischof, Johannes; Streubel, Maria Karolin; Breitenbach-Koller, Hannelore; Geisberger, Roland; Aigner, Elmar; Cadamuro, Janne; Richter, Klaus; Sopjani, Mentor; Haschke-Becher, Elisabeth; Felder, Thomas Klaus; Breitenbach, Michael

    2017-01-01

    NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis. PMID:28620580

  6. Does MW Radiation Affect Gene Expression, Apoptotic Level, and Cell Cycle Progression of Human SH-SY5Y Neuroblastoma Cells?

    PubMed

    Kayhan, Handan; Esmekaya, Meric Arda; Saglam, Atiye Seda Yar; Tuysuz, Mehmed Zahid; Canseven, Ayşe Gulnihal; Yagci, Abdullah Munci; Seyhan, Nesrin

    2016-06-01

    Neuroblastoma (NB) is a cancer that occurs in sympathetic nervous system arising from neuroblasts and nerve tissue of the adrenal gland, neck, chest, or spinal cord. It is an embryonal malignancy and affects infants and children. In this study, we investigated the effects of microwave (MW) radiation on apoptotic activity, cell viability, and cell cycle progression in human SH-SY5Y NB cells which can give information about MW radiation effects on neural cells covering the period from the embryonic stages to infants. SH-SY5Y NB cells were exposed to 2.1 GHz W-CDMA modulated MW radiation for 24 h at a specific absorption rate of 0.491 W/kg. Control samples were in the same conditions with MW-exposed samples but they were not exposed to MW radiation. The apoptotic activity of cells was measured by Annexin-V-FITC and propidium iodide staining. Moreover, mRNA levels of proliferative and cell cycle proteins were determined by real-time RT-PCR. The change in cell cycle progression was observed by using CycleTest-Plus DNA reagent. No significant change was observed in apoptotic activity of MW-exposed cells compared to control cells. The mRNA levels of c-myc and cyclin D1 were significantly reduced in MW group (p < 0.05). The percentage of MW-exposed cells in G1 phase was significantly higher than the percentage of control cells in G1 phase. MW radiation caused cell cycle arrest in G1 phase. These results showed that 2.1 GHz W-CDMA modulated MW radiation did not cause apoptotic cell death but changed cell cycle progression.

  7. Pinocembrin Attenuates Mitochondrial Dysfunction in Human Neuroblastoma SH-SY5Y Cells Exposed to Methylglyoxal: Role for the Erk1/2-Nrf2 Signaling Pathway.

    PubMed

    de Oliveira, Marcos Roberto; Peres, Alessandra; Ferreira, Gustavo Costa

    2017-04-01

    Pinocembrin (PB; 5,7-dihydroxyflavanone) is found in propolis and exhibits antioxidant activity in several experimental models. The antioxidant capacity of PB is associated with the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. The Nrf2/ARE axis mediates the expression of antioxidant and detoxifying enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), heme oxygenase-1 (HO-1), and the catalytic (GCLC) and regulatory (GCLM) subunits of the rate-limiting enzyme in the synthesis of glutathione (GSH), γ-glutamate-cysteine ligase (γ-GCL). Nonetheless, it is not clear how PB exerts mitochondrial protection in mammalian cells. Human neuroblastoma SH-SY5Y cells were pretreated (4 h) with PB (0-25 µM) and then exposed to methylglyoxal (MG; 500 µM) for further 24 h. Mitochondria were isolated by differential centrifugation. PB (25 µM) provided mitochondrial protection (decreased lipid peroxidation, protein carbonylation, and protein nitration in mitochondrial membranes; decreased mitochondrial free radical production; enhanced the content of GSH in mitochondria; rescued mitochondrial membrane potential-MMP) and blocked MG-triggered cell death by a mechanism dependent on the activation of the extracellular-related kinase (Erk1/2) and consequent upregulation of Nrf2. PB increased the levels of GPx, GR, HO-1, and mitochondrial GSH. The PB-induced effects were suppressed by silencing of Nrf2 with siRNA. Therefore, PB activated the Erk1/2-Nrf2 signaling pathway resulting in mitochondrial protection in SH-SY5Y cells exposed to MG. Our work shows that PB is a strong candidate to figure among mitochondria-focusing agents with pharmacological potential.

  8. Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways.

    PubMed

    Um, Moonkyoung; Lodish, Harvey F

    2006-03-03

    The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.

  9. L-BMAA induced ER stress and enhanced caspase 12 cleavage in human neuroblastoma SH-SY5Y cells at low nonexcitotoxic concentrations.

    PubMed

    Okle, Oliver; Stemmer, Kerstin; Deschl, Ulrich; Dietrich, Daniel R

    2013-01-01

    The cyanobacterial β-N-methylamino-L-alanine (L-BMAA) is described as a low-potency excitotoxin, possibly a factor in the increased incidence of amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) in Guam. The latter association is intensively disputed, as L-BMAA concentrations required for toxic effects exceed those assumed to occur via food. The question thus was raised whether L-BMAA leads to neurodegeneration at nonexcitotoxic conditions. Using human SH-SY5Y neuroblastoma cells, L-BMAA-transport, incorporation into proteins, and subsequent impairment of cellular protein homeostasis were investigated. Binding of L-BMAA to intracellular proteins, but no clear protein incorporation was detected in response to (14)C-L-BMAA exposures. Nevertheless, low L-BMAA concentrations (≥ 0.1mM, 48 h) increased protein ubiquitination, 20S proteasomal and caspase 12 activity, expression of the endoplasmic reticulum (ER) stress marker CHOP, and enhanced phosphorylation of elf2α in SH-SY5Y cells. In contrast, high L-BMAA concentrations (≥ 1mM, 48 h) increased reactive oxygen species and protein oxidization, which were partially ameliorated by coincubation with vitamin E. L-BMAA-mediated cytotoxicity was observable 48 h following ≥ 2mM L-BMAA treatment. Consequently, the data presented here suggest that low L-BMAA concentrations result in a dysregulation of the cellular protein homeostasis with ensuing ER stress that is independent from high-concentration effects such as excitotoxicity and oxidative stress. Thus, the latter could be a contributing factor in the onset and slow progression of ALS/PDC in Guam.

  10. Copper brain protein protection against free radical-induced neuronal death: Survival ratio in SH-SY5Y neuroblastoma cell cultures.

    PubMed

    Deloncle, Roger; Fauconneau, Bernard; Guillard, Olivier; Delaval, José; Lesage, Gérard; Pineau, Alain

    2017-01-01

    In Creutzfeldt Jakob, Alzheimer and Parkinson diseases, copper metalloproteins such as prion, amyloid protein precursor and α-synuclein are able to protect against free radicals by reduction from cupric Cu(+2) to cupreous Cu(+). In these pathologies, a regional copper (Cu) brain decrease correlated with an iron, zinc or manganese (Mn) increase has previously been observed, leading to local neuronal death and abnormal deposition of these metalloproteins in β-sheet structures. In this study we demonstrate the protective effect of Cu metalloproteins against deleterious free-radical effects. With neuroblastoma SH-SY5Y cell cultures, we show that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY5Y cells has been measured after UV irradiation (free radical production), when the incubating medium is supplemented with bovine brain homogenate in native, Cu or Mn forms. This ratio, about 28% without any addition or with bovine brain protein added in Mn form, increases by as much as 54.73% with addition to the culture medium of native bovine brain protein and by as much as 95.95% if the addition is carried out in cupric form. This protective effect of brain copper protein against free radical-induced neuronal death has been confirmed with Inductively Coupled Plasma Mass Spectrometry Mn and Cu measurement in bovine brain homogenates: respectively lower than detection limit and 9.01μg/g dry weight for native form; lower than detection limit and 825.85μg/g dry weight for Cu-supplemented form and 1.75 and 68.1μg/g dry weight in Mn-supplemented brain homogenate. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Muscarinic receptor stimulation of D-aspartate uptake into human SH-SY5Y neuroblastoma cells is attenuated by hypoosmolarity.

    PubMed

    Foster, Daniel J; Heacock, Anne M; Fisher, Stephen K

    2010-04-01

    In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250-300% of control) in the uptake of D-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in d-aspartate uptake were potently blocked by the EAAT3 inhibitor l-beta-threo-benzyl-aspartate. In hypotonic media, the ability of mAChR activation to facilitate D-aspartate uptake was significantly attenuated (40-50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated D-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in D-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca2+. In contrast, dependence on extracellular Ca2+ was observed only under isotonic conditions. The results suggest that, although the uptake of D-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity.

  12. Muscarinic Receptor Stimulation of d-Aspartate Uptake into Human SH-SY5Y Neuroblastoma Cells Is Attenuated by Hypoosmolarity

    PubMed Central

    Foster, Daniel J.; Heacock, Anne M.

    2010-01-01

    In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as d-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250–300% of control) in the uptake of d-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in d-aspartate uptake were potently blocked by the EAAT3 inhibitor l-β-threo-benzyl-aspartate. In hypotonic media, the ability of mAChR activation to facilitate d-aspartate uptake was significantly attenuated (40–50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated d-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in d-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca2+. In contrast, dependence on extracellular Ca2+ was observed only under isotonic conditions. The results suggest that, although the uptake of d-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity. PMID:20080957

  13. Impact of inhomogeneous static magnetic field (31.7-232.0 mT) exposure on human neuroblastoma SH-SY5Y cells during cisplatin administration.

    PubMed

    Vergallo, Cristian; Ahmadi, Meysam; Mobasheri, Hamid; Dini, Luciana

    2014-01-01

    Beneficial or adverse effects of Static Magnetic Fields (SMFs) are a large concern for the scientific community. In particular, the effect of SMF exposure during anticancer therapies still needs to be fully elucidated. Here, we evaluate the effects of SMF at induction levels that cisPt-treated cancer patients experience during the imaging process conducted in Low field (200-500 mT), Open field (300-700 mT) and/or inhomogeneous High field (1.5-3 T) Magnetic Resonance Imaging (MRI) machines. Human adrenergic neuroblastoma SH-SY5Y cells treated with 0.1 µM cisPt (i.e. the lowest concentration capable of inducing apoptosis) were exposed to SMF and their response was studied in vitro. Exposure of 0.1 µM cisPt-treated cells to SMF for 2 h decreased cell viability (30%) and caused overexpression of the apoptosis-related cleaved caspase-3 protein (46%). Furthermore, increase in ROS (Reactive Oxygen Species) production (23%) and reduction in the number of mitochondria vs controls were seen. The sole exposure of SMF for up to 24 h had no effect on cell viability but increased ROS production and modified cellular shape. On the other hand, the toxicity of cisPt was significantly prevented during 24 h exposure to SMF as shown by the levels of cell viability, cleaved caspase-3 and ROS production. In conclusion, due to the cytoprotective effect of 31.7-232.0 mT SMF on low-cisPt-concentration-treated SH-SY5Y cells, our data suggest that exposure to various sources of SMF in cancer patients under a cisPt regimen should be strictly controlled.

  14. Phytochemical Ginkgolide B Attenuates Amyloid-β1-42 Induced Oxidative Damage and Altered Cellular Responses in Human Neuroblastoma SH-SY5Y Cells.

    PubMed

    Gill, Iqbal; Kaur, Sukhchain; Kaur, Navrattan; Dhiman, Monisha; Mantha, Anil K

    2017-02-20

    Oxidative stress is an upsurge in reactive oxygen/nitrogen species (ROS/RNS), which aggravates damage to cellular components viz. lipids, proteins, and nucleic acids resulting in impaired cellular functions and neurological pathologies including Alzheimer's disease (AD). In the present study, we have examined amyloid-β (Aβ)-induced oxidative stress responses, a major cause for AD, in the undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Aβ1 - 42-induced oxidative damage was evaluated on lipids by lipid peroxidation; proteins by protein carbonyls; antioxidant status by SOD and GSH enzyme activities; and DNA and RNA damage levels by evaluating the number of AP sites and 8-oxo-G base damages produced. In addition, the neuro-protective role of the phytochemical ginkgolide B (GB) in countering Aβ1 - 42-induced oxidative stress was assessed. We report that the differentiated cells are highly vulnerable to Aβ1 - 42-induced oxidative stress events as exerted by the deposition of Aβ in AD. Results of the current study suggest that the pre-treatment of GB, followed by Aβ1 - 42 treatment for 24 h, displayed neuro-protective potential, which countered Aβ1 - 42-induced oxidative stress responses in both undifferentiated and differentiated SH-SY5Y neuronal cells by: 1) hampering production of ROS and RNS; 2) reducing lipid peroxidation; 3) decreasing protein carbonyl content; 4) restoring antioxidant activities of SOD and GSH enzymes; and 5) maintaining genome integrity by reducing the oxidative DNA and RNA base damages. In conclusion, Aβ1 - 42 induces oxidative damage to the cellular biomolecules, which are associated with AD pathology, and are protected by the pre-treatment of GB against Aβ-toxicity. Taken together, this study advocates for phytochemical-based therapeutic interventions against AD.

  15. TCDD-induced mitochondrial superoxide production does not lead to mitochondrial degeneration or genomic instability in human SH-SY5Y neuroblastoma cells.

    PubMed

    Luukkonen, Jukka; Höytö, Anne; Viluksela, Matti; Juutilainen, Jukka; Naarala, Jonne

    2017-10-01

    Several genotoxic and non-genotoxic agents have been reported to cause delayed genetic damage in the progeny of the exposed cells. Such induced genomic instability (IGI) may be a driving force in carcinogenesis, and it is thus highly important to understand the cellular events accompanying it. The aim of this study was to investigate whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects mitochondrial integrity and can consequently induce genomic instability. Mitochondrial integrity was evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. Micronucleus formation was used to assess immediate genetic damage and IGI. The assays were performed either immediately, 8 or 15d after the exposure. Mitochondrial superoxide production was increased by TCDD immediately after the exposure. No consistent effects on mitochondrial integrity were observed at later time points, although slightly decreased mitochondrial membrane potential at 8d and increased mitochondrial superoxide potential production at 15 after exposure were observed in the TCDD-exposed cells. TCDD did not cause immediate genetic damage, and significant IGI was not observed. In conclusion, the present results suggest that immediate TCDD-induced increase in mitochondrial superoxide level does not lead to persistent loss of mitochondrial integrity or IGI in human SH-SY5Y neuroblastoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells

    PubMed Central

    2013-01-01

    Background Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. Methods Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. Results Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC50 121.3 ± 6.97 μg/ml, while S. puniceus methanol extract was the most cytotoxic; LC50 20.75 ± 1.47 μg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. Conclusion The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties. PMID:24330357

  17. Ginsenosides Rd and Re co-treatments improve rotenone-induced oxidative stress and mitochondrial impairment in SH-SY5Y neuroblastoma cells.

    PubMed

    González-Burgos, E; Fernández-Moriano, Carlos; Lozano, R; Iglesias, I; Gómez-Serranillos, M P

    2017-08-23

    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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

    PubMed Central

    FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

    2016-01-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  19. Blood-Brain Barrier Permeability of Green Tea Catechin Metabolites and their Neuritogenic Activity in Human Neuroblastoma SH-SY5Y Cells.

    PubMed

    Unno, Keiko; Pervin, Monira; Nakagawa, Aimi; Iguchi, Kazuaki; Hara, Aya; Takagaki, Akiko; Nanjo, Fumio; Minami, Akira; Nakamura, Yoriyuki

    2017-09-11

    To understand the mechanism by which green tea lowers the risk of dementia, focus was placed on the metabolites of epigallocatechin gallate (EGCG), the most abundant catechin in green tea. Much of orally ingested EGCG is hydrolyzed to epigallocatechin (EGC) and gallic acid. In rats, EGC is then metabolized mainly to 5-(3', 5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5) and its conjugated forms, which are distributed to various tissues. Therefore, we examined the permeability of these metabolites into the blood-brain barrier (BBB), and nerve cell proliferation/differentiation in vitro. The permeability of EGC-M5, glucuronide and the sulfate of EGC-M5, pyrogallol, as well as its glucuronide into the BBB were examined using a BBB model kit. Each brain- and blood-side sample was subjected to liquid chromatography tandem-mass spectrometry analysis. BBB permeability (%, in 0.5 h) was 1.9-3.7 %. In human neuroblastoma SH-SY5Y cells, neurite length was significantly prolonged by EGC-M5, and the number of neurites was increased significantly by all metabolites examined. The permeability of EGC-M5 and its conjugated forms into the BBB suggests that they reached the brain parenchyma. In addition, the ability of EGC-M5 to affect nerve cell proliferation and neuritogenesis suggests that EGC-M5 may promote neurogenesis in the brain. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Modulation of voltage-gated ion channels on SH-SY5Y neuroblastoma by non-ionic surfactant, Cremophor EL.

    PubMed

    Noguchi, Tomohiro; Kamiyama, Naoya; Kashiwayanagi, Makoto

    2010-01-01

    Cremophor EL (CrEL) is a non-ionic surfactant widely used as a vehicle for insoluble drugs, including immunosuppressive and anticancer agents. Although CrEL has often been reported to induce sensory neuropathies, its action on voltage-gated ion channels remains unknown. We show here that CrEL modulates voltage-gated sodium current (INa) and potassium current (IK) of human neuroblastoma cells (SH-SY5Y). First, CrEL suppressed the amplitude of INa and that of IK. The suppression-concentration curve for INa was gradual but that for IK was steeper, indicating that INa remains incompletely blocked by high concentrations of CrEL, which greatly reduce IK. Thus, it is possible that CrEL paradoxically increases neuronal excitability at higher concentrations. Next, CrEL accelerated IK's inactivation process. The voltage-dependent inactivation of IK showed two time constants, τ(f) of 322±49 ms and τ(s) of 2925±184 ms, under the control condition. By applying 1000 ppm CrEL, three time constants-τ(u) of 23±2 ms, τ(f) of 196±19 ms, and τ(s) of 1396±127 ms-appeared in the inactivation process. This modified inactivation of IK probably disturbs the repolarizing phases of action potentials. These modulations of voltage-gated ion channels by CrEL may cause abnormal excitability involved in neuropathies.

  1. Differentiation impairs Bach1 dependent HO-1 activation and increases sensitivity to oxidative stress in SH-SY5Y neuroblastoma cells.

    PubMed

    Piras, Sabrina; Furfaro, Anna Lisa; Brondolo, Lorenzo; Passalacqua, Mario; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Nitti, Mariapaola

    2017-08-08

    Neuronal adaptation to oxidative stress is crucially important in order to prevent degenerative diseases. The role played by the Nrf2/HO-1 system in favoring cell survival of neuroblastoma (NB) cells exposed to hydrogen peroxide (H2O2) has been investigated using undifferentiated or all-trans retinoic acid (ATRA) differentiated SH-SY5Y cells. While undifferentiated cells were basically resistant to the oxidative stimulus, ATRA treatment progressively decreased cell viability in response to H2O2. HO-1 silencing decreased undifferentiated cell viability when exposed to H2O2, proving the role of HO-1 in cell survival. Conversely, ATRA differentiated cells exposed to H2O2 showed a significantly lower induction of HO-1, and only the supplementation with low doses of bilirubin (0,5-1 μM) restored viability. Moreover, the nuclear level of Bach1, repressor of HO-1 transcription, strongly decreased in undifferentiated cells exposed to oxidative stress, while did not change in ATRA differentiated cells. Furthermore, Bach1 was displaced from HO-1 promoter in undifferentiated cells exposed to H2O2, enabling the binding of Nrf2. On the contrary, in ATRA differentiated cells treated with H2O2, Bach1 displacement was impaired, preventing Nrf2 binding and limiting HO-1 transcription. In conclusion, our findings highlight the central role of Bach1 in HO-1-dependent neuronal response to oxidative stress.

  2. TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

    PubMed Central

    Daniele, Federica; Di Cairano, Eliana S.; Moretti, Stefania; Piccoli, Giovanni; Perego, Carla

    2015-01-01

    Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease. Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events. In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented. To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle

  3. The large conductance Ca2+ -activated K+ (BKCa) channel regulates cell proliferation in SH-SY5Y neuroblastoma cells by activating the staurosporine-sensitive protein kinases

    PubMed Central

    Curci, Angela; Mele, Antonietta; Camerino, Giulia Maria; Dinardo, Maria Maddalena; Tricarico, Domenico

    2014-01-01

    Here we investigated on the role of the calcium activated K+-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K+-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K+-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was −33 mV. IbTX(10−8 – 4 × 10−7 M) reduced the K+-current at +30 mV with an IC50 of 1.85 × 10−7 M and an Imax of −46% (slope = 2.198) (n = 21). NS1619(10–100 × 10−6 M) enhanced the K+-current of +141% (n = 6), at −10 mV(Vm). TEA(10−5–10−3 M) reduced the K+-current with an IC50 of 3.54 × 10−5 M and an Imax of −90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10−4 M). IbTX showed an MPE of +42% at 10−8 M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10−6 M) was +42%. The PKC inhibitor staurosporine (0.2–2 × 10−6 M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10−9 M), TEA (100 × 10−6 M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases. PMID:25538629

  4. TIRFM and pH-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: cell imaging and data analysis.

    PubMed

    Daniele, Federica; Di Cairano, Eliana S; Moretti, Stefania; Piccoli, Giovanni; Perego, Carla

    2015-01-29

    Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease. Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events. In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented. To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle

  5. The large conductance Ca(2+) -activated K(+) (BKCa) channel regulates cell proliferation in SH-SY5Y neuroblastoma cells by activating the staurosporine-sensitive protein kinases.

    PubMed

    Curci, Angela; Mele, Antonietta; Camerino, Giulia Maria; Dinardo, Maria Maddalena; Tricarico, Domenico

    2014-01-01

    Here we investigated on the role of the calcium activated K(+)-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K(+)-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K(+)-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was -33 mV. IbTX(10(-8) - 4 × 10(-7) M) reduced the K(+)-current at +30 mV with an IC50 of 1.85 × 10(-7) M and an Imax of -46% (slope = 2.198) (n = 21). NS1619(10-100 × 10(-6) M) enhanced the K(+)-current of +141% (n = 6), at -10 mV(Vm). TEA(10(-5)-10(-3) M) reduced the K(+)-current with an IC50 of 3.54 × 10(-5) M and an Imax of -90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10(-4) M). IbTX showed an MPE of +42% at 10(-8) M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10(-6) M) was +42%. The PKC inhibitor staurosporine (0.2-2 × 10(-6) M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10(-9) M), TEA (100 × 10(-6) M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases.

  6. Transglutaminase-2 Is Involved in All-Trans Retinoic Acid-Induced Invasion and Matrix Metalloproteinases Expression of SH-SY5Y Neuroblastoma Cells via NF-κB Pathway

    PubMed Central

    Lee, Hye Ja; Park, Mi Kyung; Bae, Hyun Cheol; Yoon, Hee Jung; Kim, Soo Youl; Lee, Chang Hoon

    2012-01-01

    All-trans retinoic acid (ATRA) is currently used in adjuvant differentiation-based treatment of residual or relapsed neuroblastoma (NB). It has been reported that short-term ATRA treatment induces migration and invasion of SH-SY5Y via transglutaminase-2 (Tgase-2). However, the detailed mechanism of Tgase-2's involvement in NB cell invasion remains unclear. Therefore we investigated the role of Tgase-2 in invasion of NB cells using SH-SY5Y cells. ATRA dose-dependently induced the invasion of SH-SY5Y cells. Cystamine (CTM), a well known tgase inhibitor suppressed the ATRA-induced invasion of SH-SY5Y cells in a dose-dependent manner. Matrix metalloproteinase-9 (MMP-9) and MMP-2, well known genes involved in invasion of cancer cells were induced in the ATRA-induced invasion of the SH-SH5Y cells. Treatment of CTM suppressed the MMP-9 and MMP-2 enzyme activities in the ATRA-induced invasion of the SH-SY5Y cells. To confirm the involvement of Tgase-2, gene silencing of Tgase-2 was performed in the ATRA-induced invasion of the SH-SH5Y cells. The siRNA of Tgase-2 suppressed the MMP-9 and MMP-2 activity of the SH-SY5Y cells. MMP-2 and MMP-9 are well known target genes of NF-κB. Therefore the relationship of Tgase-2 and NF-κB in the ATRA-induced invasion of the SH-SY5Y cells was examined using siRNA and CTM. ATRA induced the activation of NF-κB in the SH-SY5Y cells and CTM suppressed the activation of NF-κB. Gene silencing of Tgase-2 suppressed the MMP expression by ATRA. These results suggested that Tgase-2 might be a new target for controlling the ATRA-induced invasion of NBs. PMID:24130925

  7. The Interactions Between Aligned Poly(L-Lactic Acid) Nanofibers and SH-SY5Y Cells In Vitro.

    PubMed

    Yu, Yadong; Meng, Dianhuai; Man, Lili; Wang, Xin

    2016-06-01

    Aligned nanofibers have been regarded as promising nanomaterials in facilitating nerve regeneration. Investigating the interactions between aligned nanofibers and neuronal cells will be critically important for the design and application of aligned nanofibers in nerve tissue engineering. In this study, we explored the effects of electrospun Poly(L-Lactic Acid) (PLLA) aligned nanofibers on SH-SY5Y cells (a type of human neuroblastoma cell line) and specifically focused on the role of integrin in the PLLA aligned nanofiber-SH-SY5Y cell interaction. We found that PLLA aligned nanofibers could significantly guide the neurite outgrowth of SH-SY5Y cell, and promote the viability, proliferation, glucose and lactic acid metabolism of SH-SY5Y cell. This promotion effect could be alleviated when the functions of integrins on the SH-SY5Y cell membrane were hampered by pentapeptide GRGDS. Moreover, we found that PLLA aligned nanofibers could enhance the expression of phosphorylated-ERK1/2 (p-ERK1/2) in the SH-SY5Y cells and blocking the integrins would decrease p-ERK1/2 expression. These results suggested that PLLA aligned nanofibers might affect many cellular behaviors of SH-SY5Y cells via integrin mediated ERK pathway. Our findings provided more insights for understanding the interaction between aligned nanofibers and neuronal cells.

  8. Induction of genomic instability, oxidative processes, and mitochondrial activity by 50Hz magnetic fields in human SH-SY5Y neuroblastoma cells.

    PubMed

    Luukkonen, Jukka; Liimatainen, Anu; Juutilainen, Jukka; Naarala, Jonne

    2014-02-01

    Epidemiological studies have suggested that exposure to 50Hz magnetic fields (MF) increases the risk of childhood leukemia, but there is no mechanistic explanation for carcinogenic effects. In two previous studies we have observed that a 24-h pre-exposure to MF alters cellular responses to menadione-induced DNA damage. The aim of this study was to investigate the cellular changes that must occur already during the first 24h of exposure to MF, and to explore whether the MF-induced changes in DNA damage response can lead to genomic instability in the progeny of the exposed cells. In order to answer these questions, human SH-SY5Y neuroblastoma cells were exposed to a 50-Hz, 100-μT MF for 24h, followed by 3-h exposure to menadione. The main finding was that MF exposure was associated with increased level of micronuclei, used as an indicator of induced genomic instability, at 8 and 15d after the exposures. Other delayed effects in MF-exposed cells included increased mitochondrial activity at 8d, and increased reactive oxygen species (ROS) production and lipid peroxidation at 15d after the exposures. Oxidative processes (ROS production, reduced glutathione level, and mitochondrial superoxide level) were affected by MF immediately after the exposure. In conclusion, the present results suggest that MF exposure disturbs oxidative balance immediately after the exposure, which might explain our previous findings on MF altered cellular responses to menadione-induced DNA damage. Persistently elevated levels of micronuclei were found in the progeny of MF-exposed cells, indicating induction of genomic instability.

  9. Calcipotriol inhibits α-synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28k-dependent mechanism.

    PubMed

    Rcom-H'cheo-Gauthier, Alexandre N; Meedeniya, Adrian C B; Pountney, Dean L

    2017-04-01

    Many neurodegenerative diseases are characterized by the formation of microscopically visible intracellular protein aggregates. α-Synuclein is the key aggregating protein in Parkinson's disease which is characterized by neuronal cytoplasmic Lewy body inclusions. Previous studies have shown relative sparing of neurons in Parkinson's disease and dementia with Lewy bodies that are positive for the vitamin D-dependent calcium-buffering protein, calbindin-D28k, and that α-synuclein aggregates are excluded from calbindin-D28k-positive neurons. Recent cell culture studies have shown that α-synuclein aggregation can be induced by raised intracellular-free Ca(II) and demonstrated that raised intracellular calcium and oxidative stress can act synergistically to promote α-synuclein aggregation. We hypothesized that calcipotriol, a potent vitamin D analogue used pharmaceutically, may be able to suppress calcium-dependent α-synuclein aggregation by inducing calbindin-D28k expression. Immunofluorescence and western blot analysis showed that calcipotriol potently induced calbindin-D28k in a dose-dependent manner in SH-SY5Y human neuroblastoma cells. Calcipotriol significantly decreased the frequency of α-synuclein aggregate positive cells subjected to treatments that cause raised intracellular-free Ca(II) (potassium depolarization, KCl/H2 O2 combined treatment, and rotenone) in a dose-dependent manner and increased viability. Suppression of calbindin-D28k expression in calcipotriol-treated cells using calbindin-D28k-specific siRNA showed significantly higher α-synuclein aggregation levels, indicating that calcipotriol-mediated blocking of calcium-dependent α-synuclein aggregation was dependent on the induction of calbindin-D28k expression. These data indicate that targeting raised intraneuronal-free Ca(II) in the brain by promoting the expression of calbindin-D28k at the transcriptional level using calcipotriol could prevent α-synuclein aggregate formation and ameliorate

  10. Rebamipide reduces amyloid-β 1-42 (Aβ42) production and ameliorates Aβ43-lowered cell viability in cultured SH-SY5Y human neuroblastoma cells.

    PubMed

    Fukui, Kenta; Yachi, Kazuma; Yoshida, Hidemi; Tanji, Kunikazu; Matsumiya, Tomoh; Hayakari, Ryo; Tsuruga, Kazushi; Tanaka, Hiroshi; Imaizumi, Tadaatsu

    2017-06-03

    Amyloid-beta (Aβ) peptides, Aβ 1-42 (Aβ42) and Aβ43, in particular, have been implicated in the pathophysiology of neurodegenerative disease such as Alzheimer's disease (AD). Rebamipide (REB), a gastrointestinal protective drug, can cross the blood-brain barrier after oral administration; however, the effects of REB on neuronal cells have not yet been reported. In this study, we investigated the effects of REB on Aβ43-induced cytotoxicity (monomers, 10μM) in cultured SH-SY5Y human neuroblastoma cells. Addition of REB (10-1000nM) into the media partially ameliorated the reduced cell viability observed after Aβ43 treatment, which was determined by the MTT assay. REB reduced the levels of intracellular Aβ oligomers (100-150kDa) that were formed from the exogenous addition of Aβ43 monomers. In addition, REB (30nM) reduced endogenous Aβ42 secretion, which was analyzed by the enzyme-linked immunosorbent assay. Furthermore, REB enhanced the expression of tumor necrosis factor-α-converting enzyme/a disintegrin and metalloproteinase-17, neprilysin, matrix-metalloproteinase-14 (MMP-14)/membrane type-1 MMP, cyclooxygenase-2, and sirtuin 1, even in cells challenged with Aβ43. These results suggest that REB improves the cell viability by inducing genes that regulate Aβ levels and also genes that are cytoprotective. The secondary use of REB may have potential in the prevention of Aβ-mediated diseases, particularly AD. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  11. Evaluation of the Protective Effects of Sarains on H2O2-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Neuroblastoma Cells.

    PubMed

    Alvariño, Rebeca; Alonso, Eva; Tribalat, Marie-Aude; Gegunde, Sandra; Thomas, Olivier P; Botana, Luis M

    2017-05-06

    Sarains are diamide alkaloids isolated from the Mediterranean sponge Haliclona (Rhizoniera) sarai that have previously shown antibacterial, insecticidal and anti-fouling activities. In this study, we examined for the first time the neuroprotective effects of sarains 1, 2 and A against oxidative stress in a human neuronal model. SH-SY5Y cells were co-incubated with sarains at concentrations ranging from 0.01 to 10 μM, and the well-known oxidant hydrogen peroxide at 150 μM for 6 h and the protective effects of the compounds were evaluated. Among the sarains tested, sarain A was the most promising compound, improving mitochondrial function and decreasing reactive oxygen species levels in human neuroblastoma cells treated with the compound at 0.01, 0.1 and 1 μM. This compound was also able to increase the activity of the antioxidant enzymes superoxide dismutases by inducing the translocation of the nuclear factor E2-related factor 2 (Nrf2) to the nucleus at the lower concentrations tested (0.01 and 0.1 μM). Moreover, sarain A at 0.1 and 1 μM blocked the mitochondrial permeability transition pore (mPTP) opening through cyclophilin D inhibition. These results suggest that the protective effects produced by the treatment with sarain A are related with its ability to block the mPTP and to enhance the Nrf2 pathway, indicating that sarain A may be a candidate compound for further studies in neurodegenerative diseases.

  12. Chelation of Lysosomal Iron Protects Dopaminergic SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide Toxicity by Precluding Autophagy and Akt Dephosphorylation

    PubMed Central

    Castino, Roberta; Fiorentino, Ilaria; Cagnin, Monica; Giovia, Antonino; Isidoro, Ciro

    2011-01-01

    In human neuroblastoma SH-SY5Y cells, hydrogen peroxide (H2O2, 200μM) rapidly (< 5 min) induced autophagy, as shown by processing and vacuolar relocation of light chain 3(LC3). Accumulation of autophagosome peaked at 30 min of H2O2 exposure. The continuous presence of H2O2 eventually (at > 60 min) caused autophagy-dependent annexin V–positive cell death. However, the cells exposed to H2O2 for 30 min and then cultivated in fresh medium could recover and grow, despite ongoing autophagy. H2O2 rapidly (5 min) triggered the formation of dichlorofluorescein-sensitive HO·-free radicals within mitochondria, whereas the mitochondria-associated oxidoradicals revealed by MitoSox (O2·−) became apparent after 30 min of exposure to H2O2. 3-Methyladenine inhibited autophagy and cell death, but not the generation of HO·. Genetic silencing of beclin-1 prevented bax- and annexin V–positive cell death induced by H2O2, confirming the involvement of canonical autophagy in peroxide toxicity. The lysosomotropic iron chelator deferoxamine (DFO) prevented the mitochondrial generation of both HO. and O2·− and suppressed the induction of autophagy and of cell death by H2O2. Upon exposure to H2O2, Akt was intensely phosphorylated in the first 30 min, concurrently with mammalian target of rapamycin inactivation and autophagy, and it was dephosphorylated at 2 h, when > 50% of the cells were dead. DFO did not impede Akt phosphorylation, which therefore was independent of reactive oxygen species (ROS) generation but inhibited Akt dephosphorylation. In conclusion, exogenous H2O2 triggers two parallel independent pathways, one leading to autophagy and autophagy-dependent apoptosis, the other to transient Akt phosphorylation, and both are inhibited by DFO. The present work establishes HO· as the autophagy-inducing ROS and highlights the need for free lysosomal iron for its production within mitochondria in response to hydrogen peroxide. PMID:21742779

  13. P(VDF-TrFE)/BaTiO3 Nanoparticle Composite Films Mediate Piezoelectric Stimulation and Promote Differentiation of SH-SY5Y Neuroblastoma Cells.

    PubMed

    Genchi, Giada Graziana; Ceseracciu, Luca; Marino, Attilio; Labardi, Massimiliano; Marras, Sergio; Pignatelli, Francesca; Bruschini, Luca; Mattoli, Virgilio; Ciofani, Gianni

    2016-07-01

    Poly(vinylidene fluoride-trifluoroethylene, P(VDF-TrFE)) and P(VDF-TrFE)/barium titanate nanoparticle (BTNP) films are prepared and tested as substrates for neuronal stimulation through direct piezoelectric effect. Films are characterized in terms of surface, mechanical, and piezoelectric features before in vitro testing on SH-SY5Y cells. In particular, BTNPs significantly improve piezoelectric properties of the films (4.5-fold increased d31 ). Both kinds of films support good SH-SY5Y viability and differentiation. Ultrasound (US) stimulation is proven to elicit Ca(2+) transients and to enhance differentiation in cells grown on the piezoelectric substrates. For the first time in the literature, this study demonstrates the suitability of polymer/ceramic composite films and US for neuronal stimulation through direct piezoelectric effect. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells*

    PubMed Central

    Acevedo, Karla M.; Opazo, Carlos M.; Norrish, David; Challis, Leesa M.; Li, Qiao-Xin; White, Anthony R.; Bush, Ashley I.; Camakaris, James

    2014-01-01

    Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells. PMID:24610780

  15. Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.

    PubMed

    Acevedo, Karla M; Opazo, Carlos M; Norrish, David; Challis, Leesa M; Li, Qiao-Xin; White, Anthony R; Bush, Ashley I; Camakaris, James

    2014-04-18

    Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

  16. The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide.

    PubMed

    Nopparat, Chutikorn; Chantadul, Varunya; Permpoonputtana, Kannika; Govitrapong, Piyarat

    2017-04-10

    Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H2O2)-induced inflammaging state in SH-SY5Y cells. Our data showed that p21(Cip1) and p16(INK4a), cell cycle arrest markers, and the number of senescence-associated β-galactosidase (SA-βgal) staining increased significantly in H2O2-treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H2O2 treated cells. Cells pretreated with H2O2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H2O2, could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H2O2. The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells.

  17. Carnosic Acid Protects Mitochondria of Human Neuroblastoma SH-SY5Y Cells Exposed to Paraquat Through Activation of the Nrf2/HO-1Axis.

    PubMed

    de Oliveira, Marcos Roberto; Peres, Alessandra; Ferreira, Gustavo Costa; Schuck, Patrícia Fernanda; Gama, Clarissa S; Bosco, Simone Morelo Dal

    2016-09-29

    Carnosic acid (CA; C20H28O4), which is also called salvin, is a major phenolic diterpene found in Rosmarinus officinalis L. and exhibits antioxidant, anti-inflammatory, and antiproliferative properties. CA activates the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, leading to the upregulation of antioxidant and phase II detoxification enzymes, such as heme oxygenase-1 (HO-1), glutathione reductase (GR), γ-glutamate-cysteine ligase (γ-GCL), and glutathione S-transferase (GST), among others. We have previously demonstrated that CA upregulates the total and mitochondrial synthesis of glutathione (GSH), causing mitochondrial protection against paraquat (PQ) and methylglyoxal (MG). Nonetheless, the complete mechanism by which CA prevented mitochondrial dysfunction was not clear yet. Here, we examine whether HO-1 would be involved in the CA-induced mechanism of mitochondrial protection in SH-SY5Y-treated cells. SH-SY5Y cells were pretreated with CA (1 μM) for 12 h prior to a challenge with PQ at 100 μM for additional 24 h. Zinc protoporphyrin IX (ZnPP IX; a specific inhibitor of HO-1; 10 μM) was utilized prior to exposure to CA in order to investigate whether HO-1 was involved in the cytoprotective effects elicited by CA. We found that the CA-induced Nrf2-dependent HO-1 upregulation ameliorated, at least in part, the mitochondrial function in PQ-treated cells. Therefore, CA protected mitochondria of SH-SY5Y cells and exerted anti-apoptotic effects by activating the Nrf2/HO-1 axis.

  18. An apolar Pistacia lentiscus L. leaf extract: GC-MS metabolic profiling and evaluation of cytotoxicity and apoptosis inducing effects on SH-SY5Y and SK-N-BE(2)C cell lines.

    PubMed

    Piccolella, Simona; Nocera, Paola; Carillo, Petronia; Woodrow, Pasqualina; Greco, Vincenza; Manti, Lorenzo; Fiorentino, Antonio; Pacifico, Severina

    2016-09-01

    In the course of a cytotoxicity screening of Mediterranean plants vs. neuroblastoma cells, Pistacia lentiscus was of interest. Pl-C extract, prepared from dried leaves by ultrasound assisted maceration (UAM) in chloroform, was profiled through using GC-MS techniques. To evaluate Pl-C cytotoxicity towards SH-SY5Y and SK-N-BE(2)-C cell lines, MTT, SRB and LDH assays were performed. The caspase-3 activation, DNA fragmentation, as well as micronucleation, were also evaluated. The Pl-C oxidant/antioxidant ability was estimated using different methods. The extract, rich in pentacyclic triterpenes, inhibited mitochondrial redox activity and cell viability of the tested cell lines. LDH assay established that Pl-C did not affect the cell membrane integrity. Indeed, it was able to activate caspase-3 and to cause a ladder pattern of DNA. Western blotting analysis showed that Pl-C processed caspase-3 providing two cleavage products of approximately 20 and 17-kDa, whose densitometric evaluation highlighted that Pl-C was more effective than vinblastine by 3-fold. The pro-apoptotic effect could be related to a disturbance in cell redox balance. In fact, it increased intracellular ROS production, GSSG/GSH ratio and the formation of lipoperoxidation products. The data obtained prompted to further investigate and assess the in vivo efficacy of Pl-C to prevent and/or treat neuroblastoma.

  19. Caffeoylquinic Acid Derivatives Protect SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide-Induced Injury Through Modulating Oxidative Status.

    PubMed

    Jiang, Xiao-Wen; Bai, Jun-Peng; Zhang, Qiao; Hu, Xiao-Long; Tian, Xing; Zhu, Jun; Liu, Jia; Meng, Wei-Hong; Zhao, Qing-Chun

    2017-04-01

    Oxidative stress has been confirmed as a contribution to the pathogenesis and pathophysiology of many neurological disorders such as Alzheimer's disease and Parkinson's disease. Caffeoylquinic acids (CQAs) are considered to have anti-oxidative stress ability in a previous study, but the structure-activity relationships (SARs) of CQAs in neuroprotective effects are still unclear. In the present study, we primarily expound the SARs of CQAs in counteracting H2O2-induced injury in SH-SY5Y cells. We found that CQAs (1-10) represented the protection of SH-SY5Y cells against H2O2-induced injury in varying degrees and malonyl groups could obviously increase the anti-oxidative stress ability of CQAs. Intensive studies of 4,5-O-dicaffeoyl-1-O-(malic acid methyl ester)-quinic acid (MDCQA) indicated that the mechanisms could potentially involve activation of endogenous antioxidant enzymes and the regulation of the phosphorylation of MAPKs and AKT. In conclusion, MDCQA could serve as a neuroprotective agent with a potential to attenuate oxidative stress.

  20. Palmitic Acid-Induced Neuron Cell Cycle G2/M Arrest and Endoplasmic Reticular Stress through Protein Palmitoylation in SH-SY5Y Human Neuroblastoma Cells

    PubMed Central

    Hsiao, Yung-Hsuan; Lin, Ching-I; Liao, Hsiang; Chen, Yue-Hua; Lin, Shyh-Hsiang

    2014-01-01

    Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs) in the brain. An increase in SFAs, especially palmitic acid (PA), triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER) stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer’s disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction. PMID:25402647

  1. Palmitic acid-induced neuron cell cycle G2/M arrest and endoplasmic reticular stress through protein palmitoylation in SH-SY5Y human neuroblastoma cells.

    PubMed

    Hsiao, Yung-Hsuan; Lin, Ching-I; Liao, Hsiang; Chen, Yue-Hua; Lin, Shyh-Hsiang

    2014-11-13

    Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs) in the brain. An increase in SFAs, especially palmitic acid (PA), triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER) stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer's disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction.

  2. TRAIL induces pro-apoptotic crosstalk between the TRAIL-receptor signaling pathway and TrkAIII in SH-SY5Y cells, unveiling a potential therapeutic “Achilles heel” for the TrkAIII oncoprotein in neuroblastoma

    PubMed Central

    Cappabianca, Lucia; Farina, Antonietta Rosella; Ianni, Natalia Di; Mackay, Andrew Reay

    2016-01-01

    TrkAIII expression in neuroblastoma (NB) associates with advanced stage disease, worse prognosis, post therapeutic relapse, and in NB models TrkAIII exhibits oncogenic activity and promotes chemotherapeutic-resistance. Here, we report a potential therapeutic “Achilles heel” for the TrkAIII oncoprotein in a SH-SY5Y NB model that is characterised by one-way TRAIL-induced, pro-apoptotic crosstalk between the TRAIL receptor signaling pathway and TrkAIII that results in the delayed induction of apoptosis. In TrkAIII SH-SY5Y cells, blocked in the intrinsic apoptosis pathway by elevated constitutive Bcl-2, Bcl-xL and Mcl-1 expression, TRAIL induced delayed caspase-dependent apoptosis via the extrinsic pathway and completely abrogated tumourigenic capacity in vitro. This effect was initiated by TRAIL-induced SHP-dependent c-Src activation, the induction of TrkAIII/SHP-1/c-Src complexing leading to SHP-mediated TrkAIII de-phosphorylation, subsequent induction of complexing between de-phosphorylated TrkAIII and cFLIP associated with a time-dependent increase the caspase-8 to cFLIP ratio at activated death receptors, resulting in delayed caspase cleavage and caspase-dependent apoptosis. We also confirm rate-limiting roles for c-FLIP and Mcl-1 in regulating the sensitivity of TrkAIII SH-SY5Y cells to TRAIL-induced apoptosis via the extrinsic and intrinsic pathways, respectively. Our study unveils a novel mechanism for the TRAIL-induced apoptosis of TrkAIII expressing NB cells that depends upon SHP/Src-mediated crosstalk between the TRAIL-receptor signaling pathway and TrkAIII, and supports a novel potential pro-apoptotic therapeutic use for TRAIL in TrkAIII expressing NB. PMID:27821809

  3. A cell culture model for investigation of synapse influenceability: epigenetics, expression and function of gene targets important for synapse formation and preservation in SH-SY5Y neuroblastoma cells differentiated by retinoic acid.

    PubMed

    Jahn, Kirsten; Wieltsch, C; Blumer, N; Mehlich, M; Pathak, H; Khan, A Q; Hildebrandt, H; Frieling, H

    2017-09-08

    SH-SY5Y neuroblastoma cells are frequently used for different neuronal cell culture models. As there is no "gold-standard", miscellaneous protocols exist to differentiate these cells into a neuronal cell type. Here, the aim was to find a differentiation condition making cells suitable for investigation of influenceability of synapses by environmental conditions in pharmacologic experiments. For this purpose, effects on synapse molecules should be somehow rateable and cells should be usable for functional analysis like calcium imaging. A system like this is desirable for example in basic research concerning schizophrenia, depression, autism or neurodegeneration as synaptic plasticity and neuronal maturation are known to have a significant impact in these diseases. Cells grown on laminin-coated glass cover slips and treated with 50 µM retinoic acid (RA) turned out to show most convincing morphological signs of neuronal differentiation and attached strongly to the ground, thereby also fulfilling preconditions for functional analysis. Systematic characterisation of this differentiation condition in comparison to non-treated controls revealed lower methylation rates and higher expression of most candidate molecules relevant for formation, preservation and function of synapses as well as differential function. In conclusion, this combination of differentiation strategy and markers seems to be a suitable system to estimate synapse modifications in basic research as it could help to identify possible dedifferentiating effects. To our knowledge, differentiation of SH-SY5Y has not been described as systematic before regarding comprehensiveness of the set of investigated synapse molecules and coverage of applied methods spanning from epigenetics to protein function. Furthermore, this is the first time that SH-SY5Y cells were differentiated on glass cover slips to an extent making them suitable for investigation of synapse molecules as part of stable intercellular

  4. Relative expression of the p75 neurotrophin receptor, tyrosine receptor kinase A, and insulin receptor in SH-SY5Y neuroblastoma cells and hippocampi from Alzheimer's disease patients.

    PubMed

    Ito, Shingo; Ménard, Michel; Atkinson, Trevor; Brown, Leslie; Whitfield, James; Chakravarthy, Balu

    2016-12-01

    We have previously shown in SH-SY5Y human neuroblastoma cells that the expressions of basal (75 kDa) and high molecular weight (HMW; 85 kDa) isoforms of the p75 neurotrophic receptor (p75NTR) are stimulated by amyloid-β peptide1-42 oligomers (AβOs) via the insulin-like growth factor-1 receptor (IGF-1R). On the other hand, it is known that AβOs inhibit insulin receptor (IR) signaling. The purpose of the present study was to determine the involvement of IR signaling in the regulation of p75 neurotrophin receptor (p75NTR) protein isoform expression in cultured SH-SY5Y cells and in hippocampi from late-stage human Alzheimer's disease (AD) brains. Interestingly, insulin induced the expression of basal and HMW p75NTR isoforms in SH-SY5Y cells, suggesting the presence of cross-talk between the IR and IGF-1R for the regulation of p75NTR expression. Reducing IR signaling with an IR kinase inhibitor (AG 1024) or IR-targeted siRNAs increased HMW p75NTR expression and reduced tyrosine receptor kinase-A (Trk-A) expression as well as postsynaptic density protein 95 (PSD95) expression in SH-SY5Y cells. Both basal and HMW p75NTR isoforms were increased in the hippocampi of post-mortem late-stage human AD brains (relative to non-AD brains), and the protein expression of HMW p75NTR was negatively associated with Trk-A expression, PSD95 expression, and IR expression. Thus, increased p75NTR expression, specifically an increased p75NTR-to-Trk-A ratio, is likely to play a role in synaptic loss and neuronal cell death in late-stage AD. Collectively, these findings suggest that increased expression of the p75NTR due to IR signaling inhibition by AβOs might be involved in the pathology of AD.

  5. SAG protects human neuroblastoma SH-SY5Y cells against 1-methyl-4-phenylpyridinium ion (MPP+)-induced cytotoxicity via the downregulation of ROS generation and JNK signaling.

    PubMed

    Kim, Sun-Yee; Kim, Mi-Yeon; Mo, Jung-Soon; Park, Jeen-Woo; Park, Hee-Sae

    2007-02-14

    Sensitive to apoptosis gene (SAG), a novel zinc RING finger protein, exhibits anti-apoptotic and antioxidant activity against a variety of redox reagents. In the present study, we have determined that SAG suppresses 1-methyl-4-phenylpyridinium ion (MPP(+))-induced neurotoxicity via the downregulation of ROS generation and c-Jun N-terminal kinase 1 (JNK1) activity. Both transient and constitutively overexpressed SAG were found to inhibit the MPP(+)-induced neurotoxicity of SH-SY5Y neuroblastoma cells. In the SAG-expressing cells, MPP(+) induced ROS generation was suppressed to a significant degree as compared to the cells treated only with MPP(+). MPP(+)-induced JNK1 activation was also determined to be suppressed markedly by SAG. Furthermore, SAG inhibits MEKK1 dependent c-Jun transcription activity in SH-SY5Y cells. Thus, we concluded that SAG is a cellular protective molecule, which appears to function as an antioxidant, suppressing MPP(+)-induced neurotoxicity.

  6. Alpha-synuclein-induced oxidative stress correlates with altered superoxide dismutase and glutathione synthesis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Perfeito, Rita; Ribeiro, Márcio; Rego, A Cristina

    2017-03-01

    Alpha-synuclein (α-syn) is a major component of Lewy bodies found in sporadic and inherited forms of Parkinson's disease (PD). Mutations in the gene encoding α-syn and duplications and triplications of wild-type (WT) α-syn have been associated with PD. Several mechanisms have been implicated in the degeneration of dopaminergic neurons in PD, including oxidative stress and mitochondrial dysfunction. Here we defined the occurrence of oxidative stress in SH-SY5Y cells overexpressing WT α-syn in a doxycycline (Dox) regulated manner, before and after exposure to iron (500 µM), and determined the changes in proteins involved in the intracellular antioxidant defense system. Data evidenced an increase in caspase-3 activation and diminished reducing capacity of -Dox cells, associated with decreased activity of mitochondria complex I and reduced mitochondrial transcription factor A (TFAM) levels in these cells. Furthermore, total and mitochondrial reactive oxygen species levels were higher under basal conditions in cells overexpressing α-syn (-Dox) and this increase was apparently correlated with diminished levels and activities of SOD1 and SOD2 in -Dox cells. Moreover, both reduced and oxidized glutathione levels were diminished in -Dox cells under basal conditions, concomitantly with decreased activity of GCL and reduced protein levels of GCLc. The effects caused by iron (500 µM) were mostly independent of α-syn expression and triggered different antioxidant responses to possibly counterbalance higher levels of free radicals. Overall, data suggest that overexpression of α-syn modifies the antioxidant capacity of SH-SY5Y cells due to altered activity and protein levels of SOD1 and SOD2, and decreased glutathione pool.

  7. Vitamin D3 protects against Aβ peptide cytotoxicity in differentiated human neuroblastoma SH- SY5Y cells: A role for S1P1/p38MAPK/ATF4 axis.

    PubMed

    Pierucci, Federica; Garcia-Gil, Mercedes; Frati, Alessia; Bini, Francesca; Martinesi, Maria; Vannini, Eleonora; Mainardi, Marco; Luzzati, Federico; Peretto, Paolo; Caleo, Matteo; Meacci, Elisabetta

    2017-04-01

    Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH)2D3. We have found that 1,25(OH)2D3 prevents amyloid-β peptide (Aβ(1-42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo. In differentiated SH-SY5Y cells, Aβ(1-42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH)2D3 partially reverts this effect. 1,25(OH)2D3 reverts also the Aβ(1-42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH)2D3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH)2D3 prevents the reduction of S1P1 expression promoted by Aβ(1-42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH)2D3 protects from aggregated Aβ(1-42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH)2D3 and its structural analogue ZK191784 in counteracting the Aβ(1-42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells.

  8. Orexin-A Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity: Involvement of PKC and PI3K Signaling Pathways.

    PubMed

    Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi

    2017-04-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by progressive and selective death of dopaminergic neurons. Multifunctional neuropeptide orexin-A is involved in many biological events of the body. It has been shown that orexin-A has protective effects in neurodegenerative disease such as PD. However, its cellular mechanisms have not yet been fully clarified. Here, we investigated the intracellular signaling pathway of orexin-A neuroprotection in 6-hydroxydopamine (6-OHDA)-induced SH-SY5H cells damage as an in vitro model of PD. The cells were incubated with 150 μM 6-OHDA, and the viability was examined by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Mitochondrial membrane potential and intracellular calcium were measured by fluorescent probes. Western blotting was also used to determine cyclooxygenase type 2 (COX-2), nuclear factor erythroid 2 related factor 2 (Nrf2), and HSP70 protein levels. The data showed that 6-OHDA has decreasing effects on cell viability, Nrf2, and HSP70 protein expression and increases the level of mitochondrial membrane potential, intracellular calcium, and COX-2 protein. Orexin-A (500 pM) significantly attenuated the 6-OHDA-induced cell damage. Furthermore, Orexin-A significantly prevented the mentioned effects of 6-OHDA on SH-SY5Y cells. Orexin 1 receptor antagonist (SB3344867), PKC, and PI3-kinase (PI3K) inhibitors (chelerythrin and LY294002, respectively) could suppress the orexin-A neuroprotective effect. In contrast, blockage of PKA by a selective inhibitor (KT5720) had no effects on the orexin protection. The results suggest that orexin-A protective effects against 6-OHDA-induced neurotoxicity are performed via its receptors, PKC and PI3K signaling pathways.

  9. Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid).

    PubMed

    Wu, Ze-Zhi; Wang, Zheng-Wei; Zhang, Li-Guang; An, Zhi-Xing; Zhong, Dong-Huo; Huang, Qi-Ping; Luo, Mei-Rong; Liao, Yan-Jian; Jin, Liang; Li, Chen-Zhong; Kisaalita, William S

    2013-01-01

    In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

  10. Melatonin attenuates the mitochondrial translocation of mitochondrial fission proteins and Bax, cytosolic calcium overload and cell death in methamphetamine-induced toxicity in neuroblastoma SH-SY5Y cells.

    PubMed

    Parameyong, Arisa; Govitrapong, Piyarat; Chetsawang, Banthit

    2015-09-01

    Methamphetamine (METH) is an addictive drug that can cause toxicity and degeneration in the brain. Several pieces of evidence have demonstrated that METH toxicity results in increases in oxidative stress that regulate an intracellular signaling cascade that leads to cell death. Recently, several studies have emphasized that the overload of cytosolic calcium levels and mitochondrial fission into a small mitochondrial structure is involved in cell death processes. In the present study, we aimed to investigate the effects of METH toxicity on cytosolic calcium overload and mitochondrial fission in neuroblastoma SH-SY5Y cells. Additionally, the protective effect of melatonin against METH-induced toxicity was also investigated. The results of the present study demonstrated that METH significantly decreases cell viability and increases the levels of mitochondrial fission (Fis1 and Drp1) proteins and pro-apoptotic protein, Bax in isolated mitochondria. The levels of Drp1 in the cytosol of METH-treated cells had no significant differences compared to the control untreated cells. METH also significantly increased the cytosolic calcium levels. Melatonin reversed the toxic effects of METH by restoring cell viability and inhibiting the increase in mitochondrial Fis1 levels and the mitochondrial translocation of Drp1 and Bax. Additionally, melatonin was able to reduce the METH-induced increase in cytosolic calcium levels and fragmented mitochondria into small globular structures in SH-SY5Y cells. The results of the present study demonstrate the potential abilities of melatonin to maintain the homeostasis of mitochondrial dynamics and cytosolic calcium levels in METH-induced toxicity in neuronal cells.

  11. Impact of Inhomogeneous Static Magnetic Field (31.7–232.0 mT) Exposure on Human Neuroblastoma SH-SY5Y Cells during Cisplatin Administration

    PubMed Central

    Mobasheri, Hamid; Dini, Luciana

    2014-01-01

    Beneficial or adverse effects of Static Magnetic Fields (SMFs) are a large concern for the scientific community. In particular, the effect of SMF exposure during anticancer therapies still needs to be fully elucidated. Here, we evaluate the effects of SMF at induction levels that cisPt-treated cancer patients experience during the imaging process conducted in Low field (200–500 mT), Open field (300–700 mT) and/or inhomogeneous High field (1.5–3 T) Magnetic Resonance Imaging (MRI) machines. Human adrenergic neuroblastoma SH-SY5Y cells treated with 0.1 µM cisPt (i.e. the lowest concentration capable of inducing apoptosis) were exposed to SMF and their response was studied in vitro. Exposure of 0.1 µM cisPt-treated cells to SMF for 2 h decreased cell viability (30%) and caused overexpression of the apoptosis-related cleaved caspase-3 protein (46%). Furthermore, increase in ROS (Reactive Oxygen Species) production (23%) and reduction in the number of mitochondria vs controls were seen. The sole exposure of SMF for up to 24 h had no effect on cell viability but increased ROS production and modified cellular shape. On the other hand, the toxicity of cisPt was significantly prevented during 24 h exposure to SMF as shown by the levels of cell viability, cleaved caspase-3 and ROS production. In conclusion, due to the cytoprotective effect of 31.7–232.0 mT SMF on low-cisPt-concentration-treated SH-SY5Y cells, our data suggest that exposure to various sources of SMF in cancer patients under a cisPt regimen should be strictly controlled. PMID:25423171

  12. Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

    PubMed Central

    Wu, Ze-Zhi; Wang, Zheng-Wei; Zhang, Li-Guang; An, Zhi-Xing; Zhong, Dong-Huo; Huang, Qi-Ping; Luo, Mei-Rong; Liao, Yan-Jian; Jin, Liang; Li, Chen-Zhong; Kisaalita, William S

    2013-01-01

    Introduction In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds. PMID

  13. Neuroprotective Effects of Selected Microbial-Derived Phenolic Metabolites and Aroma Compounds from Wine in Human SH-SY5Y Neuroblastoma Cells and Their Putative Mechanisms of Action.

    PubMed

    Esteban-Fernández, A; Rendeiro, C; Spencer, J P E; Del Coso, D Gigorro; de Llano, M D González; Bartolomé, B; Moreno-Arribas, M V

    2017-01-01

    Moderate wine consumption has shown the potential to delay the onset of neurodegenerative diseases. This study investigates the molecular mechanisms underlying the protective effects of wine-derived phenolic and aroma compounds in a neuroinflammation model based on SIN-1 stress-induced injury in SH-SY5Y neuroblastoma cells. Cell pretreatment with microbial metabolites found in blood after wine consumption, 3,4-dihydroxyphenylacetic (3,4-DHPA), 3-hydroxyphenylacetic acids and salicylic β-d-O-glucuronide, at physiologically concentrations (0.1-10 μM) resulted in increased cell viability versus SIN-1 control group (p < 0.05). Results also showed significant decreases in mitogen-activated protein kinase (MAPK) p38 and ERK1/2 activation as well as in downstream pro-apoptotic caspase-3 activity by some of the studied compounds. Moreover, pretreatment with p38, MEK, and ERK1/2-specific inhibitors, which have a phenolic-like structure, also resulted in an increase on cell survival and a reduction on caspase-3 activity levels. Overall, these results contribute with new evidences related to the neuroprotective actions of wine, pointing out that wine-derived human metabolites and aroma compounds may be effective at protecting neuroblastoma cells from nitrosative stress injury by inhibiting neuronal MAPK p38 and ERK1/2, as well as downstream caspase 3 activity.

  14. Neuroprotective Effects of Selected Microbial-Derived Phenolic Metabolites and Aroma Compounds from Wine in Human SH-SY5Y Neuroblastoma Cells and Their Putative Mechanisms of Action

    PubMed Central

    Esteban-Fernández, A.; Rendeiro, C.; Spencer, J. P. E.; del Coso, D. Gigorro; de Llano, M. D. González; Bartolomé, B.; Moreno-Arribas, M. V.

    2017-01-01

    Moderate wine consumption has shown the potential to delay the onset of neurodegenerative diseases. This study investigates the molecular mechanisms underlying the protective effects of wine-derived phenolic and aroma compounds in a neuroinflammation model based on SIN-1 stress-induced injury in SH-SY5Y neuroblastoma cells. Cell pretreatment with microbial metabolites found in blood after wine consumption, 3,4-dihydroxyphenylacetic (3,4-DHPA), 3-hydroxyphenylacetic acids and salicylic β-d-O-glucuronide, at physiologically concentrations (0.1–10 μM) resulted in increased cell viability versus SIN-1 control group (p < 0.05). Results also showed significant decreases in mitogen-activated protein kinase (MAPK) p38 and ERK1/2 activation as well as in downstream pro-apoptotic caspase-3 activity by some of the studied compounds. Moreover, pretreatment with p38, MEK, and ERK1/2-specific inhibitors, which have a phenolic-like structure, also resulted in an increase on cell survival and a reduction on caspase-3 activity levels. Overall, these results contribute with new evidences related to the neuroprotective actions of wine, pointing out that wine-derived human metabolites and aroma compounds may be effective at protecting neuroblastoma cells from nitrosative stress injury by inhibiting neuronal MAPK p38 and ERK1/2, as well as downstream caspase 3 activity. PMID:28352628

  15. Isatin inhibits SH-SY5Y neuroblastoma cell invasion and metastasis through MAO/HIF-1α/CXCR4 signaling.

    PubMed

    Sun, Wenyan; Zhang, Li; Hou, Lin; Ju, Chuanxia; Zhao, Shengmin; Wei, Yaoyue

    2017-04-04

    Isatin was reported to possess anticancer activities through its effect on tumor proliferation, apoptosis, and metastasis in vitro and in vivo. This study aimed to elucidate the underlying mechanism behind isatin's ability to inhibit neuroblastoma cell metastasis. Our results demonstrated that isatin could inhibit neuroblastoma cell proliferation, invasion, and migration in a dose-dependent manner. Moreover, isatin inhibited the expression level of monoamine oxidase A as well as that of its downstream protein hypoxia-inducible factor 1α. Further study indicated that isatin inhibited reactive oxygen species production, extracellular signal-regulated kinase activation, vascular endothelial growth factor receptor-1 phosphorylation, and chemokine receptor type 4 expression. All results support the potential antimetastatic effect of isatin in neuroblatoma cells.

  16. Protective effects of flavonoids against oxidative stress induced by simulated microgravity in SH-SY5Y cells.

    PubMed

    Qu, Lina; Chen, Hailong; Liu, Xinmin; Bi, Lei; Xiong, Jianghui; Mao, Zebin; Li, Yinghui

    2010-09-01

    Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H(2)O(2) or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.

  17. Chikusetsu saponin V attenuates H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells through Sirt1/PGC-1α/Mn-SOD signaling pathways.

    PubMed

    Wan, Jingzhi; Deng, Lili; Zhang, Changcheng; Yuan, Qin; Liu, Jing; Dun, Yaoyan; Zhou, Zhiyong; Zhao, Haixia; Liu, Chaoqi; Yuan, Ding; Wang, Ting

    2016-09-01

    Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Chikusetsu saponin V (CsV), the most abundant member of saponins from Panax japonicus (SPJ), has attracted increasing attention for its potential to treat neurodegenerative diseases. However, the mechanisms are unclear. Our study intended to investigate the antioxidative effects of CsV in human neuroblastoma SH-SY5Y cells. Our data showed that CsV attenuated H2O2-induced cytotoxicity, inhibited ROS accumulation, increased the activities of superoxide dismutase (SOD) and GSH, and increased mitochondrial membrane potential dose-dependently. Further exploration of the mechanisms showed that CsV exhibited these effects through increasing the activation of oxidative-stress-associated factors including Sirt1, PGC-1α, and Mn-SOD. Moreover, CsV inhibited H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. In conclusion, our study demonstrated that CsV exhibited neuroprotective effects possibly through Sirt1/PGC-1α/Mn-SOD signaling pathways.

  18. Melatonin prevents cytosolic calcium overload, mitochondrial damage and cell death due to toxically high doses of dexamethasone-induced oxidative stress in human neuroblastoma SH-SY5Y cells.

    PubMed

    Suwanjang, Wilasinee; Abramov, Andrey Y; Charngkaew, Komgrid; Govitrapong, Piyarat; Chetsawang, Banthit

    2016-07-01

    Stressor exposure activates the hypothalamic-pituitary-adrenal (HPA) axis and causes elevations in the levels of glucocorticoids (GC) from the adrenal glands. Increasing evidence has demonstrated that prolonged exposure to high GC levels can lead to oxidative stress, calcium deregulation, mitochondrial dysfunction and apoptosis in a number of cell types. However, melatonin, via its antioxidant activity, exhibits a neuroprotective effect against oxidative stress-induced cell death. Therefore, in the present study, we explored the protective effect of melatonin in GC-induced toxicity in human neuroblastoma SH-SY5Y cells. Cellular treatment with the toxically high doses of the synthetic GC receptor agonist, dexamethasone (DEX) elicited marked decreases in the levels of glutathione and increases in ROS production, lipid peroxidation and cell death. DEX toxicity also induced increases in the levels of cytosolic calcium and mitochondrial fusion proteins (Mfn1 and Opa1) but decreases in the levels of mitochondrial fission proteins (Fis1 and Drp1). Mitochondrial damage was observed in large proportions of the DEX-treated cells. Pretreatment of the cells with melatonin substantially prevented the DEX-induced toxicity. These results suggest that melatonin might exert protective effects against oxidative stress, cytosolic calcium overload and mitochondrial damage in DEX-induced neurotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Treatment with the neurotoxic Aβ (25-35) peptide modulates the expression of neuroprotective factors Pin1, Sirtuin 1, and brain-derived neurotrophic factor in SH-SY5Y human neuroblastoma cells.

    PubMed

    Lattanzio, Francesca; Carboni, Lucia; Carretta, Donatella; Candeletti, Sanzio; Romualdi, Patrizia

    2016-05-01

    The deposition of Amyloid β peptide plaques is a pathological hallmark of Alzheimer's disease (AD). The Aβ (25-35) peptide is regarded as the toxic fragment of full-length Aβ (1-42). The mechanism of its toxicity is not completely understood, along with its contribution to AD pathological processes. The aim of this study was to investigate the effect of the neurotoxic Aβ (25-35) peptide on the expression of the neuroprotective factors Pin1, Sirtuin1, and Bdnf in human neuroblastoma cells. Levels of Pin1, Sirtuin 1, and Bdnf were compared by real-time PCR and Western blotting in SH-SY5Y cells treated with Aβ (25-35) or administration vehicle. The level of Pin1 gene and protein expression was significantly decreased in cells exposed to 25 μM Aβ (25-35) compared to vehicle-treated controls. Similarly, Sirtuin1 expression was significantly reduced by Aβ (25-35) exposure. In contrast, both Bdnf mRNA and protein levels were significantly increased by Aβ (25-35) treatment, suggesting the activation of a compensatory response to the insult. Both Pin1 and Sirtuin 1 exert a protective role by reducing the probability of plaque deposition, since they promote amyloid precursor protein processing through non-amyloidogenic pathways. The present results show that Aβ (25-35) peptide reduced the production of these neuroprotective proteins, thus further increasing Aβ generation.

  20. RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

    PubMed

    Lopes, Fernanda M; da Motta, Leonardo Lisbôa; De Bastiani, Marco A; Pfaffenseller, Bianca; Aguiar, Bianca W; de Souza, Luiz F; Zanatta, Geancarlo; Vargas, Daiani M; Schönhofen, Patrícia; Londero, Giovana F; de Medeiros, Liana M; Freire, Valder N; Dafre, Alcir L; Castro, Mauro A A; Parsons, Richard B; Klamt, Fabio

    2017-05-01

    Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

  1. Time-dependent effects of lithium on the agonist-stimulated accumulation of second messenger inositol 1,4,5-trisphosphate in SH-SY5Y human neuroblastoma cells.

    PubMed

    Los, G V; Artemenko, I P; Hokin, L E

    1995-10-01

    In order to approach the molecular mechanism of Li+'s mood-stabilizing action, the effect of Li+ (LiCl) on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] mass was investigated in human neuroblastoma SH-SY5Y cells, which express muscarinic M3 receptors, coupled to PtdIns hydrolysis. Stimulation of these cells, with the cholinergic agonist acetylcholine, resulted in a rapid and transient increase in Ins(1,4,5)P3 with a maximum at 10 s. This was followed by a rapid decline in Ins(1,4,5)P3 within 30 s to a plateau level above baseline, which gradually declined to reach a new steady state, which was significantly higher than resting Ins(1,4,5)P3 at 30 min. Li+ had no effect on Ins(1,4,5)P3 in resting cells, as well as on the acetylcholine-dependent peak of Ins(1,4,5)P3. However, Li+ caused a transient reduction (at 45 s), followed by a long lasting increase in the Ins(1,4,5)P3 (30 min), as compared with controls. The Li+ effects were dose-dependent and were observed at concentrations used in the treatment of bipolar disorders. Supplementation with inositol had no effect on the level of Ins(1,4,5)P3, at least over the time periods studied. Stimulation of muscarinic receptors with consequent activation of phospholipase C were necessary for the manifestation of Li+ effects in SH-SY5Y cells, Li+ did not interfere with degradation of Ins(1,4,5)P3 after receptor-blockade with atropine, suggesting that Li+ has no direct effect on the Ins(1,4,5)P3-metabolizing enzymes. A direct effect of Li+ on the phospholipase C also is unlikely. Blockade of Ca2+ entry into the cells by Ni2+, or incubation with EGTA, which reduces agonist-stimulated accumulation of Ins(1,4,5)P3, had no effect on the Li(+)-dependent increase in Ins(1,4,5)P3.

  2. Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons

    PubMed Central

    Chaban, Victor V; Cho, Taehoon; Reid, Christopher B; Norris, Keith C

    2013-01-01

    Background: Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons. Methods: We assessed intracellular calcium ([Ca2+]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention). Results: Chemosensitive receptors [Ca2+]i signaling did not differ between control 1 and 2. ATP (10 μM) and capsaicin (100nM) increased [Ca2+]i transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17β-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca2+]i transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca2+]i by >50% (p<0.05) and abolished the 17β-estradiol effect. By contrast, apoptotic DRG cells increased initial ATP-induced [Ca2+]i, flux four fold and abolished subsequent [Ca2+]i, responses to ATP stimulation (p<0.001). Capsaicin (100nM) induced [Ca2+]i responses were totally abolished. Conclusion: The local presence of apoptotic DRG or human neuroblastoma cells induced differing abnormal ATP and capsaicin-mediated [Ca2+]i fluxes in normal DRG. These findings support physically disconnected, non-diffusible cell-to-cell signaling. Further studies are needed to delineate the mechanism(s) of and model(s) of communication. PMID:23390567

  3. Differentiated SH-SY5Y human cells provide a reductionist model of HSV-1 neurotropism.

    PubMed

    Shipley, Mackenzie M; Mangold, Colleen A; Kuny, Chad V; Szpara, Moriah L

    2017-09-27

    Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats including incomplete differentiation, non-human origins, or the use of dividing cells that have neuropotential, but lack neuronal morphology. Here we use a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titer similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 hpi, suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cGAS in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in non-epithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry, and will be advantageous for the study of neurotropic viruses in vitroImportance Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and in rare cases, encephalitis. Presently, there is no cure

  4. Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Shindo, H; Thomas, T P; Larkin, D D; Karihaloo, A K; Inada, H; Onaya, T; Stevens, M J; Greene, D A

    1996-01-01

    Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes. PMID:8609230

  5. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

  6. Efficient functional coupling of the human D3 dopamine receptor to G(o) subtype of G proteins in SH-SY5Y cells.

    PubMed

    Zaworski, P G; Alberts, G L; Pregenzer, J F; Im, W B; Slightom, J L; Gill, G S

    1999-11-01

    1 The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines. 2 In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells. 4 More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.

  7. Efficient functional coupling of the human D3 dopamine receptor to Go subtype of G proteins in SH-SY5Y cells

    PubMed Central

    Zaworski, Phillip G; Alberts, Glen L; Pregenzer, Jeffrey F; Im, Wha Bin; Slightom, Jerry L; Gill, Gurnam S

    1999-01-01

    The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines.In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPγ35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes.GTPγ35S-bound Gα subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Gα-specific antibodies. Gαo subunits (not Gαi) were highly labelled with GTPγ35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells.More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor. PMID:10578130

  8. [Effects of parabolic flight on redox status in SH-SY5Y cells].

    PubMed

    Bi, Lei; Qu, Li-Na; Huang, Zeng-Ming; Wang, Chun-Yan; Li, Qi; Tan, Ying-Jun; Li, Ying-Hui

    2009-10-25

    Space flight is known to produce a number of neurological disturbances. The etiology is unknown, but it may involve increased oxidative stress. A line of experimental evidence indicates that space flight may disrupt antioxidant defense system and result in increased oxidative stress. In vitro studies found that abundant of NO was produced in rat pheochromocytoma (PC12) cells, SHSY5Y neuroblastoma cells, and protein nitration was increased in PC12 cells within a simulated microgravity rotating wall bioreactor high aspect ratio vessel system or clinostat system. In the present study, we observed the change of redox status in SH-SY5Y cells after parabolic flight, and studied the effects of key redox molecule, thioredoxin (TRX), during the altered gravity. SH-SY5Y cells were divided into four groups: control cells, control cells transfected with TRX, flight cells and flight cells transfected with TRX. The expression levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), TRX and thioredoxin reductase (TRXR) were observed by immunocytochemical method. It was shown that after parabolic flight, the staining of 3-NT and TRX were enhanced, while the expression level of TRXR was down-regulated compared with control. As for flight cells transfected with TRX, the staining of 3-NT and iNOS were weakened compared with flight cells. These results obtained suggest that altered gravity may increase protein nitration, down-regulate TRXR and elicit oxidative stress in SH-SY5Y cells, while TRX transfection could partly protect cells against oxidative stress induced by parabolic flight.

  9. Gadd153 and NF-κB Crosstalk Regulates 27-Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-SY5Y Cells

    PubMed Central

    Marwarha, Gurdeep; Raza, Shaneabbas; Prasanthi, Jaya R. P.; Ghribi, Othman

    2013-01-01

    β-amyloid (Aβ) peptide, accumulation of which is a culprit for Alzheimer’s disease (AD), is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP). We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD. PMID:23951005

  10. Retinol (Vitamin A) Increases α-Synuclein, β-Amyloid Peptide, Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line.

    PubMed

    Kunzler, Alice; Kolling, Eduardo Antônio; da Silva-Jr, Jeferson Delgado; Gasparotto, Juciano; de Bittencourt Pasquali, Matheus Augusto; Moreira, José Cláudio Fonseca; Gelain, Daniel Pens

    2017-05-11

    Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson's disease and Alzheimer's disease: α-synuclein, β-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, β-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and β-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.

  11. Opioid agonists binding and responses in SH-SY5Y cells

    NASA Technical Reports Server (NTRS)

    Costa, E. M.; Hoffmann, B. B.; Loew, G. H.

    1992-01-01

    SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

  12. Rotary bioreactor culture can discern specific behavior phenotypes in Trk-null and Trk-expressing neuroblastoma cell lines.

    PubMed

    Redden, Robert A; Iyer, Radhika; Brodeur, Garrett M; Doolin, Edward J

    2014-03-01

    Neuroblastoma is characterized by biological and genetic heterogeneity that leads to diverse, often unpredictable, clinical behavior. Differential expression of the Trk family of neurotrophin receptors strongly correlates with clinical behavior; TrkA expression is associated with favorable outcome, whereas TrkB with unfavorable outcome. Neuroblastoma cells cultured in a microgravity rotary bioreactor spontaneously aggregate into tumor-like structures, called organoids. We wanted to determine if the clinical heterogeneity of TrkA- or TrkB-expressing neuroblastomas was reflected in aggregation kinetics and organoid morphology. Trk-null SY5Y cells were stably transfected to express either TrkA or TrkB. Short-term aggregation kinetics were determined by counting the number of single (non-aggregated) viable cells in the supernatant over time. Organoids were harvested after 8 d of bioreactor culture, stained, and analyzed morphometrically. SY5Y-TrkA cells aggregated significantly slower than SY5Y and SY5Y-TrkB cells, as quantified by several measures of aggregation. SY5Y and TrkB cell lines formed irregularly shaped organoids, featuring stellate projections. In contrast, TrkA cells formed smooth (non-stellate) organoids. SY5Y organoids were slightly smaller on average, but had significantly larger average perimeter than TrkA or TrkB organoids. TrkA expression alone is sufficient to dramatically alter the behavior of neuroblastoma cells in three-dimensional, in vitro rotary bioreactor culture. This pattern is consistent with both clinical behavior and in vivo tumorigenicity, in that SY5Y-TrkA represents a more differentiated, less aggressive phenotype. The microgravity bioreactor is a useful in vitro tool to rapidly investigate the biological characteristics of neuroblastoma and potentially to assess the effect of cytotoxic as well as biologically targeted drugs.

  13. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation.

    PubMed

    Song, Jie; Li, Na; Xia, Yang; Gao, Zhong; Zou, Sa-Feng; Yan, Yu-Hui; Li, Shao-Heng; Wang, Yue; Meng, Ya-Kun; Yang, Jing-Xian; Kang, Ting-Guo

    2016-09-01

    Mechanical trauma injury is a severe insult to neural cells. Subsequent secondary injury involves the release of inflammatory factors that have dramatic consequences for undamaged cells, leading to normal cell death after the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary effects and evaluated the mechanism underlying the action of microRNA (miRNA)-199a and miRNA-16 in a mechanical trauma injury (MTI) model using SH-SY5Y cells in vitro. SH-SY5Y cells are often applied to in vitro models of neuronal function and differentiation. Recently, miRNAs have been demonstrated to play a crucial role in NF-κB and cholinergic signaling, which can regulate inflammation. The cell model was established by scratch-induced injury of human SH-SY5Y cells, which mimics the characteristics of MTI. A cell counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry were used to measure cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inflammatory cytokine and cholinesterase (CHE) content. The lactate dehydrogenase (LDH) content was measured to assess the degree of cell injury. The mRNA levels were measured by RT-PCR to analyze ARC's mechanism of action. miRNA inhibitors and mimics were used to inhibit and strengthen the expression of miRNAs. Protein expression was detected by western blotting analysis. ARC treatment reduced the TNF-α and IL-6 levels as well as the number of TUNEL+ apoptotic SH-SY5Y cells surrounding the scratch and increased the IL-10 level compared to the controls. ARC attenuated the increase of the cell damage degree and LDH content induced by scratching, indicating increased cell survival. Mechanistic studies showed that ARC upregulated the miRNA-16 and miRNA-199a levels to reduce upstream protein (IKKα and IKKβ) expression and inhibit NF-κB signaling pathway activity; moreover, the increased miRNA-199a suppresses

  14. The toxic effect of ketamine on SH-SY5Y neuroblastoma cell line and human neuron.

    PubMed

    Mak, Ying T; Lam, Wai P; Lü, Lanhai; Wong, Yeuk W; Yew, David T

    2010-03-01

    Ketamine used as an injectable anesthetic in human and animal medicine is also a recreational drug used primarily by young adults often at all night dance parties in nightclubs. The percentage of ketamine users has grown very fast in the last 5 years worldwide. However, this leads to the serious question of the long-term adverse effects of ketamine on our nervous system, particularly the brain, because ketamine as an NMDA antagonist could cause neurons to commit apoptosis. Our study therefore aimed to find out the chronic effect of ketamine on neuron using prolonged incubation (48 h) of neuronal cells with ketamine in culture. Our results showed that differentiated neuronal cells were prone to the toxicity of ketamine but probably less susceptible than undifferentiated neuronal cells and fibroblasts. This suggested that the ketamine abuse would be harmful to many other organs as well as the brain. Our results also confirmed that the toxicity of ketamine is related to apoptosis via the Bax/Bcl-2 ratio pathway and caspase-3 in the differentiated neuronal cells. Therefore, long-term ketamine treated cell or animal models should be sought to study this multiorgan effects of ketamine.

  15. Emulsion-core and polyelectrolyte-shell nanocapsules: biocompatibility and neuroprotection against SH-SY5Y cells

    NASA Astrophysics Data System (ADS)

    Piotrowski, Marek; Szczepanowicz, Krzysztof; Jantas, Danuta; Leśkiewicz, Monika; Lasoń, Władysław; Warszyński, Piotr

    2013-11-01

    The emulsion-core and polyelectrolyte-coated nanocapsules, designed as water-insoluble neuroprotective drug delivery system, were synthesized using layer-by-layer saturation method. The isopropyl myristate was used as oil phase and docusate sodium salt as emulsifier. For the polyelectrolyte shell preparation, synthetic polyelectrolytes, cationic (PDADMAC, PAH, and PLL) and anionic (PGA) were used. The particle size and zeta potential of nanocapsules were characterized by the dynamic light scattering. The average size of synthesized nanocapsules ranged from 80 to 100 nm. Zeta potential values ranged from less than approximately -30 mV for the polyanion layers to greater than approximately +30 mV for the polycation layers. Biocompatibilities of the synthesized nanocarriers were evaluated against SH-SY5Y human neuroblastoma cells using various biochemical assays. The results obtained show that synthesized nanocapsules coated with PLL and PGA were nontoxic to SH-SY5Y cells, and they were used as nanocarriers for model neuroprotective drug (a calpain inhibitor MDL 28170). The neuroprotective action of the encapsulated MDL 28170 against hydrogen peroxide-induced oxidative stress cytotoxicity was evaluated in the same cell line. The results showed that nanoencapsulated form of MDL 28170 were biocompatible and protected SH-SY5Y cells against the H2O2 (0.5 mM/24 h)-induced damage in 20-40 times lower concentrations than those of the same drug added directly to the culture medium. These data suggest that the nanoscale carriers of neuroprotective drugs might serve as novel promising therapeutic agents for oxidative stress-related neurodegenerative processes.

  16. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP(+)-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-03-30

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP(+)-induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy.

  17. Role for the PI3K/Akt/Nrf2 signaling pathway in the protective effects of carnosic acid against methylglyoxal-induced neurotoxicity in SH-SY5Y neuroblastoma cells.

    PubMed

    de Oliveira, Marcos Roberto; Ferreira, Gustavo Costa; Schuck, Patrícia Fernanda; Dal Bosco, Simone Morelo

    2015-12-05

    Glycation, a process that occurs endogenously and generates advanced glycation end products (AGEs), presents an important role in cases of neurodegeneration, as for instance Alzheimer's disease (AD). Methylglyoxal (MG), a dicarbonyl compound, is the most potent inducer of AGEs, whose levels have been found increased in samples obtained from subjects suffering from AD. Moreover, MG induces protein cross-linking and redox impairment in vitro and in vivo. Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, exerts protective effects in neuronal cells by increasing antioxidant defenses and detoxification systems. In the present work, we aimed to investigate whether there is a role for CA against MG-induced neurotoxicity. Data obtained here clearly demonstrate that CA pretreatment (1 μM for 12 h) caused cytoprotective effects and counteracted the damage elicited by MG in SH-SY5Y cells. CA inhibited loss of mitochondrial membrane polarity (MMP) and cytochrome c release from mitochondria, consequently blocking activation of pro-apoptotic caspase enzymes. Furthermore, CA alleviated MG-induced oxidative and nitrosative damage. CA prevented MG-dependent neurotoxicity by activating the PI3K/Akt/Nrf2 signaling pathway and the antioxidant enzymes modulated by Nrf2 transcription factor. Overall, the data presented here show the protective role of CA by its ability to counteract MG negative effects.

  18. The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

    PubMed

    Ruggeri, Pierdomenico; Farina, Antonietta R; Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R

    2014-01-01

    The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

  19. N-linked glycan profiling in neuroblastoma cell lines.

    PubMed

    Hu, Yunli; Mayampurath, Anoop; Khan, Saira; Cohen, Joanna K; Mechref, Yehia; Volchenboum, Samuel L

    2015-05-01

    Although MYCN amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, MYCN-nonamplified neuroblastoma from high-risk, MYCN-amplified disease are largely unknown. Genomic and proteomic studies have been limited in discerning differences in signaling pathways that account for this heterogeneity. N-Linked glycosylation is a common protein modification resulting from the attachment of sugars to protein residues and is important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proven to be useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between MYCN-nonamplified SY5Y and MYCN-amplified NLF cell lines with the aim of identifying changes in sugar abundance linked to high-risk neuroblastoma. Through a combination of liquid chromatography-mass spectrometry and bioinformatics analysis, we identified 16 glycans that show a statistically significant change in abundance between NLF and SY5Y samples. Closer examination revealed the preference for larger (in terms of total monosaccharide count) and more sialylated glycan structures in the MYCN-amplified samples in comparison to smaller, nonsialylated glycans that are more dominant in the MYCN-nonamplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis.

  20. Manganese-Induced Oxidative DNA Damage in Neuronal SH-SY5Y Cells: Attenuation of thymine base lesions by glutathione and N-acetylcysteine

    PubMed Central

    Stephenson, Adrienne P.; Schneider, Jeffrey A.; Nelson, Bryant C.; Atha, Donald H.; Jain, Ashok; Soliman, Karam F. A.; Aschner, Michael; Mazzio, Elizabeth; Reams, R. Renee

    2013-01-01

    Manganese (Mn) is an essential trace element required for normal function and development. However, exposure to this metal at elevated levels may cause manganism, a progressive neurodegenerative disorder with neurological symptoms similar to idiopathic Parkinson’s disease (IPD). Elevated body burdens of Mn from exposure to parental nutrition, vapors in mines and smelters and welding fumes have been associated with neurological health concerns. The underlying mechanism of Mn neurotoxicity remains unclear. Accordingly, the present study was designed to investigate the toxic effects of Mn2+ in human neuroblastoma SH-SY5Y cells. Mn2+ caused a concentration dependent decrease in SH-SY5Y cellular viability compared to controls. The LD50 value was 12.98 μM Mn2+ (p <0.001 for control vs. 24h Mn treatment). Both TUNEL and annexin V/propidium iodide apoptosis assays confirmed the induction of apoptosis in the cells following exposure to Mn2+ (2 μM, 62 μM or 125 μM). In addition, Mn2+ induced both the formation and accumulation of DNA single strand breaks (via alkaline comet assay analysis) and oxidatively modified thymine bases (via gas chromatography/mass spectrometry analysis). Pre-incubation of the cells with characteristic antioxidants, either 1 mM N-acetylcysteine or 1 mM glutathione reduced the level of DNA strand breaks and the formation of thymine base lesions, suggesting protection against oxidative cellular damage. Our findings indicate that 1) exposure of SH-SY5Y cells to Mn promotes both the formation and accumulation of oxidative DNA nucleotide base damage, 2) SH-SY5Y cells with accumulated DNA damage are more likely to die via an apoptotic pathway and 3) the accumulated levels of DNA damage can be abrogated by the addition of exogenous chemical antioxidants. This is the first known report of Mn2+-induction and antioxidant protection of thymine lesions in this SH-SY5Y cell line and contributes new information to the potential use of antioxidants as a

  1. Hyperosmotic Stress Induces Tau Proteolysis by Caspase-3 Activation in SH-SY5Y Cells.

    PubMed

    Olivera-Santa Catalina, Marta; Caballero-Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Cuenda, Ana; Lorenzo, María J; Centeno, Francisco

    2016-12-01

    Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor μ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.

  2. PTPN11 induces endoplasmic stress and apoptosis in SH-SY5Y cells.

    PubMed

    Chitranshi, Nitin; Dheer, Yogita; Gupta, Veer; Abbasi, Mojdeh; Mirzaei, Mehdi; You, Yuyi; Chung, Roger; Graham, Stuart L; Gupta, Vivek

    2017-09-22

    PTPN11 is associated with regulation of growth factor signalling pathways in neuronal cells. Using SH-SY5Y neuroblastoma cells, we showed that adeno-associated virus (AAV) mediated PTPN11 upregulation was associated with TrkB antagonism, reduced neuritogenesis and enhanced endoplasmic reticulum (ER) stress response leading to apoptotic changes. Genetic knock-down of PTPN11 on the other hand lead to increased TrkB phosphorylation in SH-SY5Y cells. ER stress response induced by PTPN11 upregulation was alleviated pharmacologically by a TrkB agonist. Conversely the enhanced ER stress response induced by TrkB receptor antagonism was ameliorated by PTPN11 suppression, providing evidence of cross-talk of PTPN11 effects with TrkB actions. BDNF treatment of neuronal cells with PTPN11 upregulation also resulted in reduced expression of ER stress protein markers. This study provides evidence of molecular interactions between PTPN11 and the TrkB receptor in SH-SY5Y cells. The results reinforce the role played by PTPN11 in regulating neurotrophin protective signalling in neuronal cells and highlight that PTPN11 dysregulation promotes apoptotic activation. Based on these findings we suggest that blocking PTPN11 could have potential beneficial effects to limit the progression of neuronal loss in neurodegenerative disorders. Copyright © 2017. Published by Elsevier Ltd.

  3. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells.

    PubMed

    Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

    2017-01-01

    Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease.

  4. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells

    PubMed Central

    Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

    2017-01-01

    Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson’s disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3′-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson’s disease. PMID:28250973

  5. Particulate matter cytotoxicity in cultured SH-SY5Y cells is modulated by simvastatin: Toxicological assessment for oxidative damage.

    PubMed

    Ferraro, S A; Astort, F; Yakisich, J S; Tasat, D R

    2016-03-01

    Epidemiological studies have shown a positive correlation between environmental particulate matter and adverse health effects. In particular, residual oil fly ash (ROFA) induces inflammation and reactive oxygen species (ROS), exerting not only local, but also systemic adverse effects. Previously, in an experimental animal model, we found that simvastatin (Sv) pretreatment was effective in preventing ROFA induced lung inflammation. Herein, using the human neuroblastoma SH-SY5Y cell line as a neurotoxicity in vitro model, we studied the potential Sv protective effect on ROFA cytotoxicity. We evaluated cell viability by the MTT assay, superoxide anion generation by NBT test, Nrf2 activation by immunofluorescence, apoptosis by cleaved-PARP and active-caspase 3 expressions, and senescence by β-galactosidase activity. SH-SY5Y cells exposed to ROFA (10 and 50μg/ml) for 24h showed decreased cell viability, increased superoxide anion generation, apoptosis and senescence. Pretreatment with Sv (1μM) for 6 days, restored cell viability to basal levels, reduced ROFA-induced O2(-) generation as well as the number of apoptotic and senescent cells. Sv pretreatment stimulated the basal and ROFA-induced levels of Nrf2 nuclear translocation suggesting that activation of the cellular antioxidant defense system prevented particle-induced oxidative stress. In parallel, rescue experiments with mevalonate did not modify the effects of SV pretreatment in any of the parameters evaluated in this study. We conclude that simvastatin may provide neuroprotection against air particulate matter-induced neurotoxicity independently of its ability to inhibit cholesterol synthesis.

  6. Neuroprotective effects of germinated brown rice against hydrogen peroxide induced cell death in human SH-SY5Y cells.

    PubMed

    Ismail, Norsharina; Ismail, Maznah; Fathy, Siti Farhana; Musa, Siti Nor Asma; Imam, Mustapha Umar; Foo, Jhi Biau; Iqbal, Shahid

    2012-01-01

    The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H(2)O(2)) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H(2)O(2)-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.

  7. 6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells.

    PubMed

    Gomez-Lazaro, Maria; Bonekamp, Nina A; Galindo, Maria F; Jordán, Joaquin; Schrader, Michael

    2008-06-01

    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.

  8. Neuroprotective Effects of Germinated Brown Rice against Hydrogen Peroxide Induced Cell Death in Human SH-SY5Y Cells

    PubMed Central

    Ismail, Norsharina; Ismail, Maznah; Fathy, Siti Farhana; Musa, Siti Nor Asma; Imam, Mustapha Umar; Foo, Jhi Biau; Iqbal, Shahid

    2012-01-01

    The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H2O2) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H2O2-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis. PMID:22949825

  9. Methylglyoxal increases dopamine level and leads to oxidative stress in SH-SY5Y cells.

    PubMed

    Xie, Bingjie; Lin, Fankai; Peng, Lei; Ullah, Kaleem; Wu, Hanyan; Qing, Hong; Deng, Yulin

    2014-11-01

    More and more studies have suggested that methylglyoxal (MGO) induced by type-2 diabetes is related to Parkinson's disease (PD). However, little is known about the molecular mechanism. In this study, we explored the MGO toxicity in neuroblastoma SH-SY5Y cells. Neurotoxicity of MGO was measured by mitochondrial membrane potential, malondialdehyde, and methylthiazoletetrazolium assays. The levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) were detected by liquid chromatography-mass spectrometry/mass spectrometry. The expressions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The results showed that MGO induced an increase in TH and DAT expressions in SH-SY5Y neuroblastoma cells, while the levels of dopamine, DOPAC, and endogenous neurotoxin salsolinol also increased. Aminoguanidine (AG) is an inhibitor of MGO. It was found that AG could decrease the reactive oxygen species (ROS) level induced by MGO, but could not inhibit an increase of TH, DAT and dopamine. The increase of dopamine, DOPAC and salsolinol levels could lead to high ROS and mitochondrial damage. This study suggests that ROS caused by dopamine could contribute to the damage of dopaminergic neurons when MGO is increased during the course of diabetes.

  10. Autophagy inhibits C2-ceramide-mediated cell death by decreasing the reactive oxygen species levels in SH-SY5Y cells.

    PubMed

    Fan, Chenghe; Liu, Yuanyuan; Zhao, Mingming; Zhan, Rui; Cui, Wei; Jin, Haiqiang; Teng, Yuming; Lv, Pu; Zheng, Lemin; Huang, Yining

    2017-06-09

    Ceramide has been recognized as a second messenger that regulates several intracellular processes in neuronal cells. However, its role in neuronal autophagy is not fully understood. In this study, we used a human neuroblastoma cell line (SH-SY5Y) to investigate the mechanisms underlying C2-ceramide-mediated cell death and autophagy. C2-ceramide induced caspase-3-independent cell death. In addition, C2-ceramide induced autophagy, decreased the activation of Akt and mTOR, and increased the activation of JNK and ERK1/2. However, only inhibition of ERK1/2 with PD98059 prevented C2-ceramide-induced autophagy, indicating that the ERK1/2 pathway contributes to ceramide-induced autophagy. According to the results of the flow cytometric assays, C2-ceramide-induced cell death was increased by 3-methyadenine (3-MA) and decreased by rapamycin. Furthermore, the generation of reactive oxygen species (ROS) in the cells was increased by 3-MA and decreased by rapamycin. Based on these datas, autophagy protected SH-SY5Y cells from C2-ceramide-induced cell death by decreasing ROS production. Therapeutic strategies that regulate autophagy may be used in the treatment of neurological disorders associated with ceramide-induced cell death. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Silencing of SIAH1 in SH-SY5Y affects α-synuclein degradation pathway

    PubMed Central

    Xu, Jing; Zhang, Xin-Zhi; Zhang, Yong-Jin; Li, Xiu-Ming; Cai, Zeng-Lin; Li, Xiao-Min

    2015-01-01

    Seven in absentia homolog (SIAH) is a ubiquitin ligase that monoubiquitinates α-synuclein. Lewy bodies are characteristically rich in monoubiquitinated α-synuclein. We aimed to determine the effect of siRNA-SIAH1 on α-synuclein autophagy and UPS degradation in SH-SY5Y. SIAH1 expression was measured with real-time quantitative PCR and Western Blot. Cell proliferation was measured by CCK-8 assay; cell apoptosis assayed by flow cytometry. Relative protein expressions were measured by Western Blot. mRNA levels of relative protein were measured by real-time quantitative PCR. The expression of α-synuclein, LC3-II and SIAH1 were observed by confocal microscopy. We found: (1) Transfection efficiency of SIAH1-siRNA into SH-SY5 measured approximately 89% by flow cytometry. (2) siRNA silencing of SIAH1 promoted cellular proliferation and suppressed apoptosis. (3) Protein and mRNA expression of α-synuclein, LC3-II and p53 decreased after SIAH1 knockdown. E1 protein and mRNA levels increased after SIAH1 siRNA. These data show silencing SIAH1 increased cell proliferation and inhibited apoptosis in SH-SY5Y neuroblastoma cells. SIAH1 knockdown enhanced the clearance of non-aggregated α-synuclein by UPS. SIAH1 is a potential target for treatment of Parkinson’s disease. PMID:26722480

  12. Clathrin-Dependent Uptake of Paraquat into SH-SY5Y Cells and Its Internalization into Different Subcellular Compartments.

    PubMed

    Li, Fengrui; Tian, Xiaofei; Zhan, Xiaoni; Wang, Baojie; Ding, Mei; Pang, Hao

    2017-08-01

    The herbicide paraquat (PQ) is an exogenous toxin that allows the selective activation of dopaminergic neurons in the mesencephalon to induce injury and also causes its apoptosis in vitro. However, uptake mechanisms between PQ and neurons remain elusive. To address this issue, we undertook a study of PQ endocytosis in a dopaminergic SH-SY5Y cell line as well as explored the subsequent subcellular location and potential functional analysis of PQ. The PQ was found to bind the SH-SY5Y cell membrane and then became internalized via a clathrin-dependent pathway. PQ was internalized by many subcellular organelles in a time- and dose-dependent manner. Interestingly, the taken up PQ and secretogranin III (SCG3), which became dysregulated with PQ treatment that induced SH-SY5Y apoptosis in our previous study, colocalized in cytoplasmic vesicles. Taken together, our findings indicate that PQ is endocytosed by SH-SY5Y cells and that its multiple, subcellular localizations indicate PQ may potentially be involved in subcellular-level functions. More importantly, PQ distributing preferentially into SCG3-positive vesicles demonstrates its selective targeting which may affect SCG3 and cargoes carried by SCG3-positive vesicles. Therefore, it is reasonable to infer that PQ toxic insults may potentially interfere with neurotransmitter storage and transport associated with secretory granules.

  13. Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

    PubMed Central

    Calabrò, Emanuele; Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Caccamo, Daniela; Magazù, Salvatore; Ientile, Riccardo

    2012-01-01

    AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoic-acid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves. PMID:22371824

  14. ∆(9)-Tetrahydrocannabinol decreases NOP receptor density and mRNA levels in human SH-SY5Y cells.

    PubMed

    Cannarsa, Rosalia; Carretta, Donatella; Lattanzio, Francesca; Candeletti, Sanzio; Romualdi, Patrizia

    2012-02-01

    Several studies demonstrated a cross-talk between the opioid and cannabinoid system. The NOP receptor and its endogenous ligand nociceptin/orphanin FQ represent an opioid-related functional entity that mediates some non-classical opioid effects. The relationship between cannabinoid and nociceptin/NOP system is yet poorly explored. In this study, we used the neuroblastoma SH-SY5Y cell line to investigate the effect of delta-9-tetrahydrocannabinol (∆(9)-THC) on nociceptin/NOP system. Results revealed that the exposure to ∆(9)-THC (100, 150, and 200 nM) for 24 h produces a dose-dependent NOP receptor B (max) down-regulation. Moreover, ∆(9)-THC caused a dose-dependent decrease in NOP mRNA levels. The selective cannabinoid receptor CB1 antagonist AM251 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) reduces both effects, suggesting that ∆(9)-THC activation of CB1 receptor is involved in the observed effects. These data show evidence of a cross-talk between NOP and CB1 receptors, thus suggesting a possible interplay between cannabinoid and nociceptin/NOP system.

  15. Melatonin Protects SH-SY5Y Neuronal Cells Against Methamphetamine-Induced Endoplasmic Reticulum Stress and Apoptotic Cell Death.

    PubMed

    Wongprayoon, Pawaris; Govitrapong, Piyarat

    2017-01-01

    Methamphetamine (METH), a psychostimulant with highly neurotoxic effects, has been known to induce neuronal apoptosis in part through an endoplasmic reticulum (ER) stress pathway. Melatonin is an endogenous antioxidant compound that exerts protective effects against several neurodegenerative conditions, including METH-induced neurotoxicity, via various mechanisms. However, the role of melatonin in ER stress is still relatively unclear. In the present study, we investigated ER stress and neuronal apoptosis following METH treatment and the role of melatonin in METH-mediated ER stress-induced cell death in the SH-SY5Y neuroblastoma cell line. We found that METH caused the overexpression of ER stress-related genes, including C/EBP homologous protein and spliced X-box binding protein 1, in dose- and time-dependent manners. Moreover, METH time-dependently activated caspase-12 and -3, leading to cellular apoptosis. Furthermore, we demonstrated that pretreatment with melatonin attenuated the overexpression of ER stress-related genes and the cleavages of caspase-12 and -3 caused by METH exposure. Flow cytometry revealed that METH-mediated neuronal apoptosis was also prevented by melatonin. These findings suggest the protective effects of melatonin against ER stress and apoptosis caused by METH and other harmful agents.

  16. Attenuation of rotenone toxicity in SY5Y cells by taurine and N-acetyl cysteine alone or in combination.

    PubMed

    Alkholifi, Faisal K; Albers, David S

    2015-10-05

    There is accumulating evidence that supports the involvement of reactive oxygen species (ROS), mitochondrial dysfunction and inflammation in the pathogenesis of neurodegenerative diseases. Thus, it is plausible that a multi-targeted therapeutic approach may be a more effective strategy to retard or even potentially halt the progression of the disease. Taurine is an organic acid that has a role in the regulation of oxidative stress and promoting mitochondrial normal functions, and N-Acetyl cysteine (NAC) is a well-known anti-oxidant and glutathione precursor. The main purpose of this study was to examine the cytoprotective effects of taurine alone or in combination with NAC against rotenone-induced toxicity in the SH-SY5Y neuroblastoma cell line. Taurine treatment produced a concentration-dependent reduction in rotenone-induced cell death. From this, we tested sub-effective concentrations of taurine in combination with low, sub-effective concentrations of NAC against rotenone toxicity, and found the combined treatment afforded greater cytoprotection than either treatment alone. The combined taurine/NAC treatment also attenuated rotenone-induced reductions in aconitase activity suggesting the cytoprotection afforded by the combined treatment may be associated with anti-oxidative mechanisms. Together, our data suggest that a multi-targeted approach may yield new avenues of research exploring the utility of combining therapeutic agents with different mechanisms of actions at concentrations lower than previously tested and shown to be cytoprotective. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Involvement of vimentin in neurite outgrowth damage induced by fipronil in SH-SY5Y cells.

    PubMed

    Ruangjaroon, Theetat; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Paricharttanakul, N Monique

    2017-05-06

    Fipronil, a phenylpyrazole insecticide, is more selective in its potency towards insects than humans and is thus commonly used. In this study, we demonstrated that exposure to fipronil may pose a human health risk. We observed in vitro the shortening of neurite outgrowths of SH-SY5Y neuroblastoma cells upon treatment with fipronil, even at a non-cytotoxic concentration. Fipronil induced apoptosis involving caspase-6, which is an apoptotic effector highly implicated in neurodegenerative diseases. Moreover, at a concentration that did not induce apoptosis, mitochondrial dysfunction and autophagic vacuole formation were detected. Interestingly using proteomics, we identified vimentin to be dramatically expressed by SH-SY5Y cells as a response to fipronil treatment. Not only did the expression of total vimentin increase, different isoforms were observed, indicating alterations in post-translational modifications. Vimentin was localized at the neurite outgrowth, possibly to repair the damage in cellular structure. However at high concentrations of fipronil, vimentin was found in less defined fibrils, in bridge-like formation, and dense surrounding vacuoles. In all, our results indicate that vimentin plays an important role in fipronil-induced neurotoxicity in SH-SY5Y cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The Neuroprotective Effects of Brazilian Green Propolis on Neurodegenerative Damage in Human Neuronal SH-SY5Y Cells

    PubMed Central

    Ni, Junjun; Meng, Jie; Zhu, Aiqin; Zhong, Xin; Wu, Shizheng; Nakanishi, Hiroshi

    2017-01-01

    Oxidative stress and synapse dysfunction are the major neurodegenerative damage correlated to cognitive impairment in Alzheimer's disease (AD). We have found that Brazilian green propolis (propolis) improves the cognitive functions of mild cognitive impairment patients living at high altitude; however, mechanism underlying the effects of propolis is unknown. In the present study, we investigated the effects of propolis on oxidative stress, expression of brain-derived neurotrophic factor (BDNF), and activity-regulated cytoskeleton-associated protein (Arc), the critical factors of synapse efficacy, using human neuroblastoma SH-SY5Y cells. Pretreatment with propolis significantly ameliorated the hydrogen peroxide- (H2O2-) induced cytotoxicity in SH-SY5Y cells. Furthermore, propolis significantly reduced the H2O2-generated reactive oxygen species (ROS) derived from mitochondria and 8-oxo-2′-deoxyguanosine (8-oxo-dG, the DNA oxidative damage marker) but significantly reversed the fibrillar β-amyloid and IL-1β-impaired BDNF-induced Arc expression in SH-SY5Y cells. Furthermore, propolis significantly upregulated BDNF mRNA expression in time- and dose-dependent manners. In addition, propolis induced Arc mRNA and protein expression via phosphoinositide-3 kinase (PI3K). These observations strongly suggest that propolis protects from the neurodegenerative damage in neurons through the properties of various antioxidants. The present study provides a potential molecular mechanism of Brazilian green propolis in prevention of cognitive impairment in AD as well as aging. PMID:28265338

  19. Nicotinamide N-methyltransferase increases complex I activity in SH-SY5Y cells via sirtuin 3.

    PubMed

    Liu, Karolina Y; Mistry, Rakhee J; Aguirre, Carlos A; Fasouli, Eirini S; Thomas, Martin G; Klamt, Fábio; Ramsden, David B; Parsons, Richard B

    2015-11-20

    Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to 1-methylnicotinamide. We have previously shown that NNMT is significantly overexpressed in the brains of patients who have died of Parkinson's disease, and others have shown that NNMT is significantly overexpressed in a variety of diseases ranging from cancer to hepatic cirrhosis. In vitro overexpression has revealed many cytoprotective effects of NNMT, in particular increased complex I activity and ATP synthesis. Although this appears to be mediated by an increase in 1-methylnicotinamide production, the molecular mechanisms involved remain unclear. In the present study, we have investigated the role that sirtuins 1, 2 and 3, class III DNA deacetylase enzymes known to regulate mitochondrial energy production and cell cycle, have in mediating the effects of NNMT upon complex I activity. Expression of NNMT in SH-SY5Y human neuroblastoma cells, which have no endogenous expression of NNMT, significantly increased the expression of all three sirtuins. siRNA-mediated silencing of sirtuin 3 expression decreased complex I activity in NNMT-expressing SH-SY5Y cells to that observed in wild-type SH-SY5Y, and significantly reduced cellular ATP content also. These results demonstrate that sirtuin 3 is a key mediator of NNMT-induced complex I activity and ATP synthesis. These results further reinforce a central role for NNMT in the regulation of energy homeostasis and provide further mechanistic insight into the consequences of enhanced NNMT expression.

  20. The Neuroprotective Effects of Brazilian Green Propolis on Neurodegenerative Damage in Human Neuronal SH-SY5Y Cells.

    PubMed

    Ni, Junjun; Wu, Zhou; Meng, Jie; Zhu, Aiqin; Zhong, Xin; Wu, Shizheng; Nakanishi, Hiroshi

    2017-01-01

    Oxidative stress and synapse dysfunction are the major neurodegenerative damage correlated to cognitive impairment in Alzheimer's disease (AD). We have found that Brazilian green propolis (propolis) improves the cognitive functions of mild cognitive impairment patients living at high altitude; however, mechanism underlying the effects of propolis is unknown. In the present study, we investigated the effects of propolis on oxidative stress, expression of brain-derived neurotrophic factor (BDNF), and activity-regulated cytoskeleton-associated protein (Arc), the critical factors of synapse efficacy, using human neuroblastoma SH-SY5Y cells. Pretreatment with propolis significantly ameliorated the hydrogen peroxide- (H2O2-) induced cytotoxicity in SH-SY5Y cells. Furthermore, propolis significantly reduced the H2O2-generated reactive oxygen species (ROS) derived from mitochondria and 8-oxo-2'-deoxyguanosine (8-oxo-dG, the DNA oxidative damage marker) but significantly reversed the fibrillar β-amyloid and IL-1β-impaired BDNF-induced Arc expression in SH-SY5Y cells. Furthermore, propolis significantly upregulated BDNF mRNA expression in time- and dose-dependent manners. In addition, propolis induced Arc mRNA and protein expression via phosphoinositide-3 kinase (PI3K). These observations strongly suggest that propolis protects from the neurodegenerative damage in neurons through the properties of various antioxidants. The present study provides a potential molecular mechanism of Brazilian green propolis in prevention of cognitive impairment in AD as well as aging.

  1. Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

    PubMed Central

    Mazot, Pierre; Cazes, Alex; Dingli, Florent; Degoutin, Joffrey; Irinopoulou, Théano; Boutterin, Marie-Claude; Lombard, Bérangère; Loew, Damarys; Hallberg, Bengt; Palmer, Ruth Helen; Delattre, Olivier

    2012-01-01

    Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization. PMID:22479414

  2. [The role of neuroglobin in oxygen-glucose deprivation and reoxygenation-induced mitochondrial depolarization and reactive oxygen species production in SH-SY5Y cells].

    PubMed

    Deng, S Y; Ai, Y H; Zhang, L N; Wu, L; Chen, C X; Wang, Y M; Liu, Z Y; Huang, L; Peng, Q Y

    2017-01-01

    Objective: To investigate the role of neuroglobin (NGB) in oxygen-glucose deprivation and reoxygenation (OGD/R) induced mitochondrial depolarization and reactive oxygen species (ROS)production in a human neuroblastoma cell line (SH-SY5Y). Methods: SH-SY5Y cells were transfected with lentivirus to establish a stable cell line of NGB knockdown (KD). After treated with OGD/R, cells were collected at different time points to analyze NGB mRNA and protein levels. Furthermore, cells were stained with JC-1 and DCFH-DA to evaluate mitochondrial depolarization and ROS production by inverted fluorescence microscope. Also, to determine the neurotoxicity, we measured the lactate dehydrogenase(LDH)level in the cell culture medium. Results: After the treatment of OGD/R, the NGB mRNA and protein started to elevate and peak at 4 h and 8 h (2.04±0.35 fold, 1.69±0.18 fold). Compared with the vector group, NGB KD group had much more mitochondrial depolarization [JC-1 red/green (1.10±0.10) vs (1.46±0.11), P<0.05] and ROS production [DCFH-DA fluorescence (36.30±5.32) vs (16.26±2.97), P<0.05]. Furthermore, NGB KD groups had a higher level of LDH release [(63.42±6.14)%vs (49.65±5.09)%, P<0.05]. Conclusions: NGB plays an important role in the homeostasis of mitochondria. Knockdown of NGB results in increased mitochondrial depolarization, ROS production and neurotoxicity under hypoxia circumstances.

  3. Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways.

    PubMed

    Dayem, Ahmed Abdal; Kim, BongWoo; Gurunathan, Sangiliyandi; Choi, Hye Yeon; Yang, Gwangmo; Saha, Subbroto Kumar; Han, Dawoon; Han, Jihae; Kim, Kyeongseok; Kim, Jin-Hoi; Cho, Ssang-Goo

    2014-07-01

    Nano-scale materials are noted for unique properties, distinct from those of their bulk material equivalents. In this study, we prepared spherical silver nanoparticles (AgNPs) with an average size of about 30 nm and tested their potency to induce neuronal differentiation of SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells are considered an ideal in vitro model for studying neurogenesis, as they can be maintained in an undifferentiated state or be induced to differentiate into neuron-like phenotypes in vitro by several differentiation-inducing agents. Treatment of SH-SY5Y cells by biologically synthesized AgNPs led to cell morphological changes and significant increase in neurite length and enhanced the expression of neuronal differentiation markers such as Map-2, β-tubulin III, synaptophysin, neurogenin-1, Gap-43, and Drd-2. Furthermore, we observed an increase in generation of intracellular reactive oxygen species (ROS), activation of several kinases such as ERK and AKT, and downregulation of expression of dual-specificity phosphatases (DUSPs) in AgNPs-exposed SH-SY5Y cells. Our results suggest that AgNPs modulate the intracellular signaling pathways, leading to neuronal differentiation, and could be applied as promising nanomaterials for stem cell research and therapy.

  4. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    PubMed Central

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-01-01

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells. PMID:27916824

  5. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines.

    PubMed

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-11-29

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  6. Fumonisin B1-induced apoptosis in neuroblastoma, glioblastoma and hypothalamic cell lines.

    PubMed

    Stockmann-Juvala, Helene; Naarala, Jonne; Loikkanen, Jarkko; Vähäkangas, Kirsi; Savolainen, Kai

    2006-08-15

    Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticilliodes, which commonly infects corn across the world. Fusarium fungi may also be found in moisture-damaged buildings. In this study, we investigated the role of apoptosis in the toxicity of FB(1) in four different cell lines. Activation of caspase-3-like protease, DNA fragmentation and expression of p53 and Bcl-2 family proteins were studied in mouse GT1-7 hypothalamic, rat C6 glioblastoma, human U-118MG glioblastoma, and human SH-SY5Y neuroblastoma cells exposed to 0.1-100microM FB(1) for 0-144h. Caspase-3-like protease activity increased in all cell lines, except SH-SY5Y, at 48-144h, and internucleosomal DNA fragmentation occurred in all of the cell lines, pointing to a role for apoptosis in the toxicity of FB(1). However, the expressions of p53 or pro- or antiapoptotic Bcl-2 family proteins (Bax, Bcl-2, Bcl-X(L) and Mcl-1) were not affected in any of the cell lines even after prolonged exposure to FB(1) at high doses. The results of this study, together with the results of our previous studies, provide evidence that FB(1) is a potential neurotoxin, but that the toxicity of FB(1) varies between different cell lines. The sensitivity of these cell lines towards FB(1) is as follows: U-118MG>GT1-7>C6>SH-SY5Y cells. These results are consistent with the assumption that cells of glial origin may be more sensitive towards FB(1) than cells of neural origin.

  7. Fluoxetine Increases the Expression of miR-572 and miR-663a in Human Neuroblastoma Cell Lines

    PubMed Central

    Mundalil Vasu, Mahesh; Anitha, Ayyappan; Takahashi, Taro; Thanseem, Ismail; Iwata, Keiko; Asakawa, Tetsuya; Suzuki, Katsuaki

    2016-01-01

    Evidence suggests neuroprotective effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on the developed neurons in the adult brain. In contrast, the drug may be deleterious to immature or undifferentiated neural cells, although the mechanism is unclear. Recent investigations have suggested that microRNAs (miRNA) may be critical for effectiveness of psychotropic drugs including SSRI. We investigated whether fluoxetine could modulate expressions of neurologically relevant miRNAs in two neuroblastoma SK-N-SH and SH-SY5Y cell lines. Initial screening results revealed that three (miR-489, miR-572 and miR-663a) and four (miR-320a, miR-489, miR-572 and miR-663a) miRNAs were up-regulated in SK-N-SH cells and SH-SY5Y cells, respectively, after 24 hours treatment of fluoxetine (1–25 μM). Cell viability was reduced according to the dose of fluoxetine. The upregulation of miR-572 and miR-663a was consistent in both the SH-SY5Y and SK-N-SH cells, confirmed by a larger scale culture condition. Our data is the first in vitro evidence that fluoxetine could increase the expression of miRNAs in undifferentiated neural cells, and that putative target genes of those miRNAs have been shown to be involved in fundamental neurodevelopmental processes. PMID:27716787

  8. Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

    PubMed

    Filomeni, Giuseppe; Cardaci, Simone; Da Costa Ferreira, Ana Maria; Rotilio, Giuseppe; Ciriolo, Maria Rosa

    2011-08-01

    We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

  9. Glutathione adduct of methylmercury activates the Keap1-Nrf2 pathway in SH-SY5Y cells.

    PubMed

    Yoshida, Eiko; Abiko, Yumi; Kumagai, Yoshito

    2014-10-20

    Methylmercury (MeHg) reacts readily with GSH, leading to the formation of a MeHg-SG adduct that is excreted into extracellular space through multidrug-resistance-associated protein (MRP), which is regulated by the transcription factor Nrf2. We previously reported that MeHg covalently modifies Keap1 and activates Nrf2 in human neuroblastoma SH-SY5Y cells. In the study presented here, we examined whether the MeHg-SG adduct could also modulate the Keap1-Nrf2 pathway because the formation of the Hg-S bond is believed to be reversible in the presence of a nucleophile. SH-SY5Y cells exposed to the synthetic ethyl monoester of the MeHg-SG adduct (which is hydrolyzed by cellular esterase(s) to give the MeHg-SG adduct) exhibited a concentration-dependent cellular toxicity that was enhanced by pretreatment with a specific MRP inhibitor. As expected, the MeHg-SG adduct was able to modify cellular proteins in the SH-SY5Y cells and purified Keap1. We also found that this prodrug, as well as MeHg, causes the cellular Keap1 in the cells to be modified, resulting in Nrf2 activation and, thereby, the upregulation of the downstream genes. These results suggest that the MeHg-SG adduct is not electrophilic but that it modifies protein thiols (including Keap1) through S-transmercuration and that rapid Nrf2-dependent excretion of the MeHg-SG adduct is essential in decreasing the cytotoxicity of MeHg.

  10. Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells.

    PubMed

    Yshii, Lidia M; Denadai-Souza, Alexandre; Vasconcelos, Andrea R; Avellar, Maria Christina W; Scavone, Cristoforo

    2015-10-26

    Parkinson's disease (PD) is a neurodegenerative disease with characteristics and symptoms that are well defined. Nevertheless, its aetiology remains unknown. PD is characterized by the presence of Lewy bodies inside neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD pathogenesis, given that it is an important component of Lewy bodies localized in the dopaminergic neurons of PD patients. In the present study, we investigated the influence of wild type (WT) and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the conditioned medium (CM) from primary cultures of glia challenged with lipopolysaccharide (LPS) from Escherichia coli. We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show evidence of cell death, which is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50 (NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT α-syn or control group. This effect of A30P α-syn may be due to the low MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by PD98059, decreasing LDH release by CM in SH-SY5Y cells. Our results suggest that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show cell death, which is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of activated glia, when compared to WT α-syn or control group. The mechanism underlying this process remains to be completely elucidated, but the present data suggest that MAPK42/44 phosphorylation plays an important role in this process. CRD42015020829.

  11. Notch activation induces neurite remodeling and functional modifications in SH-SY5Y neuronal cells.

    PubMed

    Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Uberti, Daniela; Napolitano, Francesco; Stante, Maria; Santoro, Federica; Minopoli, Giuseppina; Zambrano, Nicola; Russo, Tommaso; Memo, Maurizio

    2009-05-01

    Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.

  12. Alteration in MARCKS phosphorylation and expression by methylmercury in SH-SY5Y cells and rat brain.

    PubMed

    Shiraishi, Mitsuya; Hangai, Makoto; Yamamoto, Megumi; Sasaki, Masanori; Tanabe, Atsuhiro; Sasaki, Yasuharu; Miyamoto, Atsushi

    2014-05-01

    The molecular mechanisms mediating methylmercury (MeHg)-induced neurotoxicity are not completely understood. Because myristoylated alanine-rich C kinase substrate (MARCKS) plays an essential role in the differentiation and development of neuronal cells, we studied the alteration of MARCKS expression and phosphorylation in MeHg-induced neurotoxicity of neuroblastoma SH-SY5Y cells and in the rat brain. Exposure to MeHg induced a decrease in cell viability of SH-SY5Y cells, which was accompanied by a significant increase in phosphorylation and a reduction in MARCKS expression. Pretreatment of cells with a protein kinase C inhibitor or an extracellular Ca(2+) chelator suppressed MeHg-induced MARCKS phosphorylation. In MARCKS knock-down cells, MeHg-induced cell death was significantly augmented in comparison to control siRNA. In brain tissue from MeHg-treated rats, MARCKS phosphorylation was enhanced in the olfactory bulb in comparison to control rats. The present study may indicate that alteration in MARCKS expression or phosphorylation has consequences for MeHg-induced neurotoxicity.

  13. Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.

    PubMed

    Tian, Lin-Lin; Wang, Xue-Jun; Sun, Yu-Ning; Li, Chun-Rong; Xing, Ya-Ling; Zhao, Hai-Bao; Duan, Ming; Zhou, Zhe; Wang, Sheng-Qi

    2008-01-01

    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.

  14. Oxidative stress and gene expression profiling of cell death pathways in alpha-cypermethrin-treated SH-SY5Y cells.

    PubMed

    Romero, Alejandro; Ramos, Eva; Ares, Irma; Castellano, Víctor; Martínez, Marta; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Martínez, María-Aránzazu

    2017-05-01

    In this study, we investigated the induction of oxidative stress and apoptosis in human neuroblastoma cell line SH-SY5Y in response to alpha-cypermethrin (α-CYPER) exposure. MTT and LDH assays were carried out to assess the α-CYPER cytotoxicity. The IC50 value for α-CYPER was calculated to be 78.3 ± 2.98 µM for the MTT assay and 71.5 ± 3.94 µM for LDH assay. The pyrethroid α-CYPER (1-100 µM), in a dose-dependent manner, induced a significant increase in lipid peroxides measured as malondialdehyde (MDA) and in the levels of nitric oxide (NO). The neuroprotective role of three antioxidants, melatonin (MEL), Trolox and N-acetylcysteine (NAC) against α-CYPER-induced oxidative stress was examined. Compared to other antioxidants, MEL (1 µM) treatment showed the most effective protection against α-CYPER-induced lipid peroxidation and NO production. The effects of α-CYPER on gene expression profiling of cell death pathway in human neuroblastoma SH-SY5Y cells were also investigated. Of the 84 genes examined (P < 0.001; fold change >1.5), changes in mRNA levels were detected in 39 genes: 36 were up-regulated and 3 were down-regulated. A greater fold change reversion than 3.5-fold was observed on the up-regulated ATP6V1G2, BCL2, CASP9, FAS, GADD45A, SPATA2, SYCP2, ATG7, NFKB1, SNCA, ULK1 and JPH3 genes. The results demonstrated that α-CYPER alters the expression of apoptosis-, autophagy- and necrosis genes as well as induces oxidative stress which may lead to DNA damage. The detailed knowledge of the changes in gene expression obtained will provide a basis for further elucidating the molecular mechanisms of the α-CYPER-induced toxicity.

  15. [Construction of acid-sensitive potassium channel-3 eukaryotic expression plasmid and its express in SH-SY5Y cells].

    PubMed

    Wei, Lin-yu; Li, Xin-juan; Mei, Yi-wen; Wang, Guo-hong; Wang, Qi; Li, Dong-liang; Li, Chao-kun

    2015-05-01

    To construct the acid-sensitive potassium hannel-3(TASK3) eukaryotic expression plasmid and to establish a stable SH-SY5Y cell line expressing enhanced green fluorescent protein (EGFP)-tagged TASK3. TASK3 coding region was subcloned into pEGFP-N1 plasmid to construct a recombinant vector alled pEGFP-TASK3. The correct recombinant expressing plasmid was transfected with X-feet transfection reagent to SH-SY5Y cells. The cell line stably expressiing EGFP tagged-TASK3 gene was established by screening with antibiotic G418 and fluorescence microscope. The expression and localization of the EGFP tagged-TASK3 fusion protein was detected by Western blot and confocal microscope. Exposure of the SH-SY5Y cell line expressing stably TASK3-eGFP fusion proteins was exposed to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability was assessed with cell counting Kit-8 (CCK-8). All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pEGFP-TASK3 was constructed correctly. The stable SH-SY5Y cell line expressing EGFP tagged-TASK3 fusion protein was successfully established. Exposure of the wild type SH-SY5Y cells and the stable SH-SY5Y-GFP tag-TASK3 cell line to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability of two group cells significantly reduced with pH declining, and the difference was statistically significant (P < 0.05). Compared with wild type SH-SY5Y cells, the cell viability of stable SH-SYSY-GFP tag-TASK3 cell line increased significantly with the same pH media, and the difference was statistically significant (P < 0.05). The eukaryotic expression vector pEGFP-TASK3 is successfully constructed and the cell line stably expressing TASK3-eGFP fusion is established which is important for their fundamental research and potential applications.

  16. Bryostatin-1 vs. TPPB: dose-dependent APP processing and PKC-α, -δ, and -ε isoform activation in SH-SY5Y neuronal cells.

    PubMed

    Yi, P; Schrott, L; Castor, T P; Alexander, J S

    2012-09-01

    Activation of the α-secretase processing pathway of amyloid precursor protein (APP) is recognized as an important mechanism which diverts APP processing from production of beta-amyloid (Aβ) to non toxic sAPPα, decreasing Alzheimer's disease (AD) plaque formation and AD-associated cognitive deficits. Two potent classes of PKC modulators can activate the α-secretase pathway, the benzo/indolactams and bryostatin/bryologues. While both modulate PKC-dependent APP processing, no direct comparisons of their relative pharmacological potencies have been accomplished which could assist in the development of AD therapies. In this study, we measured the activation of α-secretase APP processing and PKC-α, -δ, and -ε induced by the benzolactam-APP modulator TPPB and bryostatin-1 in the neuroblastoma cell line SH-SY5Y which expresses APP and α- and β-secretase processing mechanisms. Bryostatin-1 produced a more rapid, potent, and sustained activation of α-secretase APP processing than TPPB and selectively activated PKC-δ and PKC-ε. Although TPPB also activated α-secretase, its potency was approximately 10- to 100-fold lower, possibly reflecting lower PKC-δ and -ε activation. Because bryostatin-1 is a highly potent PKC-δ and -ε activator which activates α-secretase APP processing, further characterization of bryostatin-1/bryologues may help refine their use as important tools for the clinical management of AD.

  17. Wnt co-receptor LRP5/6 overexpression confers protection against hydrogen peroxide-induced neurotoxicity and reduces tau phosphorylation in SH-SY5Y cells.

    PubMed

    Zhang, Luqi; Bahety, Priti; Ee, Pui Lai Rachel

    2015-08-01

    Emerging studies have suggested the involvement of dysregulated Wnt/β-catenin cascade in the etiology of Alzheimer's disease (AD). Recently, genetic variations in Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 6 causing reduced Wnt signaling has been linked to late-onset AD. Here, we hypothesized that overexpression of Wnt co-receptors LRP5 and LRP6 would serve as an effective new approach in reducing neurotoxicity induced by oxidative stress and decreasing tau phosphorylation in SH-SY5Y human neuroblastoma cells. Our results showed that overexpression of LRP5 and LRP6 in SH-SY5Y cells activates Wnt signaling and downstream proliferation genes, whereas knockdown of the co-receptors represses Wnt signaling and the transcription of proliferative markers. We further demonstrated that overexpression of LRP5 and LRP6 protects SH-SY5Y from cell death caused by hydrogen peroxide-induced oxidative stress, inhibits GSK3β activity and subsequently reduces tau phosphorylation. Together, our findings suggest that rescuing LRP5/6-mediated Wnt signaling improves neuronal cell survival and reduces tau phosphorylation, which support the hypothesis that Wnt signaling might be an attractive therapeutic strategy for managing AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Protection of seven dibenzocyclooctadiene lignans from Schisandra chinensis against serum and glucose deprivation injury in SH-SY5Y cells.

    PubMed

    E, Qun; Tang, Miao; Zhang, XiaoChuan; Shi, YunWei; Wang, DanDan; Gu, Yun; Li, ShiYing; Liang, XinMiao; Wang, ZhiWei; Wang, CaiPing

    2015-12-01

    Dibenzocyclooctadiene lignans, the major active components of fruit of Schisandra chinensis (Turcz.) Baill., have been found to have activities that could prevent prostate and thyroid cancer, hepatotoxicity, oxidative stress-induced cerebral injury, etc. This study was conducted to evaluate the effects of seven dibenzocyclooctadiene lignans of Schisandra chinensis and explore the possible mechanisms in the human neuroblastoma SH-SY5Y cells exposed on serum and glucose deprivation (SGD) injury. The structure-activity relationships were also analyzed. Cell viability and lactate dehydrogenase (LDH) release were determined to evaluate cell injury. Inflammation and apoptosis-related protein levels were detected to elucidate the possible mechanisms. Schisantherin A, schizandrin C, and schizandrol B were found to have stronger protective effects than schizandrin A, schizandrin B, and schisanhenol in SH-SY5Y cells against SGD injury. Moreover, the protective effects of these lignans were possibly exhibited by regulating inflammation and apoptosis-related proteins in SH-SY5Y cells after SGD injury, supporting their beneficial effects for the prevention of cell injury in the pathogenesis of the central nervous system diseases, including ischemia stroke. The number and position of hydroxyl group and methylenedioxy in these lignans may be required for their effects.

  19. Novel incretin analogues improve autophagy and protect from mitochondrial stress induced by rotenone in SH-SY5Y cells.

    PubMed

    Jalewa, Jaishree; Sharma, Mohit Kumar; Hölscher, Christian

    2016-10-01

    Currently, there is no viable treatment available for Parkinson's disease (PD) that stops or reverses disease progression. Interestingly, studies testing the glucagon-like-peptide-1 (GLP-1) mimetic Exendin-4 have shown neuroprotective/neurorestorative properties in pre-clinical tests and in a pilot clinical study of PD. Incretin analogues were originally developed to treat type 2 diabetes and several are currently on the market. In this study, we tested novel incretin analogues on the dopaminergic SH-SY5Y neuroblastoma cells against a toxic mitochondrial complex I inhibitor, Rotenone. Here, we investigate for the first time the effects of six different incretin receptor agonists - Liraglutide, D-Ser2-Oxyntomodulin, a GLP-1/GIP Dual receptor agonist, dAla(2)-GIP-GluPal, Val(8)GLP-1-GluPal and exendin-4. Post-treatment with doses of 1, 10 or 100 nM of incretin analogues for 12 h increased the survival of SH-SY5Y cells treated with 1 μM Rotenone for 12 h. Furthermore, we studied the post-treatment effect of 100 nM incretin analogues against 1 μM Rotenone stress on apoptosis, mitochondrial stress and autophagy markers. We found significant protective effects of the analogues against Rotenone stress on cell survival and on mitochondrial and autophagy-associated markers. The novel GLP-1/GIP Dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. Using the Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we further show that the neuroprotective effects are partially PI3K-independent. Our data suggest that the neuroprotective properties exhibited by incretin analogues against Rotenone stress involve enhanced autophagy, increased Akt-mediated cell survival and amelioration of mitochondrial dysfunction. These mechanisms can explain the neuroprotective effects of incretin analogues reported in clinical trials. GLP-1, GIP and dual incretin receptor agonists showed protective effects in SH-SY5Y cells

  20. Comparative non-cholinergic neurotoxic effects of paraoxon and diisopropyl fluorophosphate (DFP) on human neuroblastoma and astrocytoma cell lines

    SciTech Connect

    Qian Yongchang; Venkatraj, Jijayanagaram; Barhoumi, Rola; Pal, Ranadip; Datta, Aniruddha; Wild, James R.; Tiffany-Castiglioni, Evelyn . E-mail: ecastiglioni@cvm.tamu.edu

    2007-03-15

    The objective of this study was to evaluate the comparative non-cholinergic neurotoxic effects of paraoxon, which is acutely neurotoxic, and diisopropyl fluorophosphate (DFP), which induces OPIDN, in the human neuroblastoma SY5Y and the human astrocytoma cell line CCF-STTG1. SY5Y cells have been studied extensively as a model for OP-induced neurotoxicity, but CCF cells have not previously been studied. We conducted a preliminary human gene array assay of OP-treated SY5Y cells in order to assess at the gene level whether these cells can distinguish between OP compounds that do and do not cause OPIDN. Paraoxon and DFP induced dramatically different profiles of gene expression. Two genes were upregulated and 13 downregulated by at least 2-fold in paraoxon-treated cells. In contrast, one gene was upregulated by DFP and none was downregulated at the 2-fold threshold. This finding is consistent with current and previous observations that SY5Y cells can distinguish between OPs that do or do not induce OPIDN. We also examined gene array results for possible novel target proteins or metabolic pathways for OP neurotoxicity. Protein levels of glucose regulated protein 78 (GRP78) revealed that paraoxon exposure at 3 {mu}M for 24 h significantly reduced GRP78 levels by 30% in neuroblastoma cells, whereas DFP treatment had no effect. In comparison with SY5Y neuroblastoma cells, paraoxon and DFP (3 {mu}M for 24 h) each significantly increased GRP78 levels by 23-24% in CCF astrocytoma cells. As we have previously evaluated intracellular changes in Ca{sup 2+} levels in SY5Y cells, we investigated the effects of paraoxon and DFP on cellular Ca{sup 2+} homeostasis in CCF by studying cytosolic and mitochondrial basal calcium levels. A significant decrease in the ratio of mitochondrial to cytosolic Ca{sup 2+} fluorescence was detected in CCF cultures treated for either 1 or 3 days with 1, 3, 10, or 30 {mu}M paraoxon. In contrast, treatment with DFP for 1 day had no significant effect

  1. ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

    PubMed

    Espada, Lilia; Meo-Evoli, Nathalie; Sancho, Patricia; Real, Sebastian; Fabregat, Isabel; Ambrosio, Santiago; Tauler, Albert

    2012-01-01

    In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

  2. Dynamin-related protein 1 mediates mitochondria-dependent apoptosis in chlorpyrifos-treated SH-SY5Y cells.

    PubMed

    Park, Jae Hyeon; Ko, Juyeon; Hwang, Jungwook; Koh, Hyun Chul

    2015-12-01

    Recent studies have demonstrated that dynamin-related protein 1 (Drp1), a mitochondrial fission protein, mediates mitochondria-dependent apoptosis through mitochondrial division. However, little is known about the mechanism by which Drp1 modulates apoptosis in response to chlorpyrifos (CPF)-induced toxicity. In this study, we determined that CPF-induced mitochondrial apoptosis is mediated by Drp1 translocation in SH-SY5Y human neuroblastoma cells. Our results showed that CPF treatment induced intrinsic apoptosis by activating caspase-9, caspase-3, and cytochrome c release in SH-SY5Y cells. Cytosolic Drp1 translocated to the mitochondria in CPF-treated cells and was phosphorylated at Ser616. Treating cells with CPF induced the generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs). Inhibiting this ROS generation and MAPK activation abolished CPF-induced expression of phospho-Drp1. Furthermore, Drp1 was required for p53 to translocate to the mitochondria under CPF-induced oxidative stress. Treating cells with mitochondrial-division inhibitor-1 (mdivi-1), which blocks Drp1 translocation, increased the viability of CPF-treated cells by abrogating Drp1 translocation and caspase-3 activation. Specifically, pretreating cells with mdivi-1 inhibited Bax translocation to the mitochondria by blocking p53 signaling. Taken together, these data reveal a novel mechanism by which Drp1 activates mitochondrial-dependent apoptosis and indicate that inhibiting Dpr1 function can protect against CPF-induced cytotoxicity. We propose that inhibiting Drp1 is a possible therapeutic approach for pesticide-induced toxicity when hyperactivated Drp1 contributes to pathology.

  3. Epigenetic mechanisms of amyloid-β production in anisomycin-treated SH-SY5Y cells.

    PubMed

    Guo, X; Wu, X; Ren, L; Liu, G; Li, L

    2011-10-27

    Oxidative stress and inflammation as the pathological components of Alzheimer's disease (AD) have been well understood. Among a diversity of mitogen-activated protein kinase (MAPK) family members, JNK and p38 MAPK subfamilies are relevant to the response of environmental stress, inflammatory stimuli, or other insults. Recent studies have demonstrated that epigenetic mechanisms may play a pivotal role in AD pathogenesis and development. In the present study, we have investigated epigenetic mechanisms such as DNA methylation and histone acetylation involved in the activation of stress-related signaling pathways for amyloid-β (Aβ) production. Human neuroblastoma SH-SY5Y cells were treated by anisomycin, an activator of stress-related MAPKs (JNK and p38 MAPK). A significant increase of intracellular Aβ level in anisomycin-treated SH-SY5Y cells was observed. The expression of amyloid-β precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1), and presenilin 1 (PS1) was upregulated by demethylation in three gene promoters associated with the reduction of methyltransferases (DNMTs). Meanwhile, an enhanced level of global histone H3 acetylation accompanied with upregulation of histone acetyltransferases p300/CREB-binding protein (CBP) and downregulation of histone deacetylases (HDACs) was also observed. These findings indicated that the activation of stress-related signaling pathways could result in the increased transcription of APP, BACE1, and PS1 genes through DNMT-dependent hypomethylation and histone H3 hyperacetylation, thus leading to Aβ overproduction. Moreover, our findings provided a novel insight into epigenetic mechanisms by which oxidative stress contributes to the pathogenesis of AD. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Action of HMGB1 on miR-221/222 cluster in neuroblastoma cell lines

    PubMed Central

    Mari, Emanuela; Zicari, Alessandra; Fico, Flavia; Massimi, Isabella; Martina, Lolli; Mardente, Stefania

    2016-01-01

    microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and −222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for

  5. Action of HMGB1 on miR-221/222 cluster in neuroblastoma cell lines.

    PubMed

    Mari, Emanuela; Zicari, Alessandra; Fico, Flavia; Massimi, Isabella; Martina, Lolli; Mardente, Stefania

    2016-09-01

    microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and -222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for

  6. Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

    PubMed Central

    Korecka, Joanna A.; van Kesteren, Ronald E.; Blaas, Eva; Spitzer, Sonia O.; Kamstra, Jorke H.; Smit, August B.; Swaab, Dick F.; Verhaagen, Joost; Bossers, Koen

    2013-01-01

    Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. PMID:23724009

  7. The involvement of Eag1 potassium channels and miR-34a in rotenone-induced death of dopaminergic SH-SY5Y cells.

    PubMed

    Horst, Camila Hillesheim; Titze-de-Almeida, Ricardo; Titze-de-Almeida, Simoneide Souza

    2017-04-01

    The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH‑SY5Y cell line is a model of dopaminergic neurons, and allows for the study of dopaminergic neuronal injury. Previous studies have revealed changes in Ether à go‑go 1 (Eag1) potassium channel expression during p53-induced SH‑SY5Y apoptosis, and the regulatory involvement of microRNA‑34a (miR‑34a) was demonstrated. In the present study, the involvement of Eag1 and miR‑34a in rotenone‑induced SH‑SY5Y cell injury was investigated. Rotenone is a neurotoxin, which is often used to generate models of Parkinson's disease, since it causes the death of nigrostriatal neurons by inducing intracellular aggregation of alpha synuclein and ubiquitin. In the present study, rotenone resulted in a dose‑dependent decrease in cell viability, as revealed by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) and trypan blue cell counting assays. In addition, Eag1 was demonstrated to be constitutively expressed by SH‑SY5Y cells, and involved in cell viability. Suppression of Eag1 with astemizole resulted in a dose‑dependent decrease in cell viability, as revealed by MTT assay. Astemizole also enhanced the severity of rotenone‑induced injury in SH‑SY5Y cells. RNA interference against Eag1, using synthetic small interfering RNAs (siRNAs), corroborated this finding, as siRNAs potentiated rotenone‑induced injury. Eag1‑targeted siRNAs (kv10.1‑3 or EAG1hum_287) resulted in a statistically significant 16.4‑23.5% increase in vulnerability to rotenone. An increased number of apoptotic nuclei were observed in cells transfected with EAG1hum_287. Notably, this siRNA intensified rotenone‑induced apoptosis, as revealed by an increase in caspase 3/7 activity. Conversely, a miR‑34a inhibitor was demonstrated to exert neuroprotective effects. The viability of cells exposed to rotenone for 24 or 48 h and treated with miR‑34a

  8. Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP⁺-induced oxidative stress and cell death in SH-SY5Y cells.

    PubMed

    Chong, Cheong-Meng; Shen, Mingyun; Zhou, Zhong-Yan; Pan, Peichen; Hoi, Pui-Man; Li, Shang; Liang, Wang; Ai, Nana; Zhang, Lun-Qing; Li, Cheuk-Wing; Yu, Huidong; Hou, Tingjun; Lee, Simon Ming-Yuen

    2014-09-01

    Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP(+)-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP(+)-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP(+)-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling. Copyright © 2014. Published by Elsevier Inc.

  9. Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

    PubMed Central

    Schneider, Lonnie; Giordano, Samantha; Zelickson, Blake R.; Johnson, Michelle; Benavides, Gloria; Ouyang, Xiaosen; Fineberg, Naomi; Darley-Usmar, Victor M.; Zhang, Jianhua

    2011-01-01

    Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors, however the mechanisms remain unclear. We hypothesized that since the regulation of glycolysis and oxidative phosphorylation are modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis, 2-deoxy-D-glucose (2-DG). Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by reactive lipid species 4-hydroxynonenal (HNE) or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur

  10. Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.

    PubMed

    Dalzell, Abigail M; Mistry, Pratibha; Wright, Jayne; Williams, Faith M; Brown, Colin D A

    2015-06-15

    ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02μM (ivermectin); 0.60±0.07 and 0.56±0.02μM (emamectin) and 0.95±0.08 and 0.77±0.25μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72μM (ivermectin); 1.87±0.57 and 2.74±1.01μM (emamectin) and 2.25±0.01 and 1.68±0.63μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1μM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of

  11. 17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

    PubMed

    Adeosun, Samuel O; Albert, Paul R; Austin, Mark C; Iyo, Abiye H

    2012-05-01

    Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

  12. Extreme sensitivity of gene expression in human SH-SY5Y neurocytes to ultra-low doses of Gelsemium sempervirens

    PubMed Central

    2014-01-01

    Background Gelsemium sempervirens L. (Gelsemium s.) is a traditional medicinal plant, employed as an anxiolytic at ultra-low doses and animal models recently confirmed this activity. However the mechanisms by which it might operate on the nervous system are largely unknown. This work investigates the gene expression of a human neurocyte cell line treated with increasing dilutions of Gelsemium s. extract. Methods Starting from the crude extract, six 100 × (centesimal, c) dilutions of Gelsemium s. (2c, 3c, 4c, 5c, 9c and 30c) were prepared according to the French homeopathic pharmacopoeia. Human SH-SY5Y neuroblastoma cells were exposed for 24 h to test dilutions, and their transcriptome compared by microarray to that of cells treated with control vehicle solutions. Results Exposure to the Gelsemium s. 2c dilution (the highest dose employed, corresponding to a gelsemine concentration of 6.5 × 10-9 M) significantly changed the expression of 56 genes, of which 49 were down-regulated and 7 were overexpressed. Several of the down-regulated genes belonged to G-protein coupled receptor signaling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Fisher exact test, applied to the group of 49 genes down-regulated by Gelsemium s. 2c, showed that the direction of effects was significantly maintained across the treatment with high homeopathic dilutions, even though the size of the differences was distributed in a small range. Conclusions The study shows that Gelsemium s., a medicinal plant used in traditional remedies and homeopathy, modulates a series of genes involved in neuronal function. A small, but statistically significant, response was detected even to very low doses/high dilutions (up to 30c), indicating that the human neurocyte genome is extremely sensitive to this regulation. PMID:24642002

  13. 17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y Cells

    PubMed Central

    Adeosun, Samuel O.; Albert, Paul R.; Austin, Mark C; Iyo, Abiye H.

    2012-01-01

    NUDR/Deaf-1 (nuclear deformed epidermal autoregulatory factor-1) and Freud-1 (Five prime repressor element under dual repression) are novel transcriptional regulators of the 5-HT1A receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE2) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE2 –induced downregulation of the 5-HT1A receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10nM 17βE2 for 3- or 48-h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by Western blotting. 17βE2 treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT1A receptor showed significant decreases. Upon withdrawal of 17βE2, protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT1A receptor protein by 17βE2, which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory /homeostatic responses to changes in 5-HT1A receptor induced by 17βE2. These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT1A receptor expression. PMID:22328058

  14. Asiatic acid, a pentacyclic triterpene in Centella asiatica, attenuates glutamate-induced cognitive deficits in mice and apoptosis in SH-SY5Y cells

    PubMed Central

    Xu, Min-fang; Xiong, Yu-yun; Liu, Jian-kang; Qian, Jin-jun; Zhu, Li; Gao, Jing

    2012-01-01

    Aim: To investigate whether asiatic acid (AA), a pentacyclic triterpene in Centella asiatica, exerted neuroprotective effects in vitro and in vivo, and to determine the underlying mechanisms. Methods: Human neuroblastoma SH-SY5Y cells were used for in vitro study. Cell viability was determined with the MTT assay. Hoechst 33342 staining and flow cytometry were used to examine the apoptosis. The mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured using fluorescent dye. PGC-1α and Sirt1 levels were examined using Western blotting. Neonatal mice were given monosodium glutamate (2.5 mg/g) subcutaneously at the neck from postnatal day (PD) 7 to 13, and orally administered with AA on PD 14 daily for 30 d. The learning and memory of the mice were evaluated with the Morris water maze test. HE staining was used to analyze the pyramidal layer structure in the CA1 and CA3 regions. Results: Pretreatment of SH-SY5Y cells with AA (0.1–100 nmol/L) attenuated toxicity induced by 10 mmol/L glutamate in a concentration-dependent manner. AA 10 nmol/L significantly decreased apoptotic cell death and reduced reactive oxygen species (ROS), stabilized the mitochondrial membrane potential (MMP), and promoted the expression of PGC-1α and Sirt1. In the mice models, oral administration of AA (100 mg/kg) significantly attenuated cognitive deficits in the Morris water maze test, and restored lipid peroxidation and glutathione and the activity of SOD in the hippocampus and cortex to the control levels. AA (50 and 100 mg/kg) also attenuated neuronal damage of the pyramidal layer in the CA1 and CA3 regions. Conclusion: AA attenuates glutamate-induced cognitive deficits of mice and protects SH-SY5Y cells against glutamate-induced apoptosis in vitro. PMID:22447225

  15. Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism

    PubMed Central

    Ba, Xiao-Hong; Min, Lian-Qiu

    2015-01-01

    Objective: The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. Materials and Methods: SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Results: Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P < 0.005). Apoptosis rate was significantly lower in EGb761-pretreated groups than in OGD group (P < 0.001). Immunofluorescent staining showed that there was AIF nuclear translocation in both EGb761-pretreated groups and OGD group, but AIF nuclear translocation was less in EGb761-pretreated groups than in OGD group. Conclusion: EGb761 can reduce the apoptosis of OGD SH-SY5Y cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice. PMID:25821320

  16. Pinocembrin Suppresses H2O2-Induced Mitochondrial Dysfunction by a Mechanism Dependent on the Nrf2/HO-1 Axis in SH-SY5Y Cells.

    PubMed

    de Oliveira, Marcos Roberto; da Costa Ferreira, Gustavo; Brasil, Flávia Bittencourt; Peres, Alessandra

    2017-01-13

    Mitochondria are susceptible to redox impairment, which has been associated with neurodegeneration. These organelles are both a source and target of reactive species. In that context, there is increasing interest in finding natural compounds that modulate mitochondrial function and mitochondria-related signaling in order to prevent or to treat diseases involving mitochondrial impairment. Herein, we investigated whether and how pinocembrin (PB) would prevent mitochondrial dysfunction elicited by the exposure of human neuroblastoma SH-SY5Y cells to hydrogen peroxide (H2O2). PB (25 μM) was administrated for 4 h before H2O2 treatment (300 μM for 24 h). PB prevented H2O2-induced loss of cell viability mitochondrial depolarization in SH-SY5Y cells. PB also attenuated redox impairment in mitochondrial membranes. The production of superoxide anion radical (O2(-•)) and nitric oxide (NO(•)) was alleviated by PB in cells exposed to H2O2. PB suppressed the H2O2-induced inhibition of the tricarboxylic acid (TCA) cycle enzymes aconitase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase. Furthermore, PB induced anti-inflammatory effects by abolishing the H2O2-dependent activation of the nuclear factor-κB (NF-κB) and upregulation of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The PB-induced antioxidant and anti-inflammatory effects are dependent on the heme oxygenate-1 (HO-1) enzyme and on the activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since HO-1 inhibition (with 0.5 μM ZnPP IX) or Nrf2 silencing (with small interfering RNA (siRNA)) abolished the effects of PB. Overall, PB afforded cytoprotection by the Nrf2/HO-1 axis in H2O2-treated SH-SY5Y cells.

  17. PACAP Protects Against Salsolinol-Induced Toxicity in Dopaminergic SH-SY5Y Cells: Implication for Parkinson’s Disease

    PubMed Central

    Brown, Dwayne; Tamas, Andrea; Reglodi, Dora; Tizabi, Yousef

    2013-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino acid containing neuropeptide with various cytoprotective functions including neuroprotection. Administration of PACAP has been shown to reduce damage induced by ischemia, trauma or exogenous toxic substances. Moreover, mice deficient in PACAP are more vulnerable to damaging insults. In this study we sought to determine whether PACAP may also be protective against salsolinol-induced toxicity in SH-SY5Y cells and if so, elucidate its mechanism(s) of action. Salsolinol (SALS) is an endogenous dopamine metabolite with selective toxicity to nigral dopaminergic neurons, which are directly implicated in Parkinson’s disease (PD). SH-SY5Y cells, derived from human neuroblastoma cells express high levels of dopaminergic activity and are used extensively as a model to study these neurons. Exposure of SH-SY5Y cells to 400uM SALS for 24 h resulted in approximately 50% cell death that was mediated by apoptosis as determined by cell flow cyotmetry and increases in caspase 3 levels. Cellular toxicity was also associated with reductions in brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic AMP response element-binding (p-CREB) protein. Pretreatment with PACAP dose-dependently attenuated SALS-induced toxicity and the associated apoptosis and the chemical changes. PACAP receptor antagonist PACAP 6-38 in turn, dose-dependently blocked the effects of PACAP. Neither PACAP nor PACAP antagonist had any effect of its own on cellular viability. These results suggest protective effects of PACAP in a cellular model of PD. Hence, PACAP or its agonists could be of therapeutic benefit in PD. PMID:23625270

  18. Inhibiting gene expression of alpha3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease.

    PubMed

    Tang, Zhi; An, Yu; Qi, Xiao-Lan; Xiao, Yan; Shan, Ke-Ren; Guan, Zhi-Zhong

    2008-11-01

    In order to examine the effects of alpha3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards alpha3 nAChR. The expressions of alpha3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1muM beta-amyloid peptide (Abeta)(1-42), following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the alphaAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the alpha3 nAChR enhanced the toxicity exerted by Abeta. These results indicate that alpha3 nAChR may improve cleavage of APP by alpha-secretase, enhance antioxidation and inhibit the toxicity of Abeta, suggesting that the receptor might play an important role in AD.

  19. TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

    SciTech Connect

    Pregi, Nicolas Wenker, Shirley; Vittori, Daniela; Leiros, Claudia Perez; Nesse, Alcira

    2009-02-01

    The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-{alpha}. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-{alpha} or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-{alpha}. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-{kappa}B nuclear translocation, TNF-{alpha} induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-{kappa}B, through mechanisms involving Jak/STAT and PI3K signalling pathways.

  20. Mitochondrial respiratory dysfunction due to the conversion of substituted cathinones to methylbenzamides in SH-SY5Y cells

    PubMed Central

    den Hollander, Bjørnar; Sundström, Mira; Pelander, Anna; Siltanen, Antti; Ojanperä, Ilkka; Mervaala, Eero; Korpi, Esa R.; Kankuri, Esko

    2015-01-01

    The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo. PMID:26462443

  1. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    SciTech Connect

    Park, Jae Hyeon; Lee, Jeong Eun; Shin, In Chul; Koh, Hyun Chul

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  2. Delta-9-tetrahydrocannabinol protects against MPP+ toxicity in SH-SY5Y cells by restoring proteins involved in mitochondrial biogenesis

    PubMed Central

    Zeissler, Marie-Louise; Eastwood, Jordan; McCorry, Kieran; Hanemann, C. Oliver; Zajicek, John P.; Carroll, Camille B.

    2016-01-01

    Proliferator-activated receptor γ (PPARγ) activation can result in transcription of proteins involved in oxidative stress defence and mitochondrial biogenesis which could rescue mitochondrial dysfunction in Parkinson's disease (PD). The PPARγ agonist pioglitazone is protective in models of PD; however side effects have limited its clinical use. The cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) may have PPARγ dependent anti-oxidant properties. Here we investigate the effects of Δ9-THC and pioglitazone on mitochondrial biogenesis and oxidative stress. Differentiated SH-SY5Y neuroblastoma cells were exposed to the PD relevant mitochondrial complex 1 inhibitor 1-methyl-4-phenylpyridinium iodide (MPP+). We found that only Δ9-THC was able to restore mitochondrial content in MPP+ treated SH-SY5Y cells in a PPARγ dependent manner by increasing expression of the PPARγ co-activator 1α (PGC-1α), the mitochondrial transcription factor (TFAM) as well as mitochondrial DNA content. Co-application of Δ9-THC with pioglitazone further increased the neuroprotection against MPP+ toxicity as compared to pioglitazone treatment alone. Furthermore, using lentiviral knock down of the PPARγ receptor we showed that, unlike pioglitazone, Δ9-THC resulted in a PPARγ dependent reduction of MPP+ induced oxidative stress. We therefore suggest that, in contrast to pioglitazone, Δ9-THC mediates neuroprotection via PPARγ-dependent restoration of mitochondrial content which may be beneficial for PD treatment. PMID:27366949

  3. Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells.

    PubMed

    Park, Jae Hyeon; Park, Youn Sun; Lee, Je-Bong; Park, Kyung-Hun; Paik, Min-kyoung; Jeong, Mihye; Koh, Hyun Chul

    2016-01-01

    Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH-SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN-induced neuronal cell death. Treatment of SH-SY5Y cells with FPN induced apoptosis via activation of caspase-9 and -3, leading to nuclear condensation. In addition, FPN induced oxidative stress and increased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) via inflammatory stimulation. Pretreatment of cells with meloxicam enhanced the viability of FPN-exposed cells through attenuation of oxidative stress and inflammatory response. FPN activated mitogen activated protein kinase (MAPK) and inhibitors of MAPK abolished FPN-induced COX-2 expression. Meloxicam also attenuated FPN-induced cell death by reducing MAPK-mediated pro-inflammatory factors. Furthermore, we observed both nuclear accumulation of p53 and enhanced levels of cytosolic p53 in a concentration-dependent manner after FPN treatment. Pretreatment of cells with meloxicam blocked the translocation of p53 from the cytosol to the nucleus. Together, these data suggest that meloxicam may exert anti-apoptotic effects against FPN-induced cytotoxicity by both attenuating oxidative stress and inhibiting the inflammatory cascade via inactivation of MAPK and p53 signaling. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Decreased expression of nardilysin in SH-SY5Y cells under ethanol stress and reduced density of nardilysin-expressing neurons in brains of alcoholics.

    PubMed

    Bernstein, Hans-Gert; Stricker, Rolf; Zschiebsch, Katja; Müller, Susan; Dobrowolny, Henrik; Steiner, Johann; Bogerts, Bernhard; Reiser, Georg

    2013-03-01

    There is evidence for a genetic link between the metalloendopeptidase nardilysin and alcohol dependence, but the functional implication of the enzyme in alcoholism is unknown. Interestingly, some of the enzyme's substrates and interaction partners are altered in neural and non-neural tissues under the influence of ethanol consumption. To learn more about putative roles of nardilysin in alcohol dependence we studied the expression of the enzyme protein in human neuroblastoma cells under chronic ethanol exposure as well as in four brain regions of alcoholics and matched controls. Cultured SH-SY5Y cells were exposed for 96 h to two different concentrations of ethanol (50 and 200 mM). Nardilysin expression was determined using Western blotting with densitometric analysis. Furthermore, we morphometrically studied the cellular expression of nardilysin in postmortem brains of eight chronic alcoholics and nine controls by counting the number of nardilysin-immunopositive neurons in left frontal limbic area, Nuc. basalis of Meynert, paraventricular and supraoptic hypothalamic nuclei and calculating numerical cell densities. Nardilysin expression was significantly reduced after 96 h of SH-SY5Y cells exposure to 200 mM ethanol. In human brains nardilysin protein was localized to multiple neurons. In heavy drinkers there was a significantly reduced density of nardilysin immunoreactive neurons in Nuc. basalis of Meynert, paraventricular, and supraoptic nuclei. The alcohol-dependent reduction of nardilysin in cell culture and nervous tissue points to an implication of the enzyme in the pathophysiology of alcoholism.

  5. Inhibition of tissue transglutaminase promotes Aβ-induced apoptosis in SH-SY5Y cells

    PubMed Central

    Zhang, Ji; Ding, Yi-rong; Wang, Rui

    2016-01-01

    Aim: Tissue transglutaminase (tTG) catalyzes proteins, including β-amyloid (Aβ), to cross-link as a γ-glutamyl-ε-lysine structure isopeptide, which is highly resistant to proteolysis. Thus, tTG plays an important role in protein accumulation in Alzheimer's disease (AD). In the present study, we examined the effect of an irreversible tTG inhibitor, NTU283, on Aβ mimic-induced AD pathogenesis in SH-SY5Y cells. Methods: Western blot and in-cell Western analyses were used to detect tTG and isopeptide (representing the enzyme activity of tTG) protein levels. Moreover, Hoechst and PI co-staining was performed, and caspase-3 and caspase-7 activities and the Bax/Bcl-2 ratio were determined to evaluate the effects of NTU283 on apoptosis. Results: The results confirmed that tTG activity was inhibited by NTU283 20–500 μmol/L in a concentration-dependent manner in SH-SY5Y cells. Contrary to our expectations, however, the isopeptide bonds were increased when cells were co-treated with Aβ and NTU283. In addition, NTU283 alone did not induce apoptosis in SH-SY5Y cells. However, when co-applied with Aβ, NTU283 promoted rather than inhibited Aβ-induced apoptosis. Consistent with the apoptotic rate, pretreating cells with different concentrations of NTU283 and Aβ significantly increased the activities of caspase-3 and caspase-7 as well as the ratio of Bax/Bcl-2. Conclusion: Irreversible inhibition of tTG activity did not block but rather promoted Aβ-induced apoptosis, which indicated that tTG has complex functions in AD pathogenesis. PMID:27665848

  6. Design, synthesis and biological evaluation of coumarin derivatives as novel acetylcholinesterase inhibitors that attenuate H2O2-induced apoptosis in SH-SY5Y cells.

    PubMed

    Yao, Dahong; Wang, Jing; Wang, Guan; Jiang, Yingnan; Shang, Lei; Zhao, Yuqian; Huang, Jian; Yang, Shilin; Wang, Jinhui; Yu, Yamei

    2016-10-01

    A novel series of coumarin derivatives were designed, synthesized and investigated for inhibition of cholinesterase, including acetyl cholinesterase (AChE) and butyrylcholinesterase (BuChE). This biological study showed that these compounds containing piperazine ring had significant inhibition activities on AChE rather than BuChE. Further study suggested that 9x, as one of this kind of structure derivative, showed the strongest inhibition activity on AChE with an IC50 value of 34nM. Moreover, molecular docking, flow cytometry (FCM), and western blot assay suggested that 9x could induce cytoprotective autophagy to attenuate H2O2-induced cell death in human neuroblastoma SH-SY5Y cells. These findings highlight a new approach for the development of a novel potential neuroprotective compound targeting AChE with autophagy-inducing activity in future Alzheimer's disease (AD) therapy. Copyright © 2016. Published by Elsevier Inc.

  7. Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

    SciTech Connect

    Mouledous, Lionel

    2008-08-15

    *: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

  8. Transportation of Berberine into HepG2, HeLa and SY5Y Cells: A Correlation to Its Anti-Cancer Effect

    PubMed Central

    Pang, Yu-Nong; Liang, Yin-Wen; Feng, Tian-Shi; Zhao, Shuang; Wu, Hao; Chai, Yu-Shuang; Lei, Fan; Ding, Yi; Xing, Dong-Ming; Du, Li-Jun

    2014-01-01

    The anti-cancer activities of berberine (BBR) have been reported extensively in various cancer cell lines. However, the minimal inhibitory concentrations of BBR varied greatly among different cell lines and very few studies have been devoted to elucidate this aspect. In this study, we employed three cancer cell lines, HepG2, HeLa and SY5Y, to compare the transportation and distribution of BBR. HPLC results demonstrated that BBR was capable of penetrating all the cell lines whereas the cumulative concentrations were significantly different. HepG2 cells accumulated higher level of BBR for longer duration than the other two cell lines. Molecular docking studies revealed the BBR binding site on P-glycoprotein 1 (P-gp). In addition, we elucidated that BBR regulated P-gp at both mRNA and protein levels. BBR induced the transcription and translation of P-gp in HeLa and SY5Y cells, whereas BBR inhibited P-gp expression in HepG2 cells. Further study showed that BBR regulates P-gp expression depending on different mechanisms (or affected by different factors) in different cell lines. To summarize, our study has revealed several mechanistic aspects of BBR regulation on P-gp in different cancer cell lines and might shed some useful insights into the use of BBR in the anti-cancer drug development. PMID:25402492

  9. Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

    PubMed Central

    Ju, Xiao-Ning; Mu, Wei-Na; Liu, Yuan-Tao; Wang, Mei-Hong; Kong, Feng; Sun, Chao; Zhou, Qing-Bo

    2015-01-01

    Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. PMID:26823714

  10. Agmatine effects on mitochondrial membrane potential and NF-κB activation protect against rotenone-induced cell damage in human neuronal-like SH-SY5Y cells.

    PubMed

    Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Caccamo, Daniela; Satriano, Joseph; Ientile, Riccardo

    2011-01-01

    Agmatine, an endogenous arginine metabolite, has been proposed as a novel neuromodulator that plays protective roles in the CNS in several models of cellular damage. However, the mechanisms involved in these protective effects in neurodegenerative diseases are poorly understood. The present study was undertaken to investigate the effects of agmatine on cell injury induced by rotenone, commonly used in establishing in vivo and in vitro models of Parkinson's disease, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report that agmatine dose-dependently suppressed rotenone-induced cellular injury through a reduction of oxidative stress. Similar effects were obtained by spermine, suggesting a scavenging effect for these compounds. However, unlike spermine, agmatine also prevented rotenone-induced nuclear factor-κB nuclear translocation and mitochondrial membrane potential dissipation. Furthermore, rotenone-induced increase in apoptotic markers, such as caspase 3 activity, Bax expression and cytochrome c release, was significantly attenuated with agmatine treatment. These findings demonstrate mitochondrial preservation with agmatine in a rotenone model of apoptotic cell death, and that the neuroprotective action of agmatine appears because of suppressing apoptotic signalling mechanisms. Thus, agmatine may have therapeutic potential in the treatment of Parkinson's disease by protecting dopaminergic neurons.

  11. Neuritogenesis induced by vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and peptide histidine methionine in SH-SY5y cells is associated with regulated expression of cytoskeleton mRNAs and proteins.

    PubMed

    Héraud, Céline; Hilairet, Sandrine; Muller, Jean-Marc; Leterrier, Jean-François; Chadéneau, Corinne

    2004-02-01

    Vasoactive intestinal peptide (VIP) and the related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and peptide histidine methionine (PHM) are known to regulate proliferation and/or differentiation in normal and tumoral cells. In this study, neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium was induced by VIP, PACAP, and PHM. The establishment of this process was followed by the quantification of neurite length and branching and the expression of neurofilament mRNAs, neurofilament proteins, and other cytoskeletal protein markers of neuronal differentiation: neuron-specific MAPs and beta-tubulin III. Neurite length and branching and the expression of most markers tested were increased by VIP and PACAP in a similar, although slightly different, fashion. In contrast, neuritic elongation induced by PHM was correlated with neither an increase in branching or neurofilament mRNAs nor a clear change in the expression of cytoskeleton proteins, with the exception of the stimulation by PHM of doublecortin, a microtubule-associated marker of migrating neuroblasts. These findings are the first evidence from a human neuron-like cell line for 1) a direct regulation of the metabolism of neurofilaments by VIP and PACAP and 2) the induction by PHM of neuritic processes of an apparent immature character. Copyright 2003 Wiley-Liss, Inc.

  12. Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

    PubMed Central

    Posadas, Inmaculada; Santos, Pablo; Ceña, Valentín

    2012-01-01

    Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β. PMID:23166834

  13. Acetaminophen induces human neuroblastoma cell death through NFKB activation.

    PubMed

    Posadas, Inmaculada; Santos, Pablo; Ceña, Valentín

    2012-01-01

    Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-x(L) did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β.

  14. S-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells.

    PubMed

    Toyama, Takashi; Abiko, Yumi; Katayama, Yuko; Kaji, Toshiyuki; Kumagai, Yoshito

    2015-12-01

    Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels.

  15. Insulin-like growth factor 1 specifically up-regulates expression of modifier subunit of glutamate-cysteine ligase and enhances glutathione synthesis in SH-SY5Y cells.

    PubMed

    Takahashi, Shuhei; Hisatsune, Akinori; Kurauchi, Yuki; Seki, Takahiro; Katsuki, Hiroshi

    2016-01-15

    Glutathione is a key regulator of oxidative balance in all mammals, especially in the central nervous system. The first step of glutathione synthesis is catalyzed by glutamate-cysteine ligase (GCL), which is composed of catalytic and modifier subunits (GCLC and GCLM, respectively). In non-neural cells and tissues, insulin and insulin-like growth factor 1 (IGF-1) have been found to stimulate transcription of GCLC gene. Here we found that treatment of human neuroblastoma SH-SY5Y cells with insulin or IGF-1 increased mRNA level of GCLM, but not of GCLC, in a concentration- and time-dependent manner. In contrast, insulin did not increase GCL expression in rat C6 glioma cells. We also confirmed that IGF-1 increased protein level of GCLM and cellular glutathione content in SH-SY5Y cells. In addition, IGF-1 increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein in the nuclear fraction of SH-SY5Y cells. siRNA-mediated knockdown of Nrf2 protein expression abrogated IGF-1-induced up-regulation of GCLM mRNA expression. Finally, IGF-1-induced increase in nuclear Nrf2 protein and GCLM mRNA expression was abolished by LY294002, a phosphoinositide 3-kinase inhibitor. These results indicate that insulin and IGF-1 have the ability to enhance glutathione biosynthesis in neuronal cells via specific up-regulation of GCLM expression.

  16. Carnosic Acid Induces Anti-Inflammatory Effects in Paraquat-Treated SH-SY5Y Cells Through a Mechanism Involving a Crosstalk Between the Nrf2/HO-1 Axis and NF-κB.

    PubMed

    de Oliveira, Marcos Roberto; de Souza, Izabel Cristina Custódio; Fürstenau, Cristina Ribas

    2017-01-12

    Carnosic acid (CA) is a phenolic diterpene obtained from Rosmarinus officinalis L. and has demonstrated cytoprotective properties in several experimental models. CA exerts antioxidant effects by upregulating the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which controls the expression of antioxidant and phase II detoxification enzymes. Heme oxygenase-1 (HO-1) expression is modulated by Nrf2 and has been demonstrated as part of the mechanism underlying the CA-induced cytoprotection. Nonetheless, it remains to be studied whether and how HO-1 would mediate CA-elicited anti-inflammatory effects. Therefore, we have investigated here whether and how CA would prevent paraquat (PQ)-induced inflammation-related alterations in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated for 12 h with CA at 1 μM before exposure to PQ for further 24 h. CA suppressed the PQ-induced alterations on the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) through a mechanism involving the activation of the Nrf2/HO-1 axis. Furthermore, we observed a crosstalk between the Nrf2/HO-1 signaling pathway and the activation of the nuclear factor-κB (NF-κB) transcription factor, since administration of ZnPP IX (specific inhibitor of HO-1) or Nrf2 knockdown using small interfering RNA (siRNA) abolished the anti-inflammatory effects induced by CA. Moreover, administration of SN50 (specific inhibitor of NF-κB) inhibited the PQ-induced inflammation-related effects in SH-SY5Y cells. Therefore, CA exerted anti-inflammatory effects in SH-SY5Y cells through an Nrf2/HO-1 axis-dependent manner associated with downregulation of NF-κB.

  17. The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

    PubMed

    Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

    2013-07-01

    Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that

  18. Oxidative stress induces transient O-GlcNAc elevation and tau dephosphorylation in SH-SY5Y cells.

    PubMed

    Kátai, Emese; Pál, József; Poór, Viktor Soma; Purewal, Rupeena; Miseta, Attila; Nagy, Tamás

    2016-12-01

    O-linked β-N-acetlyglucosamine or O-GlcNAc modification is a dynamic post-translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O-GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O-GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH-SY5Y we investigated the dynamic nature of O-GlcNAc after treatment with 0.5 mM H2 O2 for 30 min. to induce oxidative stress. We found that overall O-GlcNAc quickly increased and reached peak level at around 2 hrs post-stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O-Glycosylation. In conclusion, our results show that temporary elevation in O-GlcNAc modification after H2 O2 -induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O-GlcNAc and phosphorylation on tau proteins.

  19. Synthesis of reduced-size gold nanostars and internalization in SH-SY5Y cells.

    PubMed

    Dacarro, Giacomo; Pallavicini, Piersandro; Bertani, Serena Maria; Chirico, Giuseppe; D'Alfonso, Laura; Falqui, Andrea; Marchesi, Nicoletta; Pascale, Alessia; Sironi, Laura; Taglietti, Angelo; Zuddas, Efisio

    2017-11-01

    The synthesis of large pentatwinned five-branched gold nanostars (GNS) has been modified so to obtain overall dimensions shrunk to 60% and a lower branches aspect ratio, leading to a dramatic blue shift of their two near-infrared (NIR) localized surface plasmon resonances (LSPR) absorptions but still maintaining one LSPR in the biotransparent NIR range. The interactions of polyethylene glycol (PEG) coated large and shrunk GNS with SH-SY5Y cells revealed that the large ones (DCI - diameter of the circumference in which GNS can be inscribed=76nm) are internalized more efficiently than the shrunk ones (DCI=46nm), correlating with a decreased cells surviving fraction. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Investigation of the effects of a sulfite molecule on human neuroblastoma cells via a novel oncogene URG4/URGCP.

    PubMed

    Dodurga, Yavuz; Seçme, Mücahit; Eroğlu, Canan; Gündoğdu, Gülşah; Avcı, Çığır Biray; Bağcı, Gülseren; Küçükatay, Vural; Lale Şatıroğlu-Tufan, N

    2015-12-15

    The aim of this study is to determine the anticancer effect of sulfite on SH-SY5Y neuroblastoma cells in vitro conditions and elucidate underlying molecular mechanism of sulfite and explore its therapeutic activity. In this study, cytotoxic effects of sulfite in SH-SY5Y cels were detected over time in a dose dependent manner with the IC50 doses ranging from 0.5 to 10 mM. Genotoxic effect of sulfite was shown by comet assay. IC50 doses in the SH-SY5Y cells were detected as 5 mM. Expression profiles of the target genes related to apoptosis and cell cycle control were determined by quantitative RT-PCR. Protein changes were determined by western blot analysis. URG4/URGCP, CCND1, CCND2, CDK4, CDK6, E2F4 and BCL-2 gene expression levels were significantly reduced and RB1, TP53, BAX, BID, CASP2, CASP3, CASP9 and DIABLO gene expressions were significantly increased in dose group cells. The mechanism of this result may be related to sulfite dependent inhibition of cell cycle at the G1 phase by down-regulating URG4/URGCP or CCND1, CDK4, CDK6 gene expression and stimulating apoptosis via the intrinsic pathway. Sulfite suppressed invasion and colony formation in SH-SY5Y cell line using matrigel invasion chamber and colony formation assay, respectively. It is thought that sulfite demonstrates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis s, invasion, and colony formation on SH-SY5Y cells. Sulfite may be an effective agent for treatment of neuroblastoma as a single agent or in combination with other agents.

  1. Expression patterns of antioxidant genes in human SH-SY5Y cells after treatment with methadone.

    PubMed

    Saify, Khyber; Saadat, Mostafa

    2015-11-30

    The expression levels of nine antioxidant genes in SH-SY5Y cells exposed to methadone (final concentrations 1-20µM) were investigated. Based on this study the genes could be categorized on three different groups. The number of down-regulated genes were increased as a function of exposure time (P=0.004). The methadone associated mRNA alterations were modulated by N-acetyl-cysteine. These findings suggested that different pathways for regulation of antioxidant genes could be active after exposing of SH-SY5Y cells to methadone; and also suggested that methadone might act by inducing the reactive oxygen species.

  2. Tau dephosphorylation and microfilaments disruption are upstream events of the anti-proliferative effects of DADS in SH-SY5Y cells

    PubMed Central

    Aquilano, Katia; Vigilanza, Paola; Filomeni, Giuseppe; Rotilio, Giuseppe; Ciriolo, Maria R

    2010-01-01

    Abstract Garlic organosulphur compounds have been successfully used as redox anti-proliferative agents. In this work, we dissect the effects of diallyl disulphide (DADS) focusing on the events upstream of cell cycle arrest and apoptosis induced in neuroblastoma SH-SY5Y cells. We demonstrate that DADS is able to cause early morphological changes, cytoskeleton oxidation, microfilaments reduction and depolymerization of microtubules. These events are attenuated in cells stably overexpressing the antioxidant enzyme SOD1, suggesting that superoxide plays a crucial role in destabilizing cytoskeleton. Moreover, we evidence that the main microtubules-associated protein Tau undergoes PP1-mediated dephosphorylation as demonstrated by treatment with okadaic acid as well as by immunoreaction with anti-Tau-1 antibody, which specifically recognizes its dephosphorylated forms. Tau dephosphorylation is inhibited by the two-electron reductants NAC and GSH ester but not by SOD1. The inability of DADS to induce apoptosis in neuroblastoma-differentiated cells gives emphasis to the anti-proliferative activity of DADS, which can be regarded as a promising potent anti-neuroblastoma drug by virtue of its widespread cytoskeleton disrupting action on proliferating cells. PMID:19040422

  3. Carnosic Acid Suppresses the H2O2-Induced Mitochondria-Related Bioenergetics Disturbances and Redox Impairment in SH-SY5Y Cells: Role for Nrf2.

    PubMed

    de Oliveira, Marcos Roberto; da Costa Ferreira, Gustavo; Peres, Alessandra; Bosco, Simone Morelo Dal

    2017-01-13

    The phenolic diterpene carnosic acid (CA, C20H28O4) exerts antioxidant, anti-inflammatory, anti-apoptotic, and anti-cancer effects in mammalian cells. CA activates the nuclear factor erythroid 2-related factor 2 (Nrf2), among other signaling pathways, and restores cell viability in several in vitro and in vivo experimental models. We have previously reported that CA affords mitochondrial protection against various chemical challenges. However, it was not clear yet whether CA would prevent chemically induced impairment of the tricarboxylic acid cycle (TCA) function in mammalian cells. In the present work, we found that a pretreatment of human neuroblastoma SH-SY5Y cells with CA at 1 μM for 12 h prevented the hydrogen peroxide (H2O2)-induced impairment of the TCA enzymes (aconitase, α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH)) and abolished the inhibition of the complexes I and V and restored the levels of ATP by a mechanism associated with Nrf2. CA also exhibited antioxidant abilities by enhancing the levels of reduced glutathione (GSH) and decreasing the content oxidative stress markers (cellular 8-oxo-2'-deoxyguanosine (8-oxo-dG), and mitochondrial malondialdehyde (MDA), protein carbonyl, and 3-nitrotyrosine). Silencing of Nrf2 by small interfering RNA (siRNA) abrogated the protective effects elicited by CA in mitochondria of SH-SY5Y cells. Therefore, CA prevented the H2O2-triggered mitochondrial impairment by an Nrf2-dependent mechanism. The specific role of Nrf2 in ameliorating the function of TCA enzymes function needs further research.

  4. Relationship between intracellular Ca²⁺ and ROS during fluoride-induced injury in SH-SY5Y cells.

    PubMed

    Xu, Zhixia; Xu, Bayi; Xia, Tao; He, Weihong; Gao, Ping; Guo, Lijuan; Wang, Zhenglun; Niu, Qiang; Wang, Aiguo

    2013-06-01

    The mechanisms underlying the neurotoxicology of endemic fluorosis still remain obscure. To explore lactate dehydrogenase (LDH) leakage, intracellular Ca²⁺ concentration ([Ca²⁺]i ) and reactive oxygen species (ROS) production induced by fluoride, human neuroblastoma (SH-SY5Y) cells were incubated with sodium fluoride (NaF, 20, 40, 80 mg/L) for 24 h, with 40 mg/L NaF for 3, 6, 12, 18, 24 h, and N-acetyl-L-cysteine (NAC), ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) alone or combined with fluoride (40 mg/L) respectively for 12 h in vitro. The results showed that the LDH levels in the 40 and 80 mg/L fluoride-treated groups were significantly higher than that of the control group (in the test level of 0.05, the difference were statistical significance). [Ca²⁺]i and ROS reached a peak at 3 h and 12 h respectively after exposure to 40 mg/L fluoride. Fluoride coincubated with NAC (antioxidant) dramatically decreased ROS and LDH levels compared with the fluoride only group (in the test level of 0.05, the difference were statistical significance). However, fluoride-induced increase in [Ca²⁺]i was not affected by NAC. BAPTA-AM (intracellular calcium chelator) markedly lowered fluoride-induced increase of [Ca²⁺]i , ROS and LDH levels while EGTA (extracellular calcium chelator) have no effects on them. These results indicate that fluoride-related Ca²⁺ release from the site of intracellular calcium storage causes the elevation of ROS contributing to the cytotoxicity in SH-SY5Y cells. Copyright © 2011 Wiley Periodicals, Inc.

  5. L-theanine protects the APP (Swedish mutation) transgenic SH-SY5Y cell against glutamate-induced excitotoxicity via inhibition of the NMDA receptor pathway.

    PubMed

    Di, X; Yan, J; Zhao, Y; Zhang, J; Shi, Z; Chang, Y; Zhao, B

    2010-07-14

    As a natural analogue of glutamate, l-theanine is the unique amino acid derivative in green tea. Although its underlining mechanisms are not yet clear, it has been suggested that l-theanine treatment may prove beneficial to patients with neurodegenerative diseases. In this study, we investigated the neuroprotective effect and its mechanism of l-theanine in an in vitro model of Alzheimer's disease by using the human APP (Swedish mutation) transgenic SH-SY5Y cell. Amyloid beta (Abeta) neurotoxicity was triggered by l-glutamate in this cell line. Additionally, l-theanine significantly attenuated l-glutamate-induced apoptosis at similar levels to those seen with the NMDA receptor inhibitor MK-801 in the stably expressing APP Swedish mutation SH-SY5Y cells which over-generated Abeta. Meanwhile, the activation of c-Jun N-terminal kinase and caspase-3 induced by l-glutamate was suppressed by l-theanine. We also found that cells treated with l-theanine showed decreased production of nitric oxide resulting from the down-regulated protein levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). These results indicate that the inhibition of the NMDA subtype of glutamate receptors and its related pathways is the crucial point of the neuroprotective effect of l-theanine in the cell model. Thus, our present study supports the notion that l-theanine may provide effective prophylaxis and treatment for Alzheimer's disease.

  6. Androgen Receptor Regulates the Growth of Neuroblastoma Cells in vitro and in vivo

    PubMed Central

    Sun, Junyan; Wang, Dongmei; Guo, Lianying; Fang, Shengyun; Wang, Yang; Xing, Rong

    2017-01-01

    Background: Neuroblastoma is the most common extracranial tumors in children. At present about the true etiology of neuroblastoma is unclear and many studies have tried to find effective treatments for these primary malignant tumors. Although it has been illustrated that androgen receptor (AR) was expressed in neuroblastoma cells in some former reports, the biological role of androgen receptor in the development of neuroblastoma is not fully understood. Methods: Androgen (R1881) and the antagonists of androgen receptor (MDV3100 and ARN509) were used to study the role of the androgen receptor signaling pathway in vitro and in vivo on SH-SY5Y and Neuro-2a (N2a) cell lines. Results: We found that AR expression showed an R1881 dose-dependent manner in neuroblastoma cells in vitro and R1881was able to increase, while both antagonists of androgen receptor (MDV3100 and ARN509) significantly decrease, the proliferation, migration, invasion and sphere formation of SH-SY5Y and N2a cells. Moreover, androgen promoted the growth of N2a tumor in vivo. However, when androgen receptor (AR) was effectively knocked down in the two cell lines by siRNA, either promoting or inhibiting effect of the androgen or androgen receptor antagonists, respectively, was attenuated. Conclusion: Our results suggested that androgen receptor may involve in the progression of neuroblastoma as well as provided insight into a new target for the diagnosis and treatment of neuroblastoma patients. PMID:28326012

  7. Androgen Receptor Regulates the Growth of Neuroblastoma Cells in vitro and in vivo.

    PubMed

    Sun, Junyan; Wang, Dongmei; Guo, Lianying; Fang, Shengyun; Wang, Yang; Xing, Rong

    2017-01-01

    Background: Neuroblastoma is the most common extracranial tumors in children. At present about the true etiology of neuroblastoma is unclear and many studies have tried to find effective treatments for these primary malignant tumors. Although it has been illustrated that androgen receptor (AR) was expressed in neuroblastoma cells in some former reports, the biological role of androgen receptor in the development of neuroblastoma is not fully understood. Methods: Androgen (R1881) and the antagonists of androgen receptor (MDV3100 and ARN509) were used to study the role of the androgen receptor signaling pathway in vitro and in vivo on SH-SY5Y and Neuro-2a (N2a) cell lines. Results: We found that AR expression showed an R1881 dose-dependent manner in neuroblastoma cells in vitro and R1881was able to increase, while both antagonists of androgen receptor (MDV3100 and ARN509) significantly decrease, the proliferation, migration, invasion and sphere formation of SH-SY5Y and N2a cells. Moreover, androgen promoted the growth of N2a tumor in vivo. However, when androgen receptor (AR) was effectively knocked down in the two cell lines by siRNA, either promoting or inhibiting effect of the androgen or androgen receptor antagonists, respectively, was attenuated. Conclusion: Our results suggested that androgen receptor may involve in the progression of neuroblastoma as well as provided insight into a new target for the diagnosis and treatment of neuroblastoma patients.

  8. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress.

    PubMed

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress.

  9. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

    PubMed

    Pahrudin Arrozi, Aslina; Shukri, Siti Nur Syazwani; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Ahmad Damanhuri, Mohd Hanafi; Makpol, Suzana

    2017-04-17

    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

  10. Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line.

    PubMed

    Marcuzzi, Annalisa; Zanin, Valentina; Piscianz, Elisa; Tricarico, Paola Maura; Vuch, Josef; Girardelli, Martina; Monasta, Lorenzo; Bianco, Anna Monica; Crovella, Sergio

    2012-10-01

    Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD. We demonstrate that apoptosis in neuronal lovastatin treated-cells is induced by the mitochondrial pathway, with caspase-9 as the initiator and caspase-3 as the effector caspase. The presence of geranylgeraniol modulates both the caspase-9 and caspase-3 activity in a dose-dependent way, confirming that this isoprenoid enters the mevalonate pathway, is metabolized and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. According to our findings, it should not be the time course adopted that modulates the apoptotic response but rather the isoprenoid itself. Being aware that our results have been obtained using a biochemical model of MKD, and not cells from patients with the disease, we believe our findings increase the knowledge of MA pathogenesis, and may possibly contribute to the development of novel therapeutic strategies.

  11. Modulation of μ-Opioid Receptor Signaling by RGS19 in SH-SY5Y Cells

    PubMed Central

    Wang, Qin

    2013-01-01

    Regulator of G-protein signaling protein 19 (RGS19), also known as Gα-interacting protein (GAIP), acts as a GTPase accelerating protein for Gαz as well as Gαi/o subunits. Interactions with GAIP-interacting protein N-terminus and GAIP-interacting protein C-terminus (GIPC) link RGS19 to a variety of intracellular proteins. Here we show that RGS19 is abundantly expressed in human neuroblastoma SH-SY5Y cells that also express µ- and δ- opioid receptors (MORs and DORs, respectively) and nociceptin receptors (NOPRs). Lentiviral delivery of short hairpin RNA specifically targeted to RGS19 reduced RGS19 protein levels by 69%, with a similar reduction in GIPC. In RGS19-depleted cells, there was an increase in the ability of MOR (morphine) but not of DOR [(4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80)] or NOPR (nociceptin) agonists to inhibit forskolin-stimulated adenylyl cyclase and increase mitogen-activated protein kinase (MAPK) activity. Overnight treatment with either MOR [D-Ala, N-Me-Phe, Gly-ol5-enkephalin (DAMGO) or morphine] or DOR (D-Pen5-enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a time- and concentration-dependent manner. The MOR-induced increase in RGS19 protein was prevented by pretreatment with pertussis toxin or the opioid antagonist naloxone. Protein kinase C (PKC) activation alone increased the level of RGS19 and inhibitors of PKC 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile and mitogen-activated protein kinase kinase 1 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one, but not protein kinase A (H89), completely blocked DAMGO-induced RGS19 protein accumulation. The findings show that RGS19 and GIPC are jointly regulated, that RGS19 is a GTPase accelerating protein for MOR with selectivity over DOR and NOPR, and that chronic MOR or DOR agonist treatment increases RGS19 levels by a PKC and the MAPK pathway

  12. Modulation of μ-opioid receptor signaling by RGS19 in SH-SY5Y cells.

    PubMed

    Wang, Qin; Traynor, John R

    2013-02-01

    Regulator of G-protein signaling protein 19 (RGS19), also known as Gα-interacting protein (GAIP), acts as a GTPase accelerating protein for Gαz as well as Gαi/o subunits. Interactions with GAIP-interacting protein N-terminus and GAIP-interacting protein C-terminus (GIPC) link RGS19 to a variety of intracellular proteins. Here we show that RGS19 is abundantly expressed in human neuroblastoma SH-SY5Y cells that also express µ- and δ- opioid receptors (MORs and DORs, respectively) and nociceptin receptors (NOPRs). Lentiviral delivery of short hairpin RNA specifically targeted to RGS19 reduced RGS19 protein levels by 69%, with a similar reduction in GIPC. In RGS19-depleted cells, there was an increase in the ability of MOR (morphine) but not of DOR [(4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80)] or NOPR (nociceptin) agonists to inhibit forskolin-stimulated adenylyl cyclase and increase mitogen-activated protein kinase (MAPK) activity. Overnight treatment with either MOR [D-Ala, N-Me-Phe, Gly-ol(5)-enkephalin (DAMGO) or morphine] or DOR (D-Pen(5)-enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a time- and concentration-dependent manner. The MOR-induced increase in RGS19 protein was prevented by pretreatment with pertussis toxin or the opioid antagonist naloxone. Protein kinase C (PKC) activation alone increased the level of RGS19 and inhibitors of PKC 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile and mitogen-activated protein kinase kinase 1 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one, but not protein kinase A (H89), completely blocked DAMGO-induced RGS19 protein accumulation. The findings show that RGS19 and GIPC are jointly regulated, that RGS19 is a GTPase accelerating protein for MOR with selectivity over DOR and NOPR, and that chronic MOR or DOR agonist treatment increases RGS19 levels by a PKC and the MAPK pathway

  13. Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: an in vitro model of HTLV-I-induced disease.

    PubMed

    Maldonado, Horacio; Ortiz-Riaño, Emilio; Krause, Bernardo; Barriga, Andrés; Medina, Fernando; Pando, M Elsa; Alberti, Carolina; Kettlun, Ana M; Collados, Lucía; García, Lorena; Cartier, Luis; Valenzuela, M Antonieta

    2008-01-01

    HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

  14. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells

    PubMed Central

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B.; Chow, K. Martin; Rodgers, David W.; Quintanilla-Vega, Betzabet

    2015-01-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer’s disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1–20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms. PMID:25673500

  15. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells.

    PubMed

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B; Chow, K Martin; Rodgers, David W; Quintanilla-Vega, Betzabet

    2015-05-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1-20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms.

  16. Neuroprotection by epigallo catechin gallate against bupivacaine anesthesia induced toxicity involves modulation of PI3/Akt/PTEN signalling in N2a and SH-SY5Y cells.

    PubMed

    Wang, Li-Yan; Li, Xia; Han, Yu-Zeng

    2015-01-01

    Bupivacaine, an amide type long-acting local anaesthetic is commonly employed for epidural anesthesia and as well for nerve blockades. However, studies have shown neurotoxicity following local administration of bupivacaine raising concerns over the use of the drug. Compounds that could minimize or inhibit toxic effects of bupivacaine are of high value in operative settings and in pain management. The present study aims to investigate if epigallo catechin gallate (EGCG) could inhibit or prevent bupivacaine toxicity in neuroblastoma cells (N2a and SH-SY5Y). The viability of N2a and SH-SY5Y cells following exposure to EGCG (10-50 µM) were assessed by MTT assay and Annexin V/PI staining. The influence of EGCG on ROS generation was determined. The expression of apoptotic cascade proteins (Caspases-3, -8 and -9, Bcl-xL, Bad, Bax, Bcl-2) and PI3/Akt pathway proteins (Akt, p-Akt, GSK-3β, p-GSK-3β, PTEN) were analyzed by western blotting. EGCG improved the viability of the cells and inhibited apoptosis by potentially decreasing the expression of caspases and pro-apoptotic proteins. Bupivacaine induced ROS generations were reduced on EGCG exposure. EGCG significantly promoted the phosphorylation of Akt and GSK-3β and down-regulated PTEN, thus activating PI3/Akt signalling. EGCG effectively improved the cell viability and inhibited apoptosis of N2a and SH-SY5Y cells via suppression of ROS generation and modulation of PI3K/Akt signalling cascade.

  17. The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-β peptide and β-secretase.

    PubMed

    Qi, Xiao-Lan; Zhang, Xue-Ling; Ou-Yang, Kai; Shan, Ke-Ren; Guan, Zhi-Zhong

    2013-01-01

    To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of β-secretase and the concomitant level of amyloid-β (Aβ), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the β-site of APP) and BACE2 (cleaving in the Aβ domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aβ(1-42) in culture media were determined by an Elisa procedure. In SH-SY5Y cells transfected with siRNA, the levels of α3 nAChR mRNA and protein were reduced by 96% and 88%, respectively; the levels of BACE1 mRNA and protein were significantly enhanced, while those of BACE2 were reduced; and the level of Aβ in the culture medium was elevated. In contrast, when untransfected SH-SY5Y cells were exposed to nicotine, the levels of both α3 nAChR mRNA and protein were enhanced; while the levels of BACE1 mRNA and protein were diminished and the corresponding levels of BACE2 enhanced; and the level of Aβ in the culture medium was attenuated. These results indicate that the α3 subunit of nAChR inhibits the production of Aβ by reducing the expression of BACE1 and elevating the expression of BACE2, suggesting that this subunit might play an important neuroprotective role in connection with the pathogenesis of AD.

  18. Wnt3a protects SH-SY5Y cells against 6-hydroxydopamine toxicity by restoration of mitochondria function.

    PubMed

    Wei, Lei; Ding, Li; Mo, Ming-Shu; Lei, Ming; Zhang, Limin; Chen, Kang; Xu, Pingyi

    2015-01-01

    Wnt/β-catenin signal has been reported to exert cytoprotective effects in cellular models of several diseases, including Parkinson's disease (PD). This study aimed to investigate the neuroprotective effects of actived Wnt/β-catenin signal by Wnt3a on SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA). Wnt3a-conditioned medium (Wnt3a-CM) was used to intervene dopaminegic SH-SY5Y cells treated with 6-OHDA. Cell toxicity was determined by cell viability and lactate dehydrogenase leakage (LDH) assay. The mitochondria function was measured by the mitochondrial membrane potential, while oxidative stress was monitored with intracellular reactive oxygen species (ROS). Western blot analysis was used to detect the expression of GSK3β, β-catenin as well as Akt. Our results showed that 100 μM 6-OHDA treated for 24 h significantly decreased cell viability and mitochondrial transmembrane potential, reduced the level of β-catenin and p-Akt, increased LDH leakage, ROS production and the ratio of p-GSK3β (Tyr216) to p-GSK3β (Ser9). However, Wnt3a-conditioned medium reversing SH-SY5Y cells against 6-OHDA-induced neurotoxicity by reversing these changes. Activating of Wnt/β-catenin pathway by Wnt3a-CM attenuated 6-OHDA-induced neurotoxicity significantly, which related to the inhibition of oxidative stress and maintenance of normal mitochondrial function.

  19. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    PubMed

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

  20. DJ-1-Mediated protective effect of protocatechuic aldehyde against oxidative stress in SH-SY5Y cells.

    PubMed

    Gao, Jian-Wei; Yamane, Takuya; Maita, Hiroshi; Ishikawa, Shizuma; Iguchi-Ariga, Sanae M M; Pu, Xiao-Ping; Ariga, Hiroyoshi

    2011-01-01

    DJ-1 was identified as a causal gene for a familial form of early onset Parkinson's disease (PD), park 7. DJ-1 plays roles in transcriptional regulation and the anti-oxidative stress reaction. In this study, we found that protocatechuic aldehyde (PAL), a traditional Chinese medicine compound, bound to DJ-1 in vitro and that PAL protected SH-SY5Y cells but not DJ-1-knockdown SH-SY5Y cells from oxidative stress-induced cell death, indicating that the protective effect of PAL is mediated by DJ-1. Furthermore, PAL inhibited production of reactive oxygen species and the inhibition was abated in DJ-1-knockdown cells. PAL increased and decreased phosphorylation of AKT and PTEN, respectively, in SH-SY5Y cells, suggesting that the AKT pathway is one of the specific signaling pathways in PAL-induced neuroprotection. Moreover, PAL prevented superfluous oxidation of cysteine 106 of DJ-1, an essential amino acid for DJ-1's function. The present study demonstrates that PAL has potential neuroprotective effects through DJ-1.

  1. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  2. Neuroprotective Effect of Taurine-Rich Cuttlefish (Sepia officinalis) Extract Against Hydrogen Peroxide-Induced Oxidative Stress in SH-SY5Y Cells.

    PubMed

    Kim, Yon-Suk; Kim, Eun-Kyung; Hwang, Jin-Woo; Kim, Jin-Soo; Shin, Woen-Bin; Dong, Xin; Nawarathna, Weligala Pahalagedara Amila Srilal; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2017-01-01

    Oxidative stress mediates the cell damage in several neurodegenerative diseases, some of which are Alzheimer's disease (AD), multiple sclerosis and Parkinson's disease (PD). In this study, we investigated whether the taurine-rich cuttlefish extract could exert a protective effect on damaged human neuroblastoma SH-SY5Y cells induced by hydrogen peroxide (H2O2). Our results revealed that pre-treatment with cuttlefish extract effectively increased the cell viability by protecting the cells from intracellular reactive oxygen species (ROS) induced by H2O2 exposure. Furthermore, apoptosis related proteins Bcl-2 and Bax were investigated by western-blot analysis and results indicated that cuttlefish extract promoted the expression of anti-apoptotic Bcl-2 protein while inhibiting the expression of pro-apoptotic Bax protein. Therefore, cuttlefish extract containing the ability of scavenging excessive ROS, the capacity of anti-oxidative stress, could be employed in neurodegenerative disease prevention. In conclusion, the results suggest that cuttlefish extract could be used as a potential candidate for preventing several human neurodegenerative and other disorders caused by oxidative stress.

  3. Modulation of cellular Hsp72 levels in undifferentiated and neuron-like SH-SY5Y cells determines resistance to staurosporine-induced apoptosis.

    PubMed

    Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

    2011-01-01

    Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis.

  4. Diquat causes caspase-independent cell death in SH-SY5Y cells by production of ROS independently of mitochondria.

    PubMed

    Nisar, R; Hanson, P S; He, L; Taylor, R W; Blain, P G; Morris, C M

    2015-10-01

    Evidence indicates that Parkinson's disease (PD), in addition to having a genetic aetiology, has an environmental component that contributes to disease onset and progression. The exact nature of any environmental agent contributing to PD is unknown in most cases. Given its similarity to paraquat, an agrochemical removed from registration in the EU for its suspected potential to cause PD, we have investigated the in vitro capacity of the related herbicide Diquat to cause PD-like cell death. Diquat showed greater toxicity towards SH-SY5Y neuroblastoma cells and human midbrain neural cells than paraquat and also MPTP, which was independent of dopamine transporter-mediated uptake. Diquat caused cell death independently of caspase activation, potentially via RIP1 kinase, with only a minor contribution from apoptosis, which was accompanied by enhanced reactive oxygen species production in the absence of major inhibition of complex I of the mitochondrial respiratory chain. No changes in α-synuclein expression were observed following 24-h or 4-week exposure. Diquat may, therefore, kill neural tissue by programmed necrosis rather than apoptosis, reflecting the pathological changes seen following high-level exposure, although its ability to promote PD is unclear.

  5. Protective effects of Arctium lappa L. roots against hydrogen peroxide-induced cell injury and potential mechanisms in SH-SY5Y cells.

    PubMed

    Tian, Xing; Guo, Li-Ping; Hu, Xiao-Long; Huang, Jin; Fan, Yan-Hua; Ren, Tian-Shu; Zhao, Qing-Chun

    2015-04-01

    Accumulated evidence has shown that excessive reactive oxygen species (ROS) have been implicated in neuronal cell death related with various chronic neurodegenerative disorders. This study was designed to explore neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) on hydrogen peroxide (H2O2)-induced cell injury in human SH-SY5Y neuroblastoma cells. The cell viability was significantly decreased after exposure to 200 μM H2O2, whereas pretreatment with different concentrations of EAL attenuated the H2O2-induced cytotoxicity. Hoechst 33342 staining indicated that EAL reversed nuclear condensation in H2O2-treated cells. Meanwhile, TUNEL assay with DAPI staining showed that EAL attenuated apoptosis was induced by H2O2. Pretreatment with EAL also markedly elevated activities of antioxidant enzyme (GSH-Px and SOD), reduced lipid peroxidation (MDA) production, prevented ROS formation, and the decrease of mitochondrial membrane potential. In addition, EAL showed strong radical scavenging ability in 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assays. Furthermore, EAL inhibited H2O2-induced apoptosis by increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, and attenuation of caspase-3, caspase-9 activities, and expressions. These findings suggest that EAL may be regarded as a potential antioxidant agent and possess potent neuroprotective activity against H2O2-induced injury.

  6. Oxidative stress induced by crude venom from the jellyfish Pelagia noctiluca in neuronal-like differentiated SH-SY5Y cells.

    PubMed

    Morabito, Rossana; Condello, Salvatore; Currò, Monica; Marino, Angela; Ientile, Riccardo; La Spada, Giuseppina

    2012-08-01

    Marine toxins are a suitable research model and their mechanism of action is intriguing and still under debate. Either a pore formation mechanism or oxidative stress phenomena may explain the damage induced by toxins. The effect of crude venom from isolated nematocysts of the jellyfish Pelagia noctiluca on neuronal-like cells derived from human neuroblastoma SH-SY5Y has been here studied. To prove the possible oxidative stress events, cell viability, assessed by MTT quantitative colorimetric assay, intracellular reactive oxygen species (ROS) quantified by the non-fluorescent probe H2DCF-DA and changes in mitochondrial transmembrane potential (ΔΨm) measured by the incorporation of a cationic fluorescent dye rhodamine-123 were verified on venom-treated cells (0.05-0.5μg/ml doses). A dose- and time-dependent reduction of all parameters was observed after venom treatment. NAC (N-acetyl-cysteine), antioxidant applied before crude venom application, significantly counteracted the decrease in cell viability and ROS production, while ΔΨm was only partially restored. The disruption of mitochondrial membrane by P. noctiluca crude venom may thus induce oxidative stress by inhibiting mitochondrial respiration and uncoupling oxidative phosphorylation, sensitizing mitochondria in SH-SY5H cells and facilitating membrane permeability. In sum, our findings suggest that P. noctiluca crude venom directly induces ΔΨm collapse with further generation of ROS and add novel information to the understanding of such toxins, still not completely clarified.

  7. Participation of protein kinases in cytotoxic and proapoptotic effects of ethylene glycol ethers and their metabolites in SH-SY5Y cells.

    PubMed

    Pomierny, Bartosz; Fuxe, Kjell; Krzyżanowska, Weronika; Regulska, Magdalena; Broniowska, Żaneta; Budziszewska, Bogusława

    2016-10-01

    Ethylene glycol ethers (EGEs) are compounds widely used in many branches of industry. Their toxicological profile in the peripheral tissues is relatively well described, but little is known about their action on the central nervous system (CNS). In this study, we evaluated the effect of 2-ethoxyethanol (EE), 2-butoxyethanol (BE), 2-phenoxyethanol (PHE) and their metabolites on necrotic (estimated by cell viability and lactate dehydrogenase release) and apoptotic (caspase-3 activity and mitochondrial membrane potential) processes and reactive oxygen species' (ROS) production in human neuroblastoma (SH-SY5Y) cells. We have shown that, similar to the peripheral tissues, EGE metabolites in most of the performed assays revealed greater potential to damage than the parent compounds in the CNS cells. Subsequently, we investigated the participation of some selected protein kinases in the degenerative activity of PHE and its main metabolite, phenoxyacetic acid (PHA). It has been found that a GSK3β inhibitor weakened the damaging effects of PHE and PHA in each of the performed assays. Furthermore, the kinases, p38-MAPK, JNK-MAPK and PKC, had a significant role in the cytotoxic and proapoptotic effects of PHA. These results indicate that the neurotoxic effect of EGEs may stem from their impact on many intracellular signal transduction pathways. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

    PubMed

    Tan, Charlene Siew-Hon; Ho, Christabel Fung-Yih; Heng, Swan-Ser; Wu, Jui-Sheng; Tan, Benny Kwong-Huat; Ng, Yee-Kong; Sun, Grace Y; Lin, Teng-Nan; Ong, Wei-Yi

    2016-09-01

    Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

  9. Dimerumic Acid and Deferricoprogen Activate Ak Mouse Strain Thymoma/Heme Oxygenase-1 Pathways and Prevent Apoptotic Cell Death in 6-Hydroxydopamine-Induced SH-SY5Y Cells.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-03

    Parkinson's disease (PD) is a neurodegenerative disorder, which can be modeled using the neurotoxin 6-hydroxydopamine (6-OHDA) to generate oxidative stress. Here, we studied the effects of the antioxidants deferricoprogen (DFC) and dimerumic acid (DMA), produced by rice fermented with Monascus purpureus NTU 568, on 6-OHDA-induced apoptosis in SH-SY5Y cells and their potential protective mechanisms. DMA and DFC inhibited 6-OHDA-induced apoptosis and cellular reactive oxygen species (ROS) in SH-SY5Y human neuroblastoma cells. Molecular analysis demonstrated associated upregulation of the Ak mouse strain thymoma (Akt), heme oxygenase-1 (HO-1), and signal-regulated kinase (ERK) pathways along with inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and p38 pathways and altered homodimeric glycoprotein, N-methyl-d-aspartate (NMDA) receptor, and immunoglobulin Fc receptor gene expression. These results suggested that the neuroprotection elicited by DMA and DFC against 6-OHDA-induced neurotoxicity was associated with the Akt, MAPK, and HO-1 pathways via regulating the gene expression of NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor.

  10. Metal specificity of an iron-responsive element in Alzheimer's APP mRNA 5'untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator.

    PubMed

    Bandyopadhyay, S; Huang, X; Cho, H; Greig, N H; Youdim, M B; Rogers, J T

    2006-01-01

    Iron closely regulates the expression of the Alzheimer's Amyloid Precursor Protein (APP) gene at the level of message translation by a pathway similar to iron control of the translation of the ferritin L- and H mRNAs by Iron-responsive Elements in their 5' untranslated regions (5'UTRs). Using transfection based assays in SH-SY5Y neuroblastoma cells we tested the relative efficiency by which iron, copper and zinc up-regulate IRE activity in the APP 5'UTR. Desferrioxamine (high affinity Fe3+ chelator), (ii) clioquinol (low affinity Fe/Cu/Zn chelator), (iii) piperazine-1 (oral Fe chelator), (iv) VK-28 (oral Fe chelator), were tested for their relative modulation of APP 5' UTR directed translation of a luciferase reporter gene. Iron chelation based therapeutic strategies for slowing the progression of Alzheimer's disease (and other neurological disorders that manifest iron imbalance) are discussed with regard to the relative neural toxic action of each chelator in SH-SY5Y cells and in H4 glioblastoma cells.

  11. A multidisciplinary approach to study the functional properties of neuron-like cell models constituting a living bio-hybrid system: SH-SY5Y cells adhering to PANI substrate

    NASA Astrophysics Data System (ADS)

    Caponi, S.; Mattana, S.; Ricci, M.; Sagini, K.; Juarez-Hernandez, L. J.; Jimenez-Garduño, A. M.; Cornella, N.; Pasquardini, L.; Urbanelli, L.; Sassi, P.; Morresi, A.; Emiliani, C.; Fioretto, D.; Dalla Serra, M.; Pederzolli, C.; Iannotta, S.; Macchi, P.; Musio, C.

    2016-11-01

    A living bio-hybrid system has been successfully implemented. It is constituted by neuroblastic cells, the SH-SY5Y human neuroblastoma cells, adhering to a poly-anyline (PANI) a semiconductor polymer with memristive properties. By a multidisciplinary approach, the biocompatibility of the substrate has been analyzed and the functionality of the adhering cells has been investigated. We found that the PANI films can support the cell adhesion. Moreover, the SH-SY5Y cells were successfully differentiated into neuron-like cells for in vitro applications demonstrating that PANI can also promote cell differentiation. In order to deeply characterize the modifications of the bio-functionality induced by the cell-substrate interaction, the functional properties of the cells have been characterized by electrophysiology and Raman spectroscopy. Our results confirm that the PANI films do not strongly affect the general properties of the cells, ensuring their viability without toxic effects on their physiology. Ascribed to the adhesion process, however, a slight increase of the markers of the cell suffering has been evidenced by Raman spectroscopy and accordingly the electrophysiology shows a reduction at positive stimulations in the cells excitability.

  12. "Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites.

    PubMed

    Barbosa, Daniel José; Capela, João Paulo; Silva, Renata; Ferreira, Luísa Maria; Branco, Paula Sério; Fernandes, Eduarda; Bastos, Maria Lourdes; Carvalho, Félix

    2014-02-01

    3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA's neurotoxicity.

  13. Comprehensive SNP array study of frequently used neuroblastoma cell lines; copy neutral loss of heterozygosity is common in the cell lines but uncommon in primary tumors

    PubMed Central

    2011-01-01

    Background Copy neutral loss of heterozygosity (CN-LOH) refers to a special case of LOH occurring without any resulting loss in copy number. These alterations is sometimes seen in tumors as a way to inactivate a tumor suppressor gene and have been found to be important in several types of cancer. Results We have used high density single nucleotide polymorphism arrays in order to investigate the frequency and distribution of CN-LOH and other allelic imbalances in neuroblastoma (NB) tumors and cell lines. Our results show that the frequency of these near-CN-LOH events is significantly higher in the cell lines compared to the primary tumors and that the types of CN-LOH differ between the groups. We also show that the low-risk neuroblastomas that are generally considered to have a "triploid karyotype" often present with a complex numerical karyotype (no segmental changes) with 2-5 copies of each chromosome. Furthermore a comparison has been made between the three related cell lines SK-N-SH, SH-EP and SH-SY5Y with respect to overall genetic aberrations, and several aberrations unique to each of the cell lines has been found. Conclusions We have shown that the NB tumors analyzed contain several interesting allelic imbalances that would either go unnoticed or be misinterpreted using other genome-wide techniques. These findings indicate that the genetics underlying NB might be even more complex than previously known and that SNP arrays are important analysis tools. We have also showed that these near-CN-LOH events are more frequently seen in NB cell lines compared to NB tumors and that a set of highly related cell lines have continued to evolve secondary to the subcloning event. Taken together our analysis highlights that cell lines in many cases differ substantially from the primary tumors they are thought to represent, and that caution should be taken when drawing conclusions from cell line-based studies. PMID:21899760

  14. Cold-Inducible Protein RBM3 Protects UV Irradiation-Induced Apoptosis in Neuroblastoma Cells by Affecting p38 and JNK Pathways and Bcl2 Family Proteins.

    PubMed

    Zhuang, Rui-Juan; Ma, Jian; Shi, Xiang; Ju, Fei; Ma, Shuang-Ping; Wang, Lei; Cheng, Bin-Feng; Ma, Yan-Wen; Wang, Mian; Li, Tong; Feng, Zhi-Wei; Yang, Hai-Jie

    2017-08-22

    Induced by hypothermia, cold-inducible protein RBM3 (RNA-binding protein motif 3), has been implicated in neuroprotection against various toxic insults such as hypoxia and ischemia. However, whether mild hypothermia and RBM3 prevent neural cells from UV irradiation-elicited apoptosis is unclear. In the present study, human neuroblastoma cell line SH-SY5Y was used as a cell model for neural cell death, and it was demonstrated that mild hypothermia protects SH-SY5Y cells from UV irradiation-induced apoptosis. However, the protective effect of mild hypothermia was abrogated when RBM3 was silenced. Conversely, the overexpression of RBM3 rescued SH-SY5Y cells from UV-induced apoptosis, as indicated by the decreased levels of cleaved caspase-3 and PARP, and increased cell survival. The analysis on the mechanism underlying RBM3-mediated neuroprotection against UV insult showed that RBM3 could substantially block the activation of p38 and JNK signaling pathways. In addition, the overexpression of RBM3 reduced the expression of pro-apoptotic proteins Bax and Bad, leaving the pro-survival protein Bcl-2 unaffected. In conclusion, RBM3 is the key mediator of mild hypothermia-related protection against UV in neuroblastoma cells, and the neuroprotective effect might be exerted through interfering with pro-apoptotic signaling pathways p38 and JNK and regulating pro-apoptotic proteins Bax and Bad.

  15. Inhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin.

    PubMed

    Gangoda, Lahiru; Keerthikumar, Shivakumar; Fonseka, Pamali; Edgington, Laura E; Ang, Ching-Seng; Ozcitti, Cemil; Bogyo, Matthew; Parker, Belinda S; Mathivanan, Suresh

    2015-05-10

    Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification

  16. Glycolysis-respiration relationships in a neuroblastoma cell line.

    PubMed

    Swerdlow, Russell H; E, Lezi; Aires, Daniel; Lu, Jianghua

    2013-04-01

    Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

    PubMed Central

    Armstrong, J L; Ruiz, M; Boddy, A V; Redfern, C P F; Pearson, A D J; Veal, G J

    2005-01-01

    Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 μM) or R116010 (1 or 10 μM) in combination with either 10 μM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma. PMID:15714209

  18. Pharmacological Characterization of the Mechanisms Involved in Delayed Calcium Deregulation in SH-SY5Y Cells Challenged with Methadone

    PubMed Central

    Perez-Alvarez, Sergio; Solesio, Maria E.; Cuenca-Lopez, Maria D.; Melero-Fernández de Mera, Raquel M.; Villalobos, Carlos; Kmita, Hanna; Galindo, Maria F.; Jordán, Joaquin

    2012-01-01

    Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca2+ responses to 0.5 mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, μ-opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment. PMID:22778742

  19. Neuromodulatory effects of Calyptranthes grandifolia extracts against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells.

    PubMed

    Kich, Débora Mara; Bitencourt, Shanna; Alves, Celso; Silva, Joana; Pinteus, Susete; Pedrosa, Rui; Laufer, Stefan; de Souza, Claucia Fernanda Volken; Goettert, Márcia Inês

    2016-12-01

    Alzheimer's and Parkinson's diseases are neurodegenerative disorders characterized by progressive neuronal dysfunction. Previous studies revealed that some natural products have neuroprotective properties, including species of the Myrtaceae family. However, the neuromodulatory potential of Calyptranthes grandifolia is not clear. In the present study, we examined the ability of the ethanol and hexane leaf extracts of C. grandifolia to prevent 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Initially, we investigated the potential of the extracts to inhibit the neurodegenerative-related enzymes c-Jun N-terminal kinase 3 (JNK3) and acetylcholinesterase (AChE). In addition, SH-SY5Y cell viability was assessed by MTT assay after 100μM 6-OHDA-induced cell damage. In order to verify the possible effects of both extracts on 6-OHDA-induced cell death, hydrogen peroxide generation, mitochondrial potential and caspases-3 activity were assessed. Our findings revealed that ethanol extract exhibited inhibitory activity against JNK3 and AChE. In addition, when co-treating SH-SY5Y cells with 6-OHDA and the extracts, oxidative stress was inhibited by both extracts through a decrease of mitochondrial depolarization and caspases-3 activity. In summary, ethanol and hexane extracts of C. grandifolia have some suppressive property against neurotoxicity induced by 6-OHDA. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

    PubMed Central

    Wang, Rui; Wang, Suqing; Malter, James S.; Wang, Deng-Shun

    2009-01-01

    The cerebral accumulation of β-amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer’s Disease (AD). In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβ in vivo, is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of AD patients. In order to determine if these changes affected Aβ, SH-SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1-40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1-42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared to the NEP protein level. The present study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation. PMID:19196432

  1. Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells.

    PubMed

    Ramachandran, Umamaheswari; Manavalan, Arulmani; Sundaramurthi, Husvinee; Sze, Siu Kwan; Feng, Zhi Wei; Hu, Jiang-Miao; Heese, Klaus

    2012-06-01

    Tianma (Rhizoma gastrodiae) is the dried rhizome of the plant Gastrodia elata Blume (Orchidaceae family). As a medicinal herb in traditional Chinese medicine (TCM) its functions are to control convulsions, pain, headache, dizziness, vertigo, seizure, epilepsy and others. In addition, tianma is frequently used for the treatment of neurodegenerative disorders though the mechanism of action is widely unknown. Accordingly, this study was designed to examine the effects of tianma on the proteome metabolism in differentiated human neuronal SH-SY5Y cells to explore its specific effects on neuronal signaling pathways. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 2390 modulated proteins, out of which 406 were found to be altered by tianma in differentiated human neuronal SH-SY5Y cells. Based on the observed data, we hypothesize that tianma promotes neuro-regenerative signaling cascades by controlling chaperone/proteasomal degradation pathways (e.g. CALR, FKBP3/4, HSP70/90) and mobilizing neuro-protective genes (such as AIP5) as well as modulating other proteins (RTN1/4, NCAM, PACSIN2, and PDLIM1/5) with various regenerative modalities and capacities related to neuro-synaptic plasticity.

  2. Artemether Regulates Chemosensitivity to Doxorubicin via Regulation of B7-H3 in Human Neuroblastoma Cells.

    PubMed

    Tan, Wei-Qiang; Chen, Gang; Ye, Ming; Jia, Bing

    2017-09-03

    BACKGROUND Artemether, originally used for malaria, exhibits potential therapeutic efficacy against several types of cancer, including gastric cancer, hepatocellular carcinoma, and gliomas. In this study, we investigated the role and mechanism of artemether on drug resistance of neuroblastoma cells. MATERIAL AND METHODS Cell viability and proliferation were determined by CCK-8 and EdU incorporation assay, respectively. Gene expression was measured by real-time PCR and Western blot analysis. RESULTS Our results revealed that artemether treatment remarkably inhibited the proliferation of neuroblastoma cell lines SH-SY5Y, SK-N-SH, and SK-N-BE2. In addition, co-treatment of tumor cells with artemether and doxorubicin significantly reduced cell viability and DNA synthesis compared with doxorubicin-treated cells. On the molecular level, we found that combined treatment with artemether and doxorubicin suppressed the expression of B7-H3 both at the mRNA and protein levels. In addition, artemether failed to sensitize tumor cells to doxorubicin in SH-SY5Y cells overexpressing B7-H3. CONCLUSIONS Artemether-mediated inhibition of B7-H3 may contribute to doxorubicin sensitivity in neuroblastoma cells, suggesting that artemether could serve as a potential therapeutic option for neuroblastoma.

  3. Expanded and Wild-type Ataxin-3 Modify the Redox Status of SH-SY5Y Cells Overexpressing α-Synuclein.

    PubMed

    Noronha, Carolina; Perfeito, Rita; Laço, Mário; Wüllner, Ullrich; Rego, A Cristina

    2017-05-01

    Neurodegenerative diseases are considered to be distinct clinical entities, although they share the formation of proteinaceous aggregates and several neuropathological mechanisms. Increasing evidence suggest a possible interaction between proteins that have been classically associated to distinct neurodegenerative diseases. Thus, common molecular and cellular pathways might explain similarities between disease phenotypes. Interestingly, the characteristic Parkinson's disease (PD) phenotype linked to bradykinesia is also a clinical presentation of other neurodegenerative diseases. An example is Machado-Joseph disease (MJD), with some patients presenting parkinsonism and a positive response to levodopa (L-DOPA). Protein aggregates positive for α-synuclein (α-Syn), a protein associated with PD, in the substantia nigra of MJD models made us hypothesize a putative additive biological effect induced by expression of α-Syn and ataxin-3 (Atx3), the protein affected in MJD. Hence, in this study we analysed the influence of these two proteins (α-Syn and wild-type or mutant Atx3) on modified redox signaling, a pathological process potentially linked to both diseases, and also the impact of exposure to iron and rotenone in SH-SY5Y neuroblastoma cells. Our results show that both α-Syn and mutant Atx3 overexpression per se increased oxidation of dichlorodihydrofluorescein (DCFH2), and co-expression of these proteins exhibited additive effect on intracellular oxidation, with no correlation with apoptotic features. Mutant Atx3 and α-Syn also potentiated altered redox status induced by iron and rotenone, a hint to how these proteins might influence neuronal dysfunction under pro-oxidant conditions. We further show that overexpression of wild-type Atx3 decreased intracellular DCFH2 oxidation, possibly exerting a neuroprotective role.

  4. Curcumin Rescues a PINK1 Knock Down SH-SY5Y Cellular Model of Parkinson's Disease from Mitochondrial Dysfunction and Cell Death.

    PubMed

    van der Merwe, Celia; van Dyk, Hayley Christy; Engelbrecht, Lize; van der Westhuizen, Francois Hendrikus; Kinnear, Craig; Loos, Ben; Bardien, Soraya

    2017-05-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Mutations in the PINK1 gene result in an autosomal recessive form of early-onset PD. PINK1 plays a vital role in mitochondrial quality control via the removal of dysfunctional mitochondria. The aim of the present study was to create a cellular model of PD using siRNA-mediated knock down of PINK1 in SH-SY5Y neuroblastoma cells The possible protective effects of curcumin, known for its many beneficial properties including antioxidant and anti-inflammatory effects, was tested on this model in the presence and absence of paraquat, an additional stressor. PINK1 siRNA and control cells were separated into four treatment groups: (i) untreated, (ii) treated with paraquat, (iii) pre-treated with curcumin then treated with paraquat, or (iv) treated with curcumin. Various parameters of cellular and mitochondrial function were then measured. The PINK1 siRNA cells exhibited significantly decreased cell viability, mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production, and increased apoptosis. Paraquat-treated cells exhibited decreased cell viability, increased apoptosis, a more fragmented mitochondrial network and decreased MMP. Curcumin pre-treatment followed by paraquat exposure rescued cell viability and increased MMP and mitochondrial respiration in control cells, and significantly decreased apoptosis and increased MMP and maximal respiration in PINK1 siRNA cells. These results highlight a protective effect of curcumin against mitochondrial dysfunction and apoptosis in PINK1-deficient and paraquat-exposed cells. More studies are warranted to further elucidate the potential neuroprotective properties of curcumin.

  5. Mechanical stretch exacerbates the cell death in SH-SY5Y cells exposed to paraquat: mitochondrial dysfunction and oxidative stress.

    PubMed

    Wang, Fang; Franco, Rodrigo; Skotak, Maciej; Hu, Gang; Chandra, Namas

    2014-03-01

    Recent studies suggest that traumatic brain injury (TBI) and pesticide exposure increase the risk of Parkinson's disease (PD), but the molecular mechanisms involved remain unclear. Using an in vitro model of TBI, we evaluated the role of mitochondrial membrane potential (ΔΨm) and mitochondrial reactive oxygen species (ROS) induced by stretch on dopaminergic cell death upon paraquat exposure. Human dopaminergic neuroblastoma SH-SY5Y cells grown on silicone membrane were stretched at mild (25%) and moderate (50%) strain prior to paraquat exposure. We observed that moderate stretch (50% strain) increased the vulnerability of cells to paraquat demonstrated by the loss of plasma membrane integrity (propidium iodide-uptake) and decreased mitochondrial activity (MTT assay). Mitochondrial depolarization occurred immediately after stretch, while mitochondrial ROS increased rapidly and remained elevated for up to 4h after the stretch injury. Intracellular glutathione (GSH) stores were also transiently decreased immediately after moderate stretch. Cells treated with paraquat, or moderate stretch exhibited negligible mitochondrial depolarization at 48h post treatment, whereas in cells stretched prior to paraquat exposure, a significant mitochondrial depolarization occurred compared to samples exposed to either paraquat or stretch. Moderate stretch also increased mitochondrial ROS formation, as well as exacerbated intracellular GSH loss induced by paraquat. Overexpression of manganese superoxide dismutase (MnSOD) markedly diminished the deleterious effects of stretch in paraquat neurotoxicity. Our findings demonstrate that oxidative stress induced by mitochondrial dysfunction plays a critical role in the synergistic toxic effects of stretch (TBI) and pesticide exposure. Mitigation of oxidative stress via mitochondria-targeted antioxidants appears an attractive route for treatment of neurodegeneration mediated by TBI.

  6. A pro-drug of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents differentiated SH-SY5Y cells from toxicity induced by 6-hydroxydopamine.

    PubMed

    Chao, Jianfei; Lau, Way Kwok-Wai; Huie, Michelle Justine; Ho, Yuen-Shan; Yu, Man-Shan; Lai, Cora Sau-Wan; Wang, Mingfu; Yuen, Wai-Hung; Lam, Wai Har; Chan, Tak Hang; Chang, Raymond Chuen-Chung

    2010-01-29

    Regular consumption of green tea benefits people in prevention from cardiovascular disorders, obesity as well as neurodegenerative diseases. (-)-Epigallocatechin-3-gallate (EGCG) is regarded as the most biologically active catechin in green tea. However, the stability and bioavailability of EGCG are restricted. The purpose of the present study was to investigate whether a pro-drug, a fully acetylated EGCG (pEGCG), could be more effective in neuroprotection in Parkinsonism mimic cellular model. Retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cells were pre-treated with different concentrations of EGCG and pEGCG for 30 min and followed by incubation of 25 microM 6-hydroxydopamine (6-OHDA) for 24h. We found that a broad dosage range of pEGCG (from 0.1 to 10 microM) could significantly reduce lactate dehydrogenase release. Likewise, 10 microM of pEGCG was effective in reducing caspase-3 activity, while EGCG at all concentrations tested in the model failed to attenuate caspase-3 activity induced by 6-OHDA. Furthermore, Western-blot analysis showed that Akt could be one of the specific signaling pathways stimulated by pEGCG in neuroprotection. It was demonstrated that 25 microM of 6-OHDA significantly suppressed the phosphorylation level of Akt. Only pEGCG at 10 microM markedly increased its phosphorylation level compared to 6-OHDA alone. Taken together, as pEGCG has higher stability and bioavailability for further investigation, it could be a potential neuroprotective agent and our current findings may offer certain clues for optimizing its application in future. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  7. Mechanical stretch exacerbates the cell death in SH-SY5Y cells exposed to paraquat: mitochondrial dysfunction and oxidative stress

    PubMed Central

    Wang, Fang; Franco, Rodrigo; Skotak, Maciej; Hu, Gang; Chandra, Namas

    2014-01-01

    Recent studies suggest that traumatic brain injury (TBI) and pesticide exposure increase the risk of Parkinson’s disease (PD), but the molecular mechanisms involved remain unclear. Using an in vitro model of TBI, we evaluated the role of mitochondrial membrane potential (ΔΨm) and mitochondrial reactive oxygen species (ROS) induced by stretch on dopaminergic cell death upon paraquat exposure. Human dopaminergic neuroblastoma SH-SY5Y cells grown on silicone membrane were stretched at mild (25%) and moderate (50%) strain prior to paraquat exposure. We observed that moderate stretch (50% strain) increased the vulnerability of cells to paraquat demonstrated by the loss of plasma membrane integrity (propidium iodide-uptake) and decreased mitochondrial activity (MTT assay). Mitochondrial depolarization occurred immediately after stretch, while mitochondrial ROS increased rapidly and remained elevated for up to 4 h after the stretch injury. Intracellular glutathione (GSH) stores were also transiently decreased immediately after moderate stretch. Cells treated with paraquat, or moderate stretch exhibited negligible mitochondrial depolarization at 48 h post treatment, whereas in cells stretched prior to paraquat exposure, a significant mitochondrial depolarization occurred compared to samples exposed to either paraquat or stretch. Moderate stretch also increased mitochondrial ROS formation, as well as exacerbated intracellular GSH loss induced by paraquat. Overexpression of manganese superoxide dismutase (MnSOD) markedly diminished the deleterious effects of stretch in paraquat neurotoxicity. Our findings demonstrate that oxidative stress induced by mitochondrial dysfunction plays a critical role in the synergistic toxic effects of stretch (TBI) and pesticide exposure. Mitigation of oxidative stress via mitochondria-targeted antioxidants appears an attractive route for treatment of neurodegeneration mediated by TBI. PMID:24462953

  8. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    SciTech Connect

    Lu, Zhengyu; Yang, Qi; Cui, Mei; Liu, Yanping; Wang, Tao; Zhao, Hong; Dong, Qiang

    2014-03-28

    Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.

  9. Cordyceps sinensis Oral Liquid Inhibits Damage Induced by Oxygen and Glucose Deprivation in SH-SY5Y Cells.

    PubMed

    Zou, Ying-Xin; Liu, Yu-Xiang; Ruan, Ming-Hua; Zhou, Yi; Wang, Jia-Chun; Chu, Zhi-Yong

    2016-01-01

    Cordyceps sinensis has been used in traditional Chinese medicine for thousands of years. It has been demonstrated to have a variety of biological activities, and an extract of it has been demonstrated to possess a protective effect in occlusion-induced focal cerebral ischemia of the middle cerebral artery in rats. It could be explored as an agent for treatment of ischemic stroke, and the mechanisms need to be studied further. The study intended to investigate the protective effects of the Cordyceps sinensis oral liquid (CSOL) against damage induced by oxygen and glucose deprivation (OGD) in SH-SY5Y cells. DESIGN • The research team designed an in vitro study. The study occurred at the Naval Medical Research Institute in Shanghai, China. SH-SY5Y cells were exposed to CSOL in doses of 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL, creating 5 intervention groups. The OGD condition was induced by transfer of the cells from high-glucose Dulbecco's Modified Eagle's medium (DMEM) in a box gassed with air containing 5% CO2 to glucose-free DMEM in a box gassed with 94% N2, 5% CO2, and 1% O2. Like the cells for the interventions groups, the cells for a model group were cultured with high-glucose DMEM and were transferred to the OGD, but they received no dose of COSL. Cells in a control group were cultured with high-glucose DMEM, were not transferred to the OGD condition, and did not receive any dose of COSL. Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The apoptosis and the mitochondrial membrane potential (MMP) were detected by flow cytometry, and the protein expression of caspase-3 was observed by western blot. After exposure to OGD, the cell viability of cells treated with 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL of CSOL increased in a dose-effect relationship. Compared with the cells in the model group, the treatment of CSOL at all the experimental concentrations significantly inhibited both the cell apoptosis

  10. Four new compounds from the bulbs of Lycoris aurea with neuroprotective effects against CoCl₂ and H₂O₂-induced SH-SY5Y cell injuries.

    PubMed

    Jin, An; Li, Xue; Zhu, Yun-Yun; Yu, Heng-Yi; Pi, Hui-Fang; Zhang, Peng; Ruan, Han-Li

    2014-03-01

    Three new alkaloids, 2α-hydroxy-6-O-n-butyloduline, O-n-butyllycorenine, (-)-N-(chloromethyl)lycoramine (1-3), and a new phenolic compound, ((7S)-7-(4-hydroxyphenyl)-7-hydroxypropyl)-2'-methylbenzene-3',6'-diol (14), along with ten known alkaloids (4-13), were isolated from the bulbs of Lycoris aurea collected from Huaihua County of Hunan Province, China. Their structures were elucidated by spectroscopic methods including HRESIMS, UV, IR, and NMR. All the isolated compounds were tested for their neuroprotective effects against CoCl2 and H2O2-induced SH-SY5Y cell death. Compounds 1-7 and 10 exhibited significant neuroprotective effects against CoCl2-induced SH-SY5Y cell injury, while compounds 1-5, 7, 10 and 12 showed obvious neuroprotective effects against H2O2-induced SH-SY5Y cell death.

  11. Raman micro-spectroscopy study of living SH-SY5Y cells adhering on different substrates.

    PubMed

    Caponi, S; Mattana, S; Ricci, M; Sagini, K; Urbanelli, L; Sassi, P; Morresi, A; Emiliani, C; Dalla Serra, M; Iannotta, S; Musio, C; Fioretto, D

    2016-01-01

    In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information.

  12. FOXO3 promoted mitophagy via nuclear retention induced by manganese chloride in SH-SY5Y cells.

    PubMed

    Song, Dongmei; Ma, Junxiang; Chen, Li; Guo, Caixia; Zhang, Yuanyuan; Chen, Tian; Zhang, Shixuan; Zhu, Zhonghui; Tian, Lin; Niu, Piye

    2017-09-20

    To evaluate the role of FOXO3 during the process of mitophagy induced by manganese chloride (MnCl2), mitochondrial dysfunction and mitophagy were detected before and after FOXO3 was knocked down in SH-SY5Y cells. Transmission electron microscopy (TEM), flow cytometry, confocal microscopy and a western blot were used to detect mitochondrial ultrastructure and autophagy, Ca(2+) levels, mitochondrial reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP), autophagosomes and mitophagy marker proteins (p62, LC3-II/LC3-I, Beclin-1, PINK1 and P-parkin), respectively. After SH-SY5Y cells were exposed to MnCl2, the levels of cytoplasmic Ca(2+) and mitochondrial ROS increased but the mitochondrial MMP decreased significantly compared to the control in a dose- and time-dependent manner (p < 0.05), which indicated that MnCl2 can lead to mitochondrial dysfunction. Under TEM, mitophagy and autolysosomes were observed. The WB results also showed that mitophagy marker proteins including LC3-II/LC3-I, Beclin-1, PINK1 and P-parkin except for p62 increased in a dose- and time-dependent manner, accompanied by FOXO3 nuclear retention, which indicated that MnCl2 can lead to mitophagy and FOXO3 nuclear translocation may be involved in this process. After FOXO3 was knocked down, the inverse results of mitophagy and the levels of mitochondrial ROS decreasing were observed, which showed that FOXO3 silencing could inhibit mitophagy and mitochondrial dysfunction induced by MnCl2. Our results indicated that Mn could induce mitophagy by enhancing FOXO3 nuclear retention, which might promote mitophagy induced by MnCl2.

  13. Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

    PubMed Central

    Dasgupta, Sayani; Castro, Leandro M.; Dulman, Russell; Yang, Ciyu; Schmidt, Marion; Ferro, Emer S.; Fricker, Lloyd D.

    2014-01-01

    The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell. PMID:25079948

  14. Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol.

    PubMed

    Sun, Wenjun; Chen, Liangjing; Zheng, Wei; Wei, Xiaoan; Wu, Wenqi; Duysen, Ellen G; Jiang, Wei

    2017-06-01

    Ethanol is one of the most commonly abused psychotropic substances with deleterious effects on the central nervous system. Ethanol exposure during development results in the loss of neurons in brain regions and when exposed to ethanol cultured cells undergo apoptosis. To date no information is available on whether abnormally high AChE activity is characteristic of apoptosis in animals exposed to ethanol. The aims of the present study were to determine whether induction of AChE activity is associated with ethanol-induced apoptosis and to explore the mechanism of enhanced AChE activity induced by ethanol. For this purpose, in vitro and in vivo experiments were performed. AChE activity was quantified by spectrophotometry and apoptosis by flow cytometer in SH-SY5Y cells exposed to ethanol. The results showed that cells treated with 500mM ethanol for 24h had a 9-fold increase in apoptotic cells and a 6-fold increase in AChE activity compared with controls. Mice exposed acutely to 200μl of 20% ethanol daily on days 1-4 had elevated AChE activity in plasma on days 3-7. On day 4, plasma AChE activity was 2.4-fold higher than pretreatment activity. More apoptotic cells were found in the brains of treated mice compared to controls. Cells in brain sections that were positive in the TUNEL assay stained for AChE activity. In conclusion, AChE activity and apoptosis were induced in SH-SY5Y cells and mice treated with ethanol, which may indicate that increased AChE may related to apoptosis induced by ethanol. Unusually high AChE activity may be an effect marker of exposure to ethanol. The relationship between AChE and apoptosis might represent a novel mechanism of ethanol-associated neuronal injury. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Neuroprotective Effect of SLM, a Novel Carbazole-Based Fluorophore, on SH-SY5Y Cell Model and 3xTg-AD Mouse Model of Alzheimer's Disease.

    PubMed

    Wu, Xiaoli; Kosaraju, Jayasankar; Zhou, Wei; Tam, Kin Yip

    2017-03-15

    Amyloid β (Aβ) peptide aggregating to form a neurotoxic plaque, leading to cognitive deficits, is believed to be one of the plausible mechanisms for Alzheimer's disease (AD). Inhibiting Aβ aggregation is supposed to offer a neuroprotective effect to ameliorate AD. A previous report has shown that SLM, a carbazole-based fluorophore, binds to Aβ to inhibit the aggregation. However, it is not entirely clear whether the inhibition of Aβ aggregation alone would lead to the anticipated neuroprotective effects. In the current study, we intended to examine the protective action of SLM against Aβ-induced neurotoxicity in vitro and to evaluate if SLM can decrease the cognitive and behavioral deficits observed in triple transgenic AD mouse model (3xTg-AD). In the in vitro study, neurotoxicity induced by Aβ42 in human neuroblastoma (SH-SY5Y) cells was found to be reduced through the treatment with SLM. In the in vivo study, following one month SLM intraperitoneal injection (1, 2, and 4 mg/kg), 3xTg-AD mice were tested on Morris water maze (MWM) and Y-maze for their cognitive ability and sacrificed for biochemical estimations. Results show that SLM treatment improved the learning and memory ability in 3xTg-AD mice in MWM and Y-maze tasks. SLM also mitigated the amyloid burden by decreasing brain Aβ40 and Aβ42 levels and reduced tau phosphorylation, glycogen synthase kinase-3β activity, and neuro-inflammation. From our observations, SLM shows neuroprotection in SH-SY5Y cells against Aβ42 and also in 3xTg-AD mouse model by mitigating the pathological features and behavioral impairments.

  16. Preventing expression of the nicotinic receptor subunit α7 in SH-SY5Y cells with interference RNA indicates that this receptor may protect against the neurotoxicity of Aβ.

    PubMed

    Qi, Xiao-Lan; Ou-Yang, Kai; Ren, Jia-Mou; Wu, Chang-Xue; Xiao, Yan; Li, Yi; Guan, Zhi-Zhong

    2013-05-01

    The present aim was to characterize the influence of the α7 nicotinic acetylcholine receptor (nAChR) on BACE, the enzyme that cleaves the amyloid precursor protein (APP) at the β-site, as well as on the oxidative stress induced by amyloid-β peptide (Aβ). To this end, human neuroblastoma SH-SY5Y cells were transfected with siRNAs targeting the α7 nAChR subunit and/or exposed to Aβ1-42. For α7 nAChR, BACE1 (cleaving at the β-site of APP) and BACE2 (cleaving within the Aβ domain), α-secretase (ADAM10), and the two components of γ-secretase, PS and NCT, the mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. The level of Aβ1-42 in the cell culture medium was determined by an ELISA procedure. The extent of lipid peroxidation and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were assayed spectrophotometrically. In the transfected SH-SY5Y cells, expression of α7 nAChR was reduced; the level of BACE1 increased and that of BACE2 decreased; the amount of ADAM10 lowered; and the level of PS raised. Moreover, the level of Aβ1-42 in the culture medium was elevated. Treatment of non-transfected cells with Aβ elevated the level of malondialdehyde (MDA) and lowered the activities of SOD and GSH-Px and these changes were potentiated by inhibiting expression of α7 nAChR. These results indicate that α7 nAChR plays a significant role in amyloidogenic metabolism of APP and the oxidative stress evoked by Aβ, suggesting that this receptor might help protect against the neurotoxicity of Aβ.

  17. [Protective Effects of Total Saponins from Panax japonicus on H₂O₂Induced Injury via Mitochondia Pathway in SH-SY5Y Cells].

    PubMed

    Deng, Li-li; Wan, Jing-zhi; Yuan, Ding; Zhang, Chang-cheng; Li, Geng; Wang, Ting

    2015-08-01

    To investigate the protective mechanism of total saponins from Panax japonicus (SPJ) on H₂O₂ induced injury in SH-SY5Y cells. SH-SY5Y cells were divided into three groups: blank control group, model group (600 µmol/L H₂O₂) and drug treatment groups. Different concentrations of SPJ (0.1, 1, 5 and 20 µg/mL) were incubated with SH-SY5Y cells for 12 hours prior to exposing to 600 µmol/L H₂O₂ for another 12 h. Mitochondrial membrane potential (MMP) was detected by JC-1 method. Protein expressions of Sirt1 , PGC-1α, Foxo3a, LC3-II and Beclin1 were detected by Western blotting. Compared to the H₂O₂ model group, SPJ pretreatment significantly increased MMP level and enhanced the protein expressions of Sirt1, PGC-1α, Foxo3a, LC3-II and Beclin1. SPJ exerts protective effect on H₂O₂ induced SH-SY5Y cell injury through mitochondria pathway.

  18. Antihypoxic effect of miR-24 in SH-SY5Y cells under hypoxia via downregulating expression of neurocan

    SciTech Connect

    Sun, Xingyuan Ren, Zhanjun; Pan, Yunzhi; Zhang, Chenxin

    2016-09-02

    Hypoxia-induced apoptosis-related mechanisms involved in the brain damage following cerebral ischemia injury. A subset of the small noncoding microRNA (miRNAs) is regulated by tissue oxygen levels, and miR-24 was found to be activated by hypoxic conditions. However, the roles of miR-24 and its target gene in neuron are not well understood. Here, we validated miRNA-24 is down-regulated in patients with cerebral infarction. Hypoxia suppressed the expression of miR-24, but increased the expression of neurocan in both mRNA and protein levels in SH-SY5Y cells. MiR-24 mimics reduced the expression of neurocan, suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. By luciferase reporter assay, neurocan is validated a direct target gene of miR-24. Furthermore, knockdown of neurocan suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. Taken together, miR-24 overexpression or silencing of neurocan shows an antihypoxic effect in SH-SY5Y cells. Therefore, miR-24 and neurocan play critical roles in neuron cell apoptosis and are potential therapeutic targets for ischemic brain disease. - Highlights: • miR-24 and neurocan play critical roles in neuron cell apoptosis. • miR-24 and neurocan are potential therapeutic targets for ischemic brain disease. • Antihypoxic effect of miR-24 and neurocan in SH-SY5Y cells.

  19. Noscapine induced apoptosis via downregulation of survivin in human neuroblastoma cells having wild type or null p53.

    PubMed

    Li, Shiwang; He, Jing; Li, Shuai; Cao, Guoqing; Tang, Shaotao; Tong, Qiangsong; Joshi, Harish C

    2012-01-01

    Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. Noscapine, a nontoxic natural compound, can trigger apoptosis in many cancer types. We now show that p53 is dispensable for Noscapine-induced cell death in neuroblastoma cell lines, proapoptotic response to this promising chemopreventive agent is mediated by suppression of survivin protein expression. The Noscapine treatment increased levels of total and Ser(15)-phosphorylated p53 protein in SK-SY5Y cells, but the proapoptotic response to this agent was maintained even after knockdown of the p53 protein level. Exposure of SK-SY5Y and LA1-5S cells to Noscapine resulted in a marked decrease in protein and mRNA level of survivin as early as 12 hours after treatment. Ectopic expression of survivin conferred statistically significant protection against Noscapine-mediated cytoplasmic histone-associated apoptotic DNA fragmentation. Also, the Noscapine-induced apoptosis was modestly but statistically significantly augmented by RNA interference of survivin in both cell lines. Furthermore, Noscapine-induced apoptotic cell death was associated with activation of caspase-3 and cleavage of PARP. In conclusion, the present study provides novel insight into the molecular circuitry of Noscapine-induced apoptosis to indicate suppression of survivin expression as a critical mediator of this process.

  20. Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

    PubMed Central

    Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

    2016-01-01

    Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

  1. Heterologous Regulation of Mu-Opioid (MOP) Receptor Mobility in the Membrane of SH-SY5Y Cells*

    PubMed Central

    Carayon, Kévin; Moulédous, Lionel; Combedazou, Anne; Mazères, Serge; Haanappel, Evert; Salomé, Laurence; Mollereau, Catherine

    2014-01-01

    The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane. PMID:25183007

  2. Salvianolic Acid B Inhibits Aβ Generation by Modulating BACE1 Activity in SH-SY5Y-APPsw Cells.

    PubMed

    Tang, Ying; Huang, Dan; Zhang, Mei-Hua; Zhang, Wen-Sheng; Tang, Yu-Xin; Shi, Zheng-Xiang; Deng, Li; Zhou, Dai-Han; Lu, Xin-Yi

    2016-06-01

    Alzheimer's disease (AD) is a neurodegenerative disease in humans. The accumulation of amyloid-β (Aβ) plays a critical role in the pathogenesis of AD. Previous studies indicated that Salvianolic acid B (SalB) could ameliorate Aβ-induced memory impairment. However, whether SalB could influence the generation of Aβ is unclear. Here, we show that SalB (25, 50, or 100 µM) reduces the generation of Aβ40 and Aβ42 in culture media by decreasing the protein expressions of BACE1 and sAPPβ in SH-SY5Y-APPsw cells. Meanwhile, SalB increases the levels of ADAM10 and sAPPα in the cells. However, SalB has no impact on the protein expressions of APP and PS1. Moreover, SalB attenuates oxidative stress and inhibits the activity of GSK3β, which might be related to the suppression of BACE1 expression and amyloidogenesis. Our study suggests that SalB is a promising therapeutic agent for AD by targeting Aβ generation.

  3. Salvianolic Acid B Inhibits Aβ Generation by Modulating BACE1 Activity in SH-SY5Y-APPsw Cells

    PubMed Central

    Tang, Ying; Huang, Dan; Zhang, Mei-Hua; Zhang, Wen-Sheng; Tang, Yu-Xin; Shi, Zheng-Xiang; Deng, Li; Zhou, Dai-Han; Lu, Xin-Yi

    2016-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease in humans. The accumulation of amyloid-β (Aβ) plays a critical role in the pathogenesis of AD. Previous studies indicated that Salvianolic acid B (SalB) could ameliorate Aβ-induced memory impairment. However, whether SalB could influence the generation of Aβ is unclear. Here, we show that SalB (25, 50, or 100 µM) reduces the generation of Aβ40 and Aβ42 in culture media by decreasing the protein expressions of BACE1 and sAPPβ in SH-SY5Y-APPsw cells. Meanwhile, SalB increases the levels of ADAM10 and sAPPα in the cells. However, SalB has no impact on the protein expressions of APP and PS1. Moreover, SalB attenuates oxidative stress and inhibits the activity of GSK3β, which might be related to the suppression of BACE1 expression and amyloidogenesis. Our study suggests that SalB is a promising therapeutic agent for AD by targeting Aβ generation. PMID:27258307

  4. Differential Aminoacylase Expression in Neuroblastoma

    PubMed Central

    Long, Patrick M.; Stradecki, Holly M.; Minturn, Jane E.; Wesley, Umadevi V.; Jaworski, Diane M.

    2012-01-01

    Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-risk tumors; yet, only ~25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Since aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN amplified (SK-N-AS, SK-N-SH, SH-SY5Y, and SH-EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane-associated ASPA, and nuclear ACY3). When SK-N-SH cells were treated with neural differentiation agents (e.g., retinoic acid, cAMP) in media containing 10% serum ACY1 was the only aminoacylase whose expression was up-regulated. ASPA was primarily expressed in SH-EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB-positive, MYCN-amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan-Meier survival revealed that high ACY3 expression is correlated with poor prognosis; while, low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma. PMID:21128244

  5. Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment.

    PubMed

    Giacoppo, Sabrina; Iori, Renato; Rollin, Patrick; Bramanti, Placido; Mazzon, Emanuela

    2017-07-14

    Several lines of evidence suggest the consume of natural products for cancer prevention or treatment. In particular, isothiocyanates (ITCs) exerting anti-cancer properties, have received great interest as potential chemotherapeutic agents. This study was designed to assess the anti-proliferative activities of a new preparation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl ITC (moringin) complexed with alpha-cyclodextrin (moringin + α-CD; MAC) on SH-SY5Y human neuroblastoma cells. This new formulation arises in the attempt to overcome the poor solubility and stability of moringin alone in aqueous media. SH-SY5Y cells were cultured and exposed to increasing concentrations of MAC (1.0, 2.5 and 5.0 μg). Cell proliferation was examined by MTT and cell count assays. The cytotoxic activity of the MAC complex was assessed by lactate dehydrogenase (LDH) assay and trypan blue exclusion test. In addition, western blotting analyses for the main apoptosis-related proteins were performed. Treatment of SH-SY5Y cells with the MAC complex reduced cell growth in concentration dependent manner. Specifically, MAC exhibited a potent action in inhibiting the PI3K/Akt/mTOR pathway, whose aberrant activation was found in many types of cancer. MAC was also found to induce the nuclear factor-κB (NF-κB) p65 activation by phosphorylation and its translocation into the nucleus. Moreover, treatment with MAC was able to down-regulate MAPK pathway (results focused on JNK and p38 expression). Finally, MAC was found to trigger apoptotic death pathway (based on expression levels of cleaved-caspase 3, Bax/Bcl-2 balance, p53 and p21). These findings suggest that use of MAC complex may open novel perspectives to improve the poor prognosis of patients with neuroblastoma.

  6. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors

    PubMed Central

    Klee, Werner A.; Nirenberg, Marshall

    1974-01-01

    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  7. Pyrazolo[3,4-d]pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation, characterization and cytotoxicity evaluation against neuroblastoma cell line.

    PubMed

    Fallacara, Anna Lucia; Mancini, Arianna; Zamperini, Claudio; Dreassi, Elena; Marianelli, Stefano; Chiariello, Mario; Pozzi, Gianni; Santoro, Francesco; Botta, Maurizio; Schenone, Silvia

    2017-07-15

    Pyrazolo[3,4-d]pyrimidine derivatives 1-5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1-4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Pre-exposure of neuroblastoma cell line to pulsed electromagnetic field prevents H2 O2 -induced ROS production by increasing MnSOD activity.

    PubMed

    Osera, Cecilia; Amadio, Marialaura; Falone, Stefano; Fassina, Lorenzo; Magenes, Giovanni; Amicarelli, Fernanda; Ricevuti, Giovanni; Govoni, Stefano; Pascale, Alessia

    2015-04-01

    Electromagnetic fields (EMFs) have been linked to increased risk of cancers and neurodegenerative diseases; however, EMFs can also elicit positive effects on biological systems, and redox status seems crucially involved in EMF biological effects. This study aimed to assess whether a short and repeated pulsed EMF (PEMF) could trigger adaptive responses against an oxidative insult in a neuronal cellular model. We found that a 40 min overall (four times a week, 10 min each) pre-exposure to PEMF did not affect major physiological parameters and led to a significant increase of Mn-dependent superoxide dismutase activity in the human neuroblastoma SH-SY5Y cell line. In addition, we found PEMF-pre-exposed cells exhibited decreased reactive oxygen species production following a 30 min H2 O2 challenge, with respect to non pre-exposed cells. Our findings might provide new insights on the role played by short and repeated PEMF stimulations in the enhancement of cellular defenses against oxidative insults. Although studies in normal neuronal cells would be useful to further confirm our hypothesis, we suggest that specific PEMF treatments may have potential biological repercussions in diseases where oxidative stress is implicated. © 2015 Wiley Periodicals, Inc.

  9. Fipronil sulfone induced higher cytotoxicity than fipronil in SH-SY5Y cells: Protection by antioxidants.

    PubMed

    Romero, A; Ramos, E; Ares, I; Castellano, V; Martínez, M; Martínez-Larrañaga, M R; Anadón, A; Martínez, M A

    2016-06-11

    Fipronil is a broad spectrum insecticide from the phenyl pyrazole family, which targets GABA receptor. Limited information is available about the metabolite fipronil sulfone cytotoxic actions. This study examined in vitro neurotoxicity of fipronil and fipronil sulfone and evaluated Trolox (vitamin E analog) (0.3, 1μM), N-acetyl-cysteine (0.5, 1mM), melatonin (0.1, 1μM) and Tempol (superoxide dismutase analog) (0.3, 0.5mM) protective role in SH-SY5Y cells. MTT and LDH assays were carried out to assess the cytotoxicity of fipronil and fipronil sulfone at 3-100μM concentrations. Fipronil sulfone was more toxic than fipronil. Tempol showed the best neuroprotectant profile against fipronil (50 and 150μM) and fipronil sulfone (3 and 10μM) reaching control levels. Fipronil (100μM) and fipronil sulfone (3μM) treatments induced a 4.7- and 5-fold increases in lipid peroxides measured as malondialdehyde (MDA) and a 2.2- and 2.0-fold increases in the levels of nitric oxide (NO). These results suggest that oxidative stress observed may be one of the major mechanisms of fipronil-induced neurotoxicity and it may be attributed in part to fipronil disposition and metabolism. Our results led us postulate that metabolite fipronil sulfone might be responsible for the fipronil-induced toxicity rather than fipronil itself. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Antioxidant and Proliferative Activities of Bupleuri Radix Extract Against Serum Deprivation in SH-SY5Y Cells

    PubMed Central

    Seo, Mi Kyoung; Cho, Hye Yeon; Lee, Chan Hong; Koo, Kyung Ah; Park, Yong Ki; Lee, Jung Goo; Lee, Bong Ju

    2013-01-01

    Objective Bupleuri Radix (BR) is a major component of several Oriental herbal medicines used to treat stress and mental illness. There are evidences that antidepressant drugs modulate oxidative damage implicated in the pathophysiology of neuropsychiatric disorder, including depression. The aim of the present study was to investigate antioxidant and proliferative effects of BR against oxidative stress induced by serum deprivation in SH-SY5Y cells. Methods We examined the antioxidant effects of BR on a number of measures, including cell viability, formation of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and levels of both Bcl-2 and Bax. We also investigated the effects of BR on cell proliferation using the bromodeoxyuridine (BrdU) assay, and used Western blot analysis to measure changes in expression of the cell cycle phase regulators. Results 1) Serum deprivation significantly induced the loss of cell viability, the formation of ROS, the reduction of SOD activity, down-regulation of Bcl-2 expression and up-regulation of Bax expression. However, BR extract reversed these effects in dose-dependent manner. 2) Serum deprivation significantly reduced cell proliferation. Western blot analysis revealed that serum deprivation significantly decreased cyclinD1 and phosphorylated retinoblastoma (pRb) expression, and increased p27 expression. On the other hand, BR dose dependently reversed these effects. Conclusion This study suggests that aqueous extract of BR may exert potent antioxidant effects and also play an important role in regulating cell cycle progression during neurogenesis. These effects of BR may be a potentially important mechanism of antidepressant underlying the observed antioxidant and proliferative effects. PMID:23483021

  11. Neuroblastoma

    MedlinePlus

    ... Habits for TV, Video Games, and the Internet Neuroblastoma KidsHealth > For Parents > Neuroblastoma Print A A A ... infancy, the chance of recovery is good. About Neuroblastoma Neuroblastoma most commonly starts in the tissue of ...

  12. Neuroblastoma

    MedlinePlus

    ... Old Feeding Your 1- to 2-Year-Old Neuroblastoma KidsHealth > For Parents > Neuroblastoma A A A What's ... infancy, the chance of recovery is good. About Neuroblastoma Neuroblastoma most commonly starts in the tissue of ...

  13. SIRT1 mediates salidroside-elicited protective effects against MPP(+) -induced apoptosis and oxidative stress in SH-SY5Y cells: Involvement in suppressing MAPK pathways.

    PubMed

    Wang, Chun-Yang; Sun, Zhao-Nan; Wang, Ming-Xin; Zhang, Chao

    2017-08-29

    Parkinson's disease (PD) is a progressive neurodegenerative disease, leading to tremor, rigidity, bradykinesia and gait impairment. Salidroside has been reported to exhibit antioxidative and neuroprotective properties in PD. However, the underlying neuroprotective mechanisms effects of salidroside are poorly understood. Recently, a growing body of evidences suggest that silent information regulator 1 (SIRT1) plays important roles in the pathophysiology of PD. Hence, the present study investigated the roles of SIRT1 in neuroprotective effect of salidroside against N-methyl-4-phenylpyridinium (MPP(+) )-induced SH-SY5Y cell injury. Our findings revealed that salidroside attenuates MPP(+) -induced neurotoxicity as evidenced by the increase in cell viability, and the decreases in the caspase-3 activity and apoptosis ratio. Simultaneously, salidroside pretreatment remarkably increased SIRT1 activity, SIRT1 mRNA and protein levels in MPP(+) -treated SH-SY5Y cell. However, sirtinol, a SIRT1 activation inhibitor, significantly blocked the inhibitory effects of salidroside on MPP(+) -induced cytotoxicity and apoptosis. In addition, salidroside abolished MPP(+) -induced the production of reactive oxygen species (ROS), the up-regulation of NADPH oxidase 2 (NOX2) expression, the down-regulations of superoxide dismutase (SOD) activity and glutathione (GSH) level in SH-SY5Y cells, while these effects were also blocked by sirtinol. Finally, we found that the inhibition of salidroside on MPP(+) -induced phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) were also reversed by sirtinol in SH-SY5Y cells. Taken together, these results indicated that SIRT1 contributes to the neuroprotection of salidroside against MPP(+) -induced apoptosis and oxidative stress, in part through suppressing of mitogen-activated protein kinase (MAPK) pathways. This article is protected by copyright. All rights reserved.

  14. Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

    SciTech Connect

    Li, Lingyun; Gao, Luyan; Song, Yunzhen; Qin, Zheng-Hong; Liang, Zhongqin

    2016-02-12

    Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.

  15. Dimethyl fumarate attenuates 6-OHDA-induced neurotoxicity in SH-SY5Y cells and in animal model of Parkinson's disease by enhancing Nrf2 activity.

    PubMed

    Jing, X; Shi, H; Zhang, C; Ren, M; Han, M; Wei, X; Zhang, X; Lou, H

    2015-02-12

    Oxidative stress is central to the pathology of several neurodegenerative diseases, including Parkinson's disease (PD), and therapeutics designed to enhance antioxidant potential could have clinical value. In this study, we investigated whether dimethyl fumarate (DMF) has therapeutic effects in cellular and animal model of PD, and explore the role of nuclear transcription factor related to NF-E2 (Nrf2) in this process. Treatment of animals and dopaminergic SH-SY5Y cells with DMF resulted in increased nuclear levels of active Nrf2, with subsequent upregulation of antioxidant target genes. The cytotoxicity of 6-hydroxydopamine (6-OHDA) was reduced by pre-treatment with DMF in SH-SY5Y cells. The increase in the reactive oxygen species caused by 6-OHDA treatment was also attenuated by DMF in SH-SY5Y cells. The neuroprotective effects of DMF against 6-OHDA neurotoxicity were dependent on Nrf2, since treatment with Nrf2 siRNA failed to block against 6-OHDA neurotoxicity and induce Nrf2-dependent cytoprotective genes in SH-SY5Y cells. In vivo, DMF oral administration was shown to upregulate mRNA and protein levels of Nrf2 and Nrf2-regulated cytoprotective genes, attenuate 6-OHDA induced striatal oxidative stress and inflammation in C57BL/6 mice. Moreover, DMF ameliorated dopaminergic neurotoxicity in 6-OHDA-induced PD animal models as evidenced by amelioration of locomotor dysfunction, loss in striatal dopamine, and reductions in dopaminergic neurons in the substantia nigra and striatum. Taken together, these data strongly suggest that DMF may be beneficial for the treatment of neurodegenerative diseases like PD. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

    PubMed

    Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

    2017-02-07

    High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

  17. Hydroethanolic extracts from different genotypes of açaí (Euterpe oleracea) presented antioxidant potential and protected human neuron-like cells (SH-SY5Y).

    PubMed

    Torma, Priscila do Carmo Marchioro Raupp; Brasil, Allana Von Sulzback; Carvalho, Ana Vânia; Jablonski, André; Rabelo, Thallita Kelly; Moreira, José Cláudio Fonseca; Gelain, Daniel Pens; Flôres, Simone Hickmann; Augusti, Paula Rossini; Rios, Alessandro de Oliveira

    2017-05-01

    Fruit breeding programs have resulted in bioactive compounds increase and health effects. Thus, this study aimed to evaluate the antioxidant activity and neuroprotective effects of the hydroethanolic extracts from six açaí (Euterpe oleracea) genotypes using ABTS, deoxyribose, and glutathione oxidation assays, as well as, SH-SY5Y cells insulted with H2O2. L22P13 genotype showed the highest total content of anthocyanins, while L06P13 showed a high content of total carotenoids. However, the genotypes showed no difference in the antioxidant activity by ABTS and deoxyribose assays. The hydroethanolic extracts from different genotypes of açaí showed a protective effect (13-62%) on SH-SY5Y cells insulted by H2O2 at a concentration of 50μg/mL by DCFH-DA assay. Except L04P16, no genotypes showed cytotoxicity in the SRB assay. These results indicate that açaí genotypes have antioxidant effect against reactive species generated in SH-SY5Y cells, suggesting a neuroprotective effect of the hydroethanolic extracts from these fruits. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Myricitrin alleviates methylglyoxal-induced mitochondrial dysfunction and AGEs/RAGE/NF-κB pathway activation in SH-SY5Y cells.

    PubMed

    Wang, Yue-Hua; Yu, Hai-Tao; Pu, Xiao-Ping; Du, Guan-Hua

    2014-08-01

    Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of neurodegenerative diseases. Methylglyoxal (MGO), a dicarbonyl metabolite, is a major precursor of AGEs which have been linked to the development of neurodegenerative diseases. Myricitrin, a flavanoid isolated from the root bark of Myrica cerifera, attenuated 6-OHDA-induced mitochondrial dysfunction and had a potential anti-Parkinson's disease in our previous investigation. The aims of this study were to investigate the protective effects of myricitrin against MGO-induced injury in SH-SY5Y cells and also to look for the possible mechanisms. The results showed that exposure of SH-SY5Y cells to MGO caused decreases of cell viability, intracellular ATP, mitochondrial redox activity, and mitochondrial membrane potential and an increase in reactive oxygen species generation. However, these mitochondrial dysfunctions were alleviated by co-treatment with myricitrin. Additionally, myricitrin was capable of inhibiting AGEs formation, blocking RAGE expression, and inhibiting NF-κB activation and translocation triggered by MGO in SH-SY5Y cells. Our results suggest that myricitrin alleviates MGO-induced mitochondrial dysfunction, and the possible mechanism is through modulating the AGEs/RAGE/NF-κB pathway. In summary, myricitrin might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive dicarbonyl compounds, providing a potential benefit agent with age-related neurodegenerative diseases.

  19. Identification of chaperones in a MPP+-induced and ATRA/TPA-differentiated SH-SY5Y cell PD model

    PubMed Central

    Xie, Hongrong; Hu, Hui; Chang, Ming; Huang, Dongya; Gu, Xiaobo; Xiong, Xinli; Xiong, Ran; Hu, Linsen; Li, Gang

    2016-01-01

    Parkinson’s disease (PD) is characterized by the pathological accumulation of misfolded proteins. Molecular chaperones assist in the proper folding of proteins and removal of irreversibly misfolded proteins. This study aims to identify potential chaperones associated with protein misfolding and accumulation in PD. ATRA/TPA-differentiated SH-SY5Y cells were treated with 1 mM of MPP+ for 48 hours. Proteins were analyzed by 2D-DIGE followed by MALDI-ToF MS. The treatment of differentiated SH-SY5Y cells by MPP+ led to the unambiguous identification of 10 protein spots, which corresponds to six proteins. Among these six proteins, four were chaperone proteins including nucleophosmin (NPM1), chaperonin-containing TCP-1 subunit 2 (CCT2 or CCTβ), heat shock 90 kDa protein 1 beta (HSP90AB1 or HSP90-β), and tyrosin3/tryptopha5-monoxygenase activation protein, zeta polypeptide (14-3-3ζ, gene symbol: Ywhaz). To our knowledge, this is the first report that linked the upregulation of chaperones after MPP+ treatment with SH-SY5Y cells. However, the NPM1 protein was identified for the first time in the PD model. The upregulation of four chaperone proteins provided evidence that these chaperones have a complementary effect on protein misfolding in the pathogenesis of PD, and hold promise as a good therapeutic target for PD treatment. PMID:28078037

  20. The neuroprotective effects of Lonicera japonica THUNB. against hydrogen peroxide-induced apoptosis via phosphorylation of MAPKs and PI3K/Akt in SH-SY5Y cells.

    PubMed

    Kwon, Seung-Hwan; Hong, Sa-Ik; Kim, Ji-Ah; Jung, Yang-Hee; Kim, Sun-Yeou; Kim, Hyoung-Chun; Lee, Seok-Yong; Jang, Choon-Gon

    2011-04-01

    We investigated the neuroprotective effects of Lonicera japonica THUNB. (Caprifoliaceae) (LJ) extract against hydrogen peroxide (H(2)O(2)), a toxin created by oxidative stress and implicated in neurodegenerative diseases, in human SH-SY5Y neuroblastoma cells. We examined the effects of LJ against H(2)O(2)-induced cytotoxicity, apoptosis, the production of reactive oxygen species (ROS), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, and cleaved caspase-3. Moreover, we attempted to determine whether LJ suppressed the phosphorylation of Akt, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). We found that LJ improved cell viability, inhibited cytotoxicity and apoptosis, and attenuated elevations in ROS and nuclear condensation. In addition, LJ showed radical scavenging ability in 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Western blot data revealed that LJ inhibited H(2)O(2)-induced up- and down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. Furthermore, LJ significantly attenuated the H(2)O(2)-induced phosphorylation of Akt, JNK, p38 MAPK, and ERK1/2. These results demonstrate that LJ possesses potent neuroprotective activity. Its potential to treat neurodegenerative diseases warrants further research. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  1. L- and T-type calcium channel blockers protect against the inhibitory effects of mipafox on neurite outgrowth and plasticity-related proteins in SH-SY5Y cells.

    PubMed

    Fernandes, Laís Silva; Dos Santos, Neife Aparecida G; Emerick, Guilherme Luz; Santos, Antonio Cardozo Dos

    2017-09-01

    Some organophosphorus compounds (OP), including the pesticide mipafox, produce late onset distal axonal degeneration, known as organophosphorus-induced delayed neuropathy (OPIDN). The underlying mechanism involves irreversible inhibition of neuropathy target esterase (NTE) activity, elevated intracellular calcium levels, increased activity of calcium-activated proteases and impaired neuritogenesis. Voltage-gated calcium channels (VGCC) appear to play a role in several neurologic disorders, including OPIDN. Therefore, this study aimed to examine and compare the neuroprotective effects of T-type (amiloride) and L-type (nimodipine) VGCC blockers induced by the inhibitory actions of mipafox on neurite outgrowth and axonal proteins of retinoic-acid-stimulated SH-SY5Y human neuroblastoma cells, a neuronal model widely employed to determine the neurotoxicity attributed to OP. Both nimodipine and amiloride significantly blocked augmentation of intracellular calcium levels and activity of calpains, as well as decreased neurite length, number of differentiated cells, and lowered concentrations of growth-associated protein 43 (GAP-43) and synapsin induced by mipafox. Only nimodipine inhibited reduction of synaptophysin levels produced by mipafox. These findings demonstrate a role for calcium and VGCC in the impairment of neuronal plasticity mediated by mipafox. Data also demonstrated the neuroprotective potential of T-type and L-type VGCC blockers to inhibit OP-mediated actions, which may be beneficial to counteract cases of pesticide poisoning.

  2. Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells.

    PubMed

    Feio-Azevedo, R; Costa, V M; Ferreira, L M; Branco, P S; Pereira, F C; Bastos, M L; Carvalho, F; Capela, J P

    2017-03-05

    Amphetamine (AMPH) is a psychostimulant used worldwide by millions of patients in the clinical treatment of attention deficit hyperactivity disorder, narcolepsy or even obesity, and is also a drug of abuse. 4-Hydroxynorephedrine (4-OHNE) and 4-hydroxyamphetamine (4-OHAMPH) are two major metabolites known to persist in the brain longer than AMPH. The contribution of AMPH metabolites for its neurotoxicity is undetermined. We evaluated the toxicity of AMPH and its metabolites 4-OHNE and 4-OHAMPH, obtained by chemical synthesis, in human dopaminergic differentiated SH-SY5Y neurons. Cells were exposed to AMPH (concentration range 0-5mM) or 4-OHAMPH or 4-OHNE (concentration range 0-10mM) for 24 or 48h, and the viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays. Results showed that for both AMPH and the metabolites a concentration-dependent toxicity was observed. The toxic concentration 50% (TC50) for AMPH and 4-OHNE following 24h exposure was circa 3.5mM and 8mM, respectively. For 4-OHAMPH the TC50 was not reached in the tested concentration range. N-acetyl cysteine, cycloheximide, l-carnitine, and methylphenidate were able to reduce cell death induced by AMPH TC50. Acridine orange/ethidium bromide staining showed evident signs of late apoptotic cells and necrotic cells following 24h exposure to AMPH 3.50mM. The 4-OHAMPH metabolite at 8.00mM originated few late apoptotic cells, whereas 4-OHNE at 8.00mM resulted in late apoptotic cells and necrotic cells, in a scenario similar to AMPH. In conclusion, the AMPH metabolite 4-OHNE is more toxic than 4-OHAMPH, nonetheless both are less toxic than the parent compound in vitro. The most toxic metabolite 4-OHNE has longer permanence in the brain, rendering likely its contribution for AMPH neurotoxicity.

  3. Glyceryl 1,3-Dipalmitate Produced from Lactobacillus paracasei subspecies. paracasei NTU 101 Inhibits Oxygen-Glucose Deprivation and Reperfusion-Induced Oxidative Stress via Upregulation of Peroxisome Proliferator-Activated Receptor γ in Neuronal SH-SY5Y Cells.

    PubMed

    Cheng, Meng-Chun; Pan, Tzu-Ming

    2017-09-13

    Glyceryl 1,3-dipalmitate (GD) purified from Lactobacillus paracasei subsp. paracasei NTU 101-fermented products has been demonstrated to possess neuroprotective properties. We determined the effect of GD on oxygen-glucose deprivation and reperfusion (OGD/R)-induced SH-SY5Y neuroblastoma cell death. GD ameliorated OGD/R-induced apoptosis by elevating the protein expression of nuclear peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor erythroid 2-related factor 2 (Nrf2), thereby attenuating reactive oxygen species (ROS) generation. Pretreatment with GD reduced nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) expression from 1.54 ± 0.27 to 0.84 ± 0.46, thereby attenuating the induction of pro-inflammatory mediators, and increased the plasma membrane Ca(2+) ATPase (PMCA) levels from 0.81 ± 0.02 to 1.08 ± 0.06, thus reducing the levels of cytosolic Ca(2+); this also correlated with reduced cell death. We conclude that GD prevents SH-SY5Y cells from injury after OGD/R insult, possibly by modulating oxidative stress and inflammatory response.

  4. The Co-chaperone BAG2 Mediates Cold-Induced Accumulation of Phosphorylated Tau in SH-SY5Y Cells.

    PubMed

    de Paula, Cesar Augusto Dias; Santiago, Fernando Enrique; de Oliveira, Adriele Silva Alves; Oliveira, Fernando Augusto; Almeida, Maria Camila; Carrettiero, Daniel Carneiro

    2016-05-01

    Inclusions of phosphorylated tau (p-tau) are a hallmark of many neurodegenerative disorders classified as "tauopathy," of which Alzheimer's disease is the most prevalent form. Dysregulation of tau phosphorylation disrupts neuron structure and function, and hyperphosphorylated tau aggregates to form neurotoxic inclusions. The abundance of ubiquitin in tau inclusions suggests a defect in ubiquitin-mediated tau protein degradation by the proteasome. Under the temperature of 37 °C, the co-chaperone BAG2 protein targets phosphorylated tau for degradation via by a more-efficient, ubiquitin-independent pathway. In both in vivo and in vitro studies, cold exposure induces the accumulation of phosphorylated tau protein. The SH-SY5Y cell line differentiates into neuron-like cells on treatment with retinoic acid and is an established model for research on the effects of cold on tau phosphorylation. The aim of the present study was to investigate whether BAG2 mediates the cold-induced accumulation of phosphorylated tau protein. Our findings show that cold exposure causes a decrease in BAG2 expression in undifferentiated cells. Conversely, BAG2 expression is increased in differentiated cells exposed to cold. Further, undifferentiated cells exposed to cold had an increased proportion of p-tau to total tau, suggesting an accumulation of p-tau that is consistent with decreased levels of BAG2. Overexpression of BAG2 in cold-exposed undifferentiated cells restored levels of p-tau to those of 37 °C undifferentiated control. Interestingly, although BAG2 expression increased in differentiated cells, this increase was not accompanied by a decrease in the proportion of p-tau to total tau. Further, overexpression of BAG2 in cold exposed differentiated cells showed no significant difference in p-tau levels compared to 37 °C controls. Taken together, these data show that expression of BAG2 is differently regulated in a differentiation-dependent context. Our results suggest that

  5. Amyloid Beta Peptides Affect Pregnenolone and Pregnenolone Sulfate Levels in PC-12 and SH-SY5Y Cells Depending on Cholesterol.

    PubMed

    Calan, Ozlem Gursoy; Akan, Pinar; Cataler, Aysenur; Dogan, Cumhur; Kocturk, Semra

    2016-07-01

    Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25-35, AB 1-40 and AB 1-42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose-and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25-35 were higher than those in AB 1-40 and AB 1-42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9 mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase

  6. Targeting Notch pathway induces growth inhibition and differentiation of neuroblastoma cells.

    PubMed

    Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Uberti, Daniela; Buizza, Laura; Bettinsoli, Paola; Poliani, Pietro Luigi; Facchetti, Fabio; Memo, Maurizio

    2010-12-01

    High-risk neuroblastoma is a severe pediatric tumor characterized by poor prognosis. Understanding the molecular mechanisms involved in tumor development and progression is strategic for the improvement of pharmacological therapies. Notch was recently proposed as a pharmacological target for the therapy of several cancers and is emerging as a new neuroblastoma-related molecular pathway. However, the precise role played by Notch in this cancer remains to be studied extensively. Here, we show that Notch activation by the Jagged1 ligand enhances the proliferation of neuroblastoma cells, and we propose the possible use of Notch-blocking γ-secretase inhibitors (GSIs) in neuroblastoma therapy. Two different GSIs, Compound E and DAPT, were tested alone or in combination with 13-cis retinoic acid (RA) on neuroblastoma cell lines. SH-SY5Y and IMR-32 cells were chosen as paradigms of lower and higher malignancy, respectively. Used alone, GSIs induced complete cell growth arrest, promoted neuronal differentiation, and significantly reduced cell motility. The combination of GSIs and 13-cis RA resulted in the enhanced growth inhibition, differentiation, and migration of neuroblastoma cells. In summary, our data suggest that a combination of GSIs with 13-cis RA offers a therapeutic advantage over a single agent, indicating a potential novel therapy for neuroblastoma.

  7. Neuroblastoma

    MedlinePlus

    Cancer - neuroblastoma ... Neuroblastoma can occur in many areas of the body. It develops from the tissues that form the ... pressure, digestion, and levels of certain hormones. Most neuroblastomas begin in the abdomen, in the adrenal gland, ...

  8. The protective effect of geniposide on human neuroblastoma cells in the presence of formaldehyde

    PubMed Central

    2013-01-01

    Background Formaldehyde can induce misfolding and aggregation of Tau protein and β amyloid protein, which are characteristic pathological features of Alzheimer’s disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline. Methods In this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored. Results Cell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment. Conclusions Our results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione

  9. Oxidized LDL lipids increase β-amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation.

    PubMed

    Dias, Irundika H K; Mistry, Jayna; Fell, Shaun; Reis, Ana; Spickett, Corinne M; Polidori, Maria C; Lip, Gregory Y H; Griffiths, Helen R

    2014-10-01

    Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4μg oxLDL and 25µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  10. An Extract from Shrimp Processing By-Products Protects SH-SY5Y Cells from Neurotoxicity Induced by Aβ25–35

    PubMed Central

    Zhang, Yongping; Jiao, Guangling; Song, Cai; Gu, Shelly; Brown, Richard E.; Zhang, Junzeng; Zhang, Pingcheng; Gagnon, Jacques; Locke, Steven; Stefanova, Roumiana; Pelletier, Claude; Zhang, Yi; Lu, Hongyu

    2017-01-01

    Increased evidence suggests that marine unsaturated fatty acids (FAs) can protect neurons from amyloid-β (Aβ)-induced neurodegeneration. Nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and gas chromatography (GC) assays showed that the acetone extract 4-2A obtained from shrimp Pandalus borealis industry processing wastes contained 67.19% monounsaturated FAs and 16.84% polyunsaturated FAs. The present study evaluated the anti-oxidative and anti-inflammatory effects of 4-2A in Aβ25–35-insulted differentiated SH-SY5Y cells. Cell viability and cytotoxicity were measured by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Quantitative PCR and Western blotting were used to study the expression of neurotrophins, pro-inflammatory cytokines and apoptosis-related genes. Administration of 20 μM Aβ25–35 significantly reduced SH-SY5Y cell viability, the expression of nerve growth factor (NGF) and its tyrosine kinase TrkA receptor, as well as the level of glutathione, while increased reactive oxygen species (ROS), nitric oxide, tumor necrosis factor (TNF)-α, brain derived neurotrophic factor (BDNF) and its TrkB receptor. Aβ25–35 also increased the Bax/Bcl-2 ratio and Caspase-3 expression. Treatment with 4-2A significantly attenuated the Aβ25–35-induced changes in cell viability, ROS, GSH, NGF, TrkA, TNF-α, the Bax/Bcl-2 ratio and Caspase-3, except for nitric oxide, BDNF and TrKB. In conclusion, 4-2A effectively protected SH-SY5Y cells against Aβ-induced neuronal apoptosis/death by suppressing inflammation and oxidative stress and up-regulating NGF and TrKA expression. PMID:28327516

  11. The mixture of "ecstasy" and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations.

    PubMed

    Barbosa, Daniel José; Capela, João Paulo; Silva, Renata; Vilas-Boas, Vânia; Ferreira, Luísa Maria; Branco, Paula Sério; Fernandes, Eduarda; Bastos, Maria de Lourdes; Carvalho, Félix

    2014-02-01

    The neurotoxicity of "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca(2+) raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.

  12. 1,25-Dyhydroxyvitamin D₃ attenuates rotenone-induced neurotoxicity in SH-SY5Y cells through induction of autophagy.

    PubMed

    Jang, Wooyoung; Kim, Hee Ju; Li, Huan; Jo, Kwang Deog; Lee, Moon Kyu; Song, Sun Hong; Yang, Hyun Ok

    2014-08-15

    Dysregulation of the autophagy pathway has been suggested as an important mechanism in the pathogenesis of Parkinson's disease (PD). Therefore, modulation of autophagy may be a novel strategy for the treatment of PD. Recently, an active form of vitamin D₃ has been reported to have neuroprotective properties. Therefore, we investigated the protective, autophagy-modulating effects of 1,25-dyhydroxyvitamin D₃ (calcitriol) in an in vitro model of Parkinson's disease. An in vitro model of Parkinson's disease, the rotenone-induced neurotoxicity model in SH-SY5Y cells was adapted. We measured cell viability using an MTT assay, Annexin V/propidium iodide assay, and intracellular reactive oxygen species levels and analyzed autophagy-associated intracellular signaling proteins by Western blotting. Rotenone treatment of SH-SY5Y cells reduced their viability. This treatment also increased reactive oxygen species levels and decreased levels of intracellular signaling proteins associated with cell survival; simultaneous exposure to calcitriol significantly reversed these effects. Additionally, calcitriol increased levels of autophagy markers, including LC3, beclin-1, and AMPK. Rotenone inhibited autophagy, as indicated by decreased beclin-1 levels and increased mTOR levels, and this effect was reversed by calcitriol treatment. Calcitriol protects against rotenone-induced neurotoxicity in SH-SY5Y cells by enhancing autophagy signaling pathways such as those involving LC3 and beclin-1. These neuroprotective effects of calcitriol against rotenone-induced dopaminergic neurotoxicity provide an experimental basis for its clinical use in the treatment of PD. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Tumor necrosis factor expressed by primary hippocampal neurons and SH-SY5Y cells is regulated by alpha(2)-adrenergic receptor activation.

    PubMed

    Renauld, A E; Spengler, R N

    2002-01-15

    Neuron expression of the cytokine tumor necrosis factor-alpha (TNF), and the regulation of the levels of TNF by alpha(2)-adrenergic receptor activation were investigated. Adult rat hippocampal neurons and phorbol ester (PMA)-differentiated SH-SY5Y cells were examined. Intracellular levels of TNF mRNA accumulation, as well as TNF protein and that released into the supernatant were quantified by in situ hybridization, immunocytochemistry and bioanalysis, respectively. Both neuron cultures demonstrated constitutive production of TNF. Activation of the alpha(2)-adrenergic receptor increased intracellular levels of TNF mRNA and protein in SH-SY5Y cells after addition of graded concentrations of the selective agonist, Brimonidine (UK-14304) to parallel cultures. Intracellular levels of mRNA were increased in a concentration-dependent fashion within 15 min of UK-14304 addition and were sustained during 24 hr of receptor activation. In addition, the levels of TNF in the supernatant were increased in both types of neuron cultures within 15 min of alpha(2)-adrenergic receptor activation. Furthermore, levels of TNF significantly increased in the supernatants of both neuron cultures after potassium-induced depolarization. A reduction in this depolarization-induced release occurred in hippocampal neuron cultures after exposure to the sympathomimetic tyramine with media replacement to deplete endogenous catecholamines. This finding reveals a role for endogenous catecholamines in the regulation of TNF production. Potassium-induced depolarization resulted in the release of TNF in hippocampal neuron cultures within 15 min but not until 24 hr in SH-SY5Y cultures demonstrating a temporally mediated event dependent upon cell type. Neuron expression of TNF, regulated by alpha(2)-adrenergic receptor activation demonstrates not only how a neuron controls its own production of this pleiotropic cytokine, but also displays a normal role for neurons in directing the many functions of TNF.

  14. An Extract from Shrimp Processing By-Products Protects SH-SY5Y Cells from Neurotoxicity Induced by Aβ25-35.

    PubMed

    Zhang, Yongping; Jiao, Guangling; Song, Cai; Gu, Shelly; Brown, Richard E; Zhang, Junzeng; Zhang, Pingcheng; Gagnon, Jacques; Locke, Steven; Stefanova, Roumiana; Pelletier, Claude; Zhang, Yi; Lu, Hongyu

    2017-03-22

    Increased evidence suggests that marine unsaturated fatty acids (FAs) can protect neurons from amyloid-β (Aβ)-induced neurodegeneration. Nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and gas chromatography (GC) assays showed that the acetone extract 4-2A obtained from shrimp Pandalus borealis industry processing wastes contained 67.19% monounsaturated FAs and 16.84% polyunsaturated FAs. The present study evaluated the anti-oxidative and anti-inflammatory effects of 4-2A in Aβ25-35-insulted differentiated SH-SY5Y cells. Cell viability and cytotoxicity were measured by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Quantitative PCR and Western blotting were used to study the expression of neurotrophins, pro-inflammatory cytokines and apoptosis-related genes. Administration of 20 μM Aβ25-35 significantly reduced SH-SY5Y cell viability, the expression of nerve growth factor (NGF) and its tyrosine kinase TrkA receptor, as well as the level of glutathione, while increased reactive oxygen species (ROS), nitric oxide, tumor necrosis factor (TNF)-α, brain derived neurotrophic factor (BDNF) and its TrkB receptor. Aβ25-35 also increased the Bax/Bcl-2 ratio and Caspase-3 expression. Treatment with 4-2A significantly attenuated the Aβ25-35-induced changes in cell viability, ROS, GSH, NGF, TrkA, TNF-α, the Bax/Bcl-2 ratio and Caspase-3, except for nitric oxide, BDNF and TrKB. In conclusion, 4-2A effectively protected SH-SY5Y cells against Aβ-induced neuronal apoptosis/death by suppressing inflammation and oxidative stress and up-regulating NGF and TrKA expression.

  15. FLZ, a novel HSP27 and HSP70 inducer, protects SH-SY5Y cells from apoptosis caused by MPP(+).

    PubMed

    Kong, Xiang-Chen; Zhang, Dan; Qian, Cheng; Liu, Geng-Tao; Bao, Xiu-Qi

    2011-04-06

    Heat shock proteins (HSPs) play an essential role in various neurodegenerative diseases. Manipulation of upregulation of HSPs in cells has been demonstrated to provide a therapeutic strategy to counteract the misfolding and aggregation of proteins that resulted in neurodegenerative disease. Our previous studies have shown that FLZ, a synthetic novel derivative of squamosamide from a Chinese herb, had potent neuroprotective effect against several experimental Parkinson's disease (PD) models. However, the mechanism of its neuroprotective effect is still not clarified. The present study demonstrated that FLZ induced HSP27 and HSP70 proteins and mRNA expression in a time- and dose-dependent manner in SH-SY5Y cells. Further studies showed that FLZ treatment stimulated the activation of heat shock factor 1 (HSF1) and its regulatory kinase Akt. Inactivation of Akt pathway by the PI3K inhibitor LY294002 blocked the expression of HSP27 and HSP70 induced by FLZ. Moreover, the inducing effects of FLZ on HSP27, HSP70, and HSF1 were all blocked by quercetin, an inhibitor of HSP biosynthesis. The cytoprotective effect of HSP27/HSP70 induced by FLZ against MPP(+) was assessed in SH-SY5Y cells. The pretreatment of FLZ significantly induced the accumulations of HSP27/HSP70 and suppressed the apoptosis caused by MPP(+) in SH-SY5Y cells. However, the protective effects of FLZ against MPP(+) were significantly blocked by quercetin, which indicated that the cytoprotective action of FLZ against MPP(+)-induced apoptosis is at least partially mediated by its induction of HSP27/HSP70. These results provide new evidence for elucidating the mechanism of the neuroprotective effect of FLZ against PD.

  16. DHA, EPA and their combination at various ratios differently modulated Aβ25-35-induced neurotoxicity in SH-SY5Y cells.

    PubMed

    Zhang, Yong-Ping; Brown, Richard E; Zhang, Ping-Cheng; Zhao, Yun-Tao; Ju, Xiang-Hong; Song, Cai

    2017-07-14

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been reported to prevent neurodegenerative diseases such as Alzheimer's disease (AD) in both experimental and clinical/epidemiological studies. However, whether DHA and EPA from natural products exert similar or different neuroprotective effects and how these n-3 PUFAs target cellular and molecular mechanisms associated with neurodegenerative disease pathogenesis are unknown. In the present study, we used amyloid-β (Aβ)25-35-treated differentiated SH-SY5Y cells as a model of AD to compare the neuroprotective effect of DHA, EPA and their combination at various ratios. Administration of 20μM Aβ25-35 significantly decreased SH-SY5Y cell viability, the expression of nerve growth factor (NGF), its TrkA receptor, and the level of glutathione (GSH) and increased reactive oxygen species (ROS), nitric oxide, tumor necrosis factor (TNF)-α, brain derived neurotrophic factor (BDNF) and its TrkB receptor. Aβ25-35 also increased the Bax/Bcl-2 ratio and the expression of Caspase-3 in these cells. Compared with the Aβ group, pretreatment with DHA/EPA significantly reduced cell death, especially at ratio of 1:1 and 2:1 DHA/EPA or pure DHA. However, the most efficient ratio for reducing changes in ROS and GSH and for decreasing TNF-α appeared at ratio of 1:2 and 1:1, respectively. The ratio of 1:1, 2:1 and pure DHA resulted in significant increase in the level of NGF. Furthermore, pure DHA was the most efficient for reducing Bax/Bcl ratio and Caspase-3 expression. In conclusion, DHA, EPA and their combination differently modulated Aβ25-35-induced neurotoxicity in SH-SY5Y cells by exerting anti-oxidative, anti-inflammatory and neurotrophic effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Interleukin-18 alters protein expressions of neurodegenerative diseases-linked proteins in human SH-SY5Y neuron-like cells

    PubMed Central

    Sutinen, Elina M.; Korolainen, Minna A.; Häyrinen, Jukka; Alafuzoff, Irina; Petratos, Steven; Salminen, Antero; Soininen, Hilkka; Pirttilä, Tuula; Ojala, Johanna O.

    2014-01-01

    Chronic inflammation and oxidative stress (OS) are present in Alzheimer's disease (AD) brains in addition to neuronal loss, Amyloid-β (Aβ) plaques and hyperphosphorylated tau-protein neurofibrillary tangles (NFTs). Previously we showed that levels of the pro-inflammatory cytokine, interleukin-18 (IL-18), are elevated in post-mortem AD brains. IL-18 can modulate the tau kinases, Cdk5 and GSK3β, as well as Aβ-production. IL-18 levels are also increased in AD risk diseases, including type-2 diabetes and obesity. Here, we explored other IL-18 regulated proteins in neuron-like SH-SY5Y cells. Differentiated SH-SY5Y cells, incubated with IL-18 for 24, 48, or 72 h, were analyzed by two-dimensional gel electrophoresis (2D-DIGE). Specific altered protein spots were chosen and identified with mass spectrometry (MS) and verified by western immunoblotting (WIB). IL-18 had time-dependent effects on the SH-SY5Y proteome, modulating numerous protein levels/modifications. We concentrated on those related to OS (DDAH2, peroxiredoxins 2, 3, and 6, DJ-1, BLVRA), Aβ-degradation (MMP14, TIMP2), Aβ-aggregation (Septin-2), and modifications of axon growth and guidance associated, collapsin response mediator protein 2 (CRMP2). IL-18 significantly increased antioxidative enzymes, indicative of OS, and altered levels of glycolytic α- and γ-enolase and multifunctional 14-3-3γ and -ε, commonly affected in neurodegenerative diseases. MMP14, TIMP2, α-enolase and 14-3-3ε, indirectly involved in Aβ metabolism, as well as Septin-2 showed changes that increase Aβ levels. Increased 14-3-3γ may contribute to GSK3β driven tau hyperphosphorylation and CRMP2 Thr514 and Ser522 phosphorylation with the Thr555-site, a target for Rho kinase, showing time-dependent changes. IL-18 also increased caspase-1 levels and vacuolization of the cells. Although our SH-SY5Y cells were not aged, as neurons in AD, our work suggests that heightened or prolonged IL-18 levels can drive protein changes of

  18. Nitric oxide-mediated toxicity in paraquat-exposed SH-SY5Y cells: a protective role of 7-nitroindazole.

    PubMed

    Ortiz-Ortiz, Miguel A; Morán, José M; González-Polo, Rosa A; Niso-Santano, Mireia; Soler, Germán; Bravo-San Pedro, José M; Fuentes, José M

    2009-08-01

    The precise mechanism underlying the role of nitric oxide (NO) or nitric oxide synthases (NOSs) in paraquat-mediated toxicity is yet to be fully elucidated. The importance of the NADPH-diaphorase activity of NOSs in paraquat toxicity, in addition to the production of NO, has previously been reported as a mechanism of toxicity. However, other studies have highlighted the toxicity of NO alone and, conversely a protective role of NO in paraquat-mediated toxicity has also been described. The goal of this study was to clarify the involvement of NO and NOS in paraquat-mediated toxicity in an SH-SY5Y cell system, and to evaluate the putative role of 7-nitroindazole as a protective agent in human neural cells. Our results indicate that the three previously described isoforms of NOS are expressed in SH-SY5Y cells, with the data showing that these synthases act as paraquat diaphorases. While this process could occur at the expense of NO production, NO alone does play a toxic role, with its production leading to the formation of the toxicant peroxynitrite. Although the efficacies of the different inhibitors tested cannot be directly compared because the various NOS forms were probably inhibited to differing extents, the results support the idea that endogenous and inducible NO is a neurotoxic mediator of the effects of paraquat. The NADPH-diaphorase activity of NOS and NO production are therefore factors implicated in the toxicity mediated by the herbicide paraquat.

  19. Quantitative proteomics study of the neuroprotective effects of B12 on hydrogen peroxide-induced apoptosis in SH-SY5Y cells.

    PubMed

    Zhong, Lijun; Zhou, Juntuo; Chen, Xi; Lou, Yaxin; Liu, Dan; Zou, Xiajuan; Yang, Bin; Yin, Yuxin; Pan, Yan

    2016-03-08

    B12 belongs to the coumarin class of compounds that have been shown to have various physiological and pharmacological activities including anti-inflammatory, antibacterial, and antioxidant. In the present study, we characterised the neuroprotective effects of B12 against H2O2-induced neuronal cell damage in SH-SY5Y cells. Protein expression profiling in combination with pathway analysis was deployed to investigate the molecular events associated with the neuroprotective effects in human neuronal cells using a label-free quantitative proteomics approach. A total of 22 proteins were significantly differentially expressed in H2O2-damaged cells with or without B12 treatment. Bioinformatics analysis using the Cytoscape platform indicated that poly pyrimidine tract binding protein 1 (PTBP1) was highly associated with the protective effect, and western blotting verified that PTBP1 was up-regulated in H2O2 + B12 treatment group, compared with the H2O2 treated group. PTBP RNAi experiments knocked down PTBP expression, which cancelled out the protective effect of B12 on cell viability. Thus, we infer that B12 neuroprotective activity involves up-regulation of PTBP1 and its associated signalling networks following H2O2-induced apoptosis in SH-SY5Y cells. B12 or related compounds may prove to be useful therapeutic agents for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's.

  20. Inhibition of amyloid β aggregation and protective effect on SH-SY5Y cells by triterpenoid saponins from the cactus Polaskia chichipe.

    PubMed

    Fujihara, Koji; Koike, Shin; Ogasawara, Yuki; Takahashi, Kunio; Koyama, Kiyotaka; Kinoshita, Kaoru

    2017-07-01

    Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R0). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aβ aggregation and their protective effects on SH-SY5Y cells against Aβ-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aβ aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aβ-associated toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson’s disease patients

    PubMed Central

    Dieriks, Birger Victor; Park, Thomas I-H.; Fourie, Chantelle; Faull, Richard L. M.; Dragunow, Mike; Curtis, Maurice A.

    2017-01-01

    Parkinson’s disease (PD) is characterized by the presence of inclusions known as Lewy bodies, which mainly consist of α-synuclein (α-syn) aggregates. There is growing evidence that α-syn self-propagates in non-neuronal cells, thereby contributing to the progression and spread of PD pathology in the brain. Tunneling nanotubes (TNTs) are long, thin, F-actin-based membranous channels that connect cells and have been proposed to act as conduits for α-syn transfer between cells. SH-SY5Y cells and primary human brain pericytes, derived from postmortem PD brains, frequently form TNTs that allow α-syn transfer and long-distance electrical coupling between cells. Pericytes in situ contain α-syn precipitates like those seen in neurons. Exchange through TNTs was rapid, but dependent on the size of the protein. Proteins were able to spread throughout a network of cells connected by TNTs. Transfer through TNTs was not restricted to α-syn; fluorescent control proteins and labeled membrane were also exchanged through TNTs. Most importantly the formation of TNTs and transfer continued during mitosis. Together, our results provide a detailed description of TNTs in SH-SY5Y cells and human brain PD pericytes, demonstrating their role in α-syn transfer and further emphasize the importance that non-neuronal cells, such as pericytes play in disease progression. PMID:28230073

  2. Effects of low intensity static magnetic field on FTIR spectra and ROS production in SH-SY5Y neuronal-like cells.

    PubMed

    Calabrò, Emanuele; Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Caccamo, Daniela; Magazù, Salvatore; Ientile, Riccardo

    2013-12-01

    Biological effects of man-made electromagnetic fields (EMFs) have been studied so far by experimental approaches exposing animals and cell cultures to EMFs. However, the evidence for cell toxicity induced by static magnetic field (SMF) is still uncertain. We investigated the effects produced by the exposure of human SH-SY5Y neuronal-like cells to a uniform magnetic field at intensities of 2.2 mT, which is less than the recommended public exposure limits set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). A decrease of membrane mitochondrial potential up to 30% was measured after 24 h of exposure to SMF in SH-SY5Y cells, and this effect was associated with reactive oxygen species production increase. Fourier transform infrared spectroscopy (FTIR) analysis showed that exposure to a static magnetic intensity around 2.2 mT changed the secondary structure of cellular proteins and lipid components. The vibration bands relative to the methylene group increased significantly after 4 h of exposure, whereas further exposure up to 24 h produced evident shifts of amide I and II modes and a relative increase in β-sheet contents with respect to α-helix components. Our study demonstrated that a moderate SMF causes alteration in cell homeostasis, as indicated by FTIR spectroscopy observations of changes in protein structures that are part of cell response to magnetic field exposure.

  3. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    SciTech Connect

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira . E-mail: akiranak@chiba-cc.jp

    2007-03-23

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53{delta}C) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53{delta}C was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.

  4. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells

    PubMed Central

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F.; He, Rong-Qiao

    2016-01-01

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation. PMID:27029216

  5. Protective effects of acetyl-L-carnitine on cisplatin cytotoxicity and oxidative stress in neuroblastoma.

    PubMed

    Altun, Zekiye Sultan; Güneş, Dilek; Aktaş, Safiye; Erbayraktar, Zübeyde; Erbayrktar, Zübeyde; Olgun, Nur

    2010-03-01

    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.

  6. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death

    SciTech Connect

    Guida, Natascia; Laudati, Giusy; Galgani, Mario; Santopaolo, Marianna; Montuori, Paolo; Triassi, Maria; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2014-10-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1–100 μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48 h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination. - Highlights: • Di(2-ethylhexyl)phthalate (DEHP) is cytotoxic to SH-SY5Y cells and cortical neurons. • DEHP-induced cytotoxicity is mediated by apoptosis. • DEHP-induced apoptotic cell death is inhibited by class II HDAC MC-1568. • DEHP neurotoxicity is caused by HDAC4-mediated Sp3 degradation by ubiquitin.

  7. Haptoglobin increases with age in rat hippocampus and modulates Apolipoprotein E mediated cholesterol trafficking in neuroblastoma cell lines

    PubMed Central

    Spagnuolo, Maria Stefania; Maresca, Bernardetta; Mollica, Maria Pina; Cavaliere, Gina; Cefaliello, Carolina; Trinchese, Giovanna; Esposito, Maria Grazia; Scudiero, Rosaria; Crispino, Marianna; Abrescia, Paolo; Cigliano, Luisa

    2014-01-01

    Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain

  8. Curcumin inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells.

    PubMed

    Uğuz, Abdülhadi Cihangir; Öz, Ahmi; Nazıroğlu, Mustafa

    2016-08-01

    Neurological diseases such as Alzheimer's and Parkinson's diseases are incurable progressive neurological disorders caused by the degeneration of neuronal cells and characterized by motor and non-motor symptoms. Curcumin, a turmeric product, is an anti-inflammatory agent and an effective reactive oxygen and nitrogen species scavenging molecule. Hydrogen peroxide (H2O2) is the main source of oxidative stress, which is claimed to be the major source of neurological disorders. Hence, in this study we aimed to investigate the effect of curcumin on Ca(2+) signaling, oxidative stress parameters, mitochondrial depolarization levels and caspase-3 and -9 activities that are induced by the H2O2 model of oxidative stress in SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were divided into four groups namely, the control, curcumin, H2O2, and curcumin + H2O2 groups. The dose and duration of curcumin and H2O2 were determined from published data. The cells in the curcumin, H2O2, and curcumin + H2O2 groups were incubated for 24 h with 5 µM curcumin and 100 µM H2O2. Lipid peroxidation and cytosolic free Ca(2+) concentrations were higher in the H2O2 group than in the control group; however, their levels were lower in the curcumin and curcumin + H2O2 groups than in the H2O2 group alone. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) values were lower in the H2O2 group although they were higher in the curcumin and curcumin + H2O2 groups than in the H2O2 group. Caspase-3 activity was lower in the curcumin group than in the H2O2 group. In conclusion, curcumin strongly induced modulator effects on oxidative stress, intracellular Ca(2+) levels, and the caspase-3 and -9 values in an experimental oxidative stress model in SH-SY5Y cells.

  9. The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells.

    PubMed

    Okamoto, Akihisa; Tanaka, Masahiro; Sumi, Chisato; Oku, Kanako; Kusunoki, Munenori; Nishi, Kenichiro; Matsuo, Yoshiyuki; Takenaga, Keizo; Shingu, Koh; Hirota, Kiichi

    2016-10-24

    The local anesthetic lidocaine can affect intra- and extra-cellular signaling pathways in both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions, including cell growth and death. Indeed, lidocaine was shown to induce necrosis and apoptosis in vitro. While several studies have suggested that lidocaine-induced apoptosis is mitochondrial pathway-dependent, it remains unclear whether reactive oxygen species (ROS) are involved in this process and whether the observed cell death can be prevented by antioxidant treatment. The effects of lidocaine and antioxidants on cell viability and death were evaluated using SH-SY5Y cells, HeLa cells, and HeLa cell derivatives. Cell viability was examined via MTS/PES ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]/phenazine ethosulfate) assay. Meanwhile, cell apoptosis and necrosis were evaluated using a cell death detection assay with Annexin V-FITC and PI staining, as well as by assaying for caspase-3/7 and caspase-9 activity, and by measuring the release of lactate dehydrogenase, respectively. Mitochondrial transmembrane potential (ΔΨm) was assessed using the fluorescent probe tetramethylrhodamine ethyl ester. Lidocaine treatment resulted in suppression of the mitochondrial electron transport chain and subsequent attenuation of mitochondrial membrane potential, as well as enhanced ROS production, activation of caspase-3/7 and caspase-9, and induction of apoptosis and necrosis in SH-SY5Y cells in a dose- and time-dependent manner. Likewise, the anesthetics mepivacaine and bupivacaine also induced apoptosis in SH-SY5Y cells. Notably, the antioxidants N-acetyl cysteine (NAC) and Trolox successfully scavenged the mitochondria-derived ROS and suppressed local lidocaine-induced cell death. Our findings demonstrate that the local anesthetics lidocaine, mepivacaine, and bupivacaine inhibited the activity of mitochondria and induced apoptosis

  10. Valproate Attenuates Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells via the AKT/GSK3β Signaling Pathway

    PubMed Central

    Li, Zhengmao; Wu, Fenzan; Zhang, Xie; Chai, Yi; Chen, Daqing; Yang, Yuetao; Xu, Kebin; Yin, Jiayu; Li, Rui; Shi, Hongxue; Wang, Zhouguang; Li, Xiaokun; Xiao, Jian; Zhang, Hongyu

    2017-01-01

    Endoplasmic reticulum (ER) stress-induced apoptosis plays an important role in a range of neurological disorders, such as neurodegenerative diseases, spinal cord injury, and diabetic neuropathy. Valproate (VPA), a typical antiepileptic drug, is commonly used in the treatment of bipolar disorder and epilepsy. Recently, VPA has been reported to exert neurotrophic effects and promote neurite outgrowth, but its molecular mechanism is still unclear. In the present study, we investigated whether VPA inhibited ER stress and promoted neuroprotection and neuronal restoration in SH-SY5Y cells and in primary rat cortical neurons, respectively, upon exposure to thapsigargin (TG). In SH-SY5Y cells, cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and the expression of ER stress-related apoptotic proteins such as glucose‑regulated protein (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase-12/-3 were analyzed with Western blot analyses and immunofluorescence assays. To explore the pathway involved in VPA-induced cell proliferation, we also examined p-AKT, GSK3β, p-JNK and MMP-9. Moreover, to detect the effect of VPA in primary cortical neurons, immunofluorescence staining of β-III tubulin and Anti-NeuN was analyzed in primary cultured neurons exposed to TG. Our results demonstrated that VPA administration improved cell viability in cells exposed to TG. In addition, VPA increased the levels of GRP78 and p-AKT and decreased the levels of ATF6, XBP-1, GSK3β, p-JNK and MMP-9. Furthermore, the levels of the ER stress-induced apoptosis response proteins CHOP, cleaved caspase-12 and cleaved caspase-3 were inhibited by VPA treatment. Meanwhile, VPA administration also increased the ratio of Bcl-2/Bax. Moreover, VPA can maintain neurite outgrowth of primary cortical neurons. Collectively, the neurotrophic effect of VPA is related to the inhibition of ER stress-induced apoptosis in SH-SY5Y cells and the

  11. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

    PubMed

    Harenza, Jo Lynne; Diamond, Maura A; Adams, Rebecca N; Song, Michael M; Davidson, Heather L; Hart, Lori S; Dent, Maiah H; Fortina, Paolo; Reynolds, C Patrick; Maris, John M

    2017-03-28

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

  12. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines

    PubMed Central

    Harenza, Jo Lynne; Diamond, Maura A.; Adams, Rebecca N.; Song, Michael M.; Davidson, Heather L.; Hart, Lori S.; Dent, Maiah H.; Fortina, Paolo; Reynolds, C. Patrick; Maris, John M.

    2017-01-01

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma. PMID:28350380

  13. Establishment of functional clonal lines of neurons from mouse neuroblastoma.

    PubMed

    Augusti-Tocco, G; Sato, G

    1969-09-01

    Clonal lines of neurons were obtained in culture from a mouse neuroblastoma. The neuroblastoma cells were adapted to culture growth by the animal-culture alternate passage technique and cloned after single-cell plating. The clonal lines retained the ability to form tumors when injected back into mice. A striking morphological change was observed in the cells adapted to culture growth; they appeared as mature neurons, while the cells of the tumor appeared as immature neuroblasts. Acetylcholinesterase and the enzymes for the synthesis of neurotransmitters, cholineacetylase and tyrosine hydroxylase were assayed in the tumor and compared with brain levels; tyrosine hydroxylase was found to be particularly high, as described previously in human neuroblastomas. The three enzymes were found in the clonal cultures at levels comparable to those found in the tumors. Similarly, there were no remarkable differences between the three clones examined.

  14. Synergistic efficacy of sorafenib and genistein in growth inhibition by down regulating angiogenic and survival factors and increasing apoptosis through upregulation of p53 and p21 in malignant neuroblastoma cells having N-Myc amplification or non-amplification.

    PubMed

    Roy Choudhury, Subhasree; Karmakar, Surajit; Banik, Naren L; Ray, Swapan K

    2010-12-01

    Neuroblastoma is an extracranial, solid, and heterogeneous malignancy in children. The conventional therapeutic modalities are mostly ineffective and thus new therapeutic strategies for malignant neuroblastoma are urgently warranted. We examined the synergistic efficacy of combination of sorafenib (SF) and genistein (GST) in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. MTT assay showed dose-dependent decrease in cell viability and the combination therapy more prominently inhibited the cell proliferation in both cell lines than either treatment alone. Apoptosis was confirmed morphologically by Wright staining. Flow cytometric analysis of cell cycle phase distribution and Annexin V-FITC/PI staining showed increase in subG1 DNA content and early apoptosis, respectively, after treatment with the combination of drugs. Apoptosis was further confirmed by scanning electron microscopy. Combination therapy showed activation of caspase-8, cleavage of Bid to tBid, increase in p53 and p21 expression, down regulation of anti-apoptotic Mcl-1, and increase in Bax:Bcl-2 ratio to trigger apoptosis. Down regulation of MDR, hTERT, N-Myc, VEGF, FGF-2, NF-κB, p-Akt, and c-IAP2 indicated suppression of angiogenic and survival pathways. Mitochondrial release of cytochrome c and Smac into cytosol indicated involvement of mitochondia in apoptosis. Increases in proteolytic activities of calpain and caspase-3 were also confirmed. Our results suggested that combination of SF and GST inhibited angiogenic and survival factors and increased apoptosis via receptor and mitochondria mediated pathways in both neuroblastoma SK-N-DZ and SH-SY5Y cell lines. Thus, this combination of drugs could be a potential therapeutic strategy against human malignant neuroblastoma cells having N-Myc amplification or non-amplific