Science.gov

Sample records for neuroblastoma cells due

  1. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  2. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2014-07-01

    AD_________________ Award Number: W81XWH-10-1-0425 TITLE: Improve T Cell Therapy in Neuroblastoma ...2014 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Improve T Cell Therapy in Neuroblastoma 5b. GRANT NUMBER W81XWH-10-1-0425 5c. PROGRAM...AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Neuroblastoma (NB) is

  3. Neuroblastoma

    MedlinePlus

    ... Old Feeding Your 1- to 2-Year-Old 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 ...

  4. 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 ...

  5. Sublethal irradiation promotes invasiveness of neuroblastoma cells

    SciTech Connect

    Schweigerer, Lothar; Rave-Fraenk, Margret; Schmidberger, Heinz; Hecht, Monica . E-mail: monica.hecht@med.uni-goettingen.de

    2005-05-13

    Neuroblastoma is the most frequent extracranial solid tumour of childhood. Despite multiple clinical efforts, clinical outcome has remained poor. Neuroblastoma is considered to be radiosensitive, but some clinical studies including the German trial NB90 failed to show a clinical benefit of radiation therapy. The mechanisms underlying this apparent discrepancy are still unclear. We have therefore investigated the effects of radiation on neuroblastoma cell behaviour in vitro. We show that sublethal doses of irradiation up-regulated the expression of the hepatocyte growth factor (HGF) and its receptor c-Met in some neuroblastoma cell lines. The increase in HGF/c-Met expression was correlated with enhanced invasiveness and activation of proteases degrading the extracellular matrix. Thus, irradiation at sublethal doses may promote the metastatic dissemination of neuroblastoma cells through activating the HGF/c-Met pathway and triggering matrix degradation.

  6. 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, ...

  7. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2015-09-01

    antibodies and T-cell immune responses elicited by tumor vaccine , we have combined the beneficial effects of both humoral and cell-mediated components of...the anti-neuroblastoma activity of CTLs expressing the GD2-specific CAR in two xenograft mouse models of neuroblastoma. Figure 1: Vaccination with...K562-derived whole-cell vaccine expressing CMV-pp65, CD40L and OX40L enhances antitumor effects of CAR-CMV- CTLs in vivo. Panel A. NOG/SCID/γc

  8. What Is Neuroblastoma?

    MedlinePlus

    ... Research and Treatment? Neuroblastoma About Neuroblastoma What Is Neuroblastoma? Cancer starts when cells in the body begin ... see the section, Signs and Symptoms of Neuroblastoma ). Neuroblastomas Neuroblastomas are cancers that start in early nerve ...

  9. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    DTIC Science & Technology

    2014-09-01

    AD_________________ Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma ...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT We identified 14 microRNA candidates that induce neuroblastoma cell differentiation based on a high...content screening of neurite outgrowth — the morphological differentiation marker of neuroblastoma cells. We further validated that the identified

  10. PHOX2B Is Associated with Neuroblastoma Cell Differentiation.

    PubMed

    Yang, Liqun; Ke, Xiao-Xue; Xuan, Fan; Tan, Juan; Hou, Jianbing; Wang, Mei; Cui, Hongjuan; Zhang, Yundong

    2016-03-01

    Neuroblastoma is a common pediatric malignancy that accounts for ∼15% of tumor-related deaths in children. The tumor is generally believed to originate from neural crest cells during early sympathetic neurogenesis. As the degree of neuroblastoma differentiation has been correlated with clinical outcome, clarifying the molecular mechanisms that drive neuroblastoma progression and differentiation is important for increasing the survival of these patients. In a previous study, the authors identified paired-like homeobox 2b (PHOX2B) as a key mediator of neuroblastoma pathogenesis in a TH-MYCN mouse model. In the present study, they aimed to define whether PHOX2B is also associated with proliferation and differentiation of human neuroblastoma cells. PHOX2B expression in neuroblastoma cells was evaluated by immunoblot analyses, and the effects of PHOX2B on the proliferation of neuroblastoma cells in vitro were determined using clonogenic and sphere formation assays. Xenograft experiments in NOD/SCID mice were used to examine the in vivo response to PHOX2B knockdown. Their data demonstrated that PHOX2B acts as a prognostic marker in neuroblastoma and that retinoic acid-induced neuronal differentiation downregulates PHOX2B expression, thereby suppressing the self-renewal capacity of neuroblastoma cells and inhibiting tumorigenicity. These findings confirmed that PHOX2B is a key regulator of neuroblastoma differentiation and stemness maintenance and indicated that PHOX2B might serve as a potential therapeutic target in neuroblastoma patients.

  11. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    DTIC Science & Technology

    2015-10-01

    Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma Cell Differentiation PRINCIPAL INVESTIGATOR: Dr. Liqin Du...inducing miRNA, miR- 449a. We examined the differentiation-inducing function of miR-449a in multiple neuroblastoma cell lines. We have demonstrated that...miR-449a functions as an inducer of cell differentiation in neuroblastoma cell lines with distinct genetic backgrounds, including the MYCN

  12. Dye-mediated photosensitization of murine neuroblastoma cells

    SciTech Connect

    Sieber, F.; Sieber-Blum, M.

    1986-04-01

    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  13. Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma.

    PubMed

    Spel, Lotte; Boelens, Jaap-Jan; van der Steen, Dirk M; Blokland, Nina J G; van Noesel, Max M; Molenaar, Jan J; Heemskerk, Mirjam H M; Boes, Marianne; Nierkens, Stefan

    2015-11-03

    Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20-40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses.

  14. Polysialic acid in human neuroblastoma cells

    SciTech Connect

    Livingston, B.D.; Jacobs, J.; Shaw, G.W.; Glick, M.C.; Troy, F.A.

    1987-05-01

    Prokaryotic-derived probes that specifically detect ..cap alpha..-2,8-linked polysialic acid (PSA) units on embryonic neural cell adhesion molecules (N-CAM) were used to show that membrane glycoproteins (GPs) from metastatic human neuroblastoma cells (CHP-134) also contain these unique carbohydrate moieties. This conclusion was based on the following evidence: (1) membranes from CHP-134 cells served as an exogenous acceptor of (/sup 14/C)NeuNAc units in an E. coli K1 sialyltransferase (ST) assay. The bacterial ST is specific for the transfer of (/sup 14/C)NeuNAc to exogenous acceptors containing at least 3 sialyl units (DP3); (2) in SDS-PAGE, the (/sup 14/C)NeuNAc-labeled CHP-134 membranes showed a major peak of radioactivity that was polydisperse. N-CAM shows a similar Mr heterogeneity; (3) treatment of the high Mr CHP-134 product with Endo-N-acetylneuraminidase (Endo-N) released the (/sup 14/C)NeuNAc label as a DP4. Endo-N is specific for hydrolysing ..cap alpha..-2,8-linked PSA chains containing a minimum of 5 sialyl residues; (4) treatment of the DP4 with sialidase converted the label to (/sup 14/C)NeuNAc, thus proving the tetramer contained sialic acid; (5) CHP-134 cells were labeled in vivo with (/sup 3/H)GlcN. A glycopeptide fraction representing ca. 1% of the (/sup 3/H)GlcN incorporated was isolated. Based on Endo-N sensitivity, this glycopeptide contained at least 15-20% of the (/sup 3/H)GlcN label as PSA. Endo-N digestion of the (/sup 3/H)-labeled glycopeptide released (/sup 3/H)-DP4. These results suggest that the surface expression of PSA-containing GPs may be important in neuroblastoma metastasis.

  15. Natural Killer Cells to the Attack: Combination Therapy against Neuroblastoma.

    PubMed

    Zenarruzabeitia, Olatz; Vitallé, Joana; Astigarraga, Itziar; Borrego, Francisco

    2017-02-01

    TGFβ in the tumor microenvironment diminishes natural killer (NK) cell-mediated anti-disialoganglioside (anti-GD2) mAb elimination of neuroblastoma cells. Consequently, blockade of TGFβ signaling with galunisertib in combination with the anti-GD2 mAb dinutuximab plus adoptively transferred NK cells is a promising tool for the treatment of neuroblastoma. Clin Cancer Res; 23(3); 615-7. ©2016 AACRSee related article by Tran et al., p. 804.

  16. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    SciTech Connect

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  17. Cellular Immunotherapy for Neuroblastoma: A Review of Current Vaccine and Adoptive T Cell Therapeutics

    PubMed Central

    Louis, C.U.; Brenner, M.K.

    2014-01-01

    Immunotherapy is an attractive option for patients with high risk neuroblastoma due to their poor long-term survival rates after conventional treatment. Neuroblastoma cells are derived from the embryonic neural crest and therefore express tumor antigens not widely seen in normal cells, making them potential targets for immunologic attack. There is already considerable experience with monoclonal antibodies that target these tumor associated antigens, and in this review we focus on more exploratory approaches, using tumor vaccines and adoptive transfer of tumor-directed T cells. PMID:19199969

  18. Probenecid Sensitizes Neuroblastoma Cancer Stem Cells to Cisplatin.

    PubMed

    Campos-Arroyo, Denise; Maldonado, Vilma; Bahena, Ivan; Quintanar, Valeria; Patiño, Nelly; Carlos Martinez-Lazcano, Juan; Melendez-Zajgla, Jorge

    2016-01-01

    We used both in vitro cultures of neuroblastoma cell lines and nude-mice xenotransplants to explore the effects of co-administration of cisplatin and probenecid. Probenecid sensitized neuroblastoma cells, including tumor cells with stem features, to the effects of cisplatin, both in vitro and in vivo. This effect was mediated by an increase in the apoptotic cell death and a concomitant decrease in cell proliferation. This effect is accompanied by modulation of the mRNA and protein of the drug efflux transporters MDR1, MRP2, and BCRP. The co-administration of probenecid with cisplatin should be explored as a possible therapeutic strategy.

  19. 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

  20. Cytotoxic effects of catechol to neuroblastoma N2a cells.

    PubMed

    Lima, Rute M F; Alvarez, Lisandro D G; Costa, Maria F D; Costa, Silvia L; Clarêncio, Jorge; El-Bachá, Ramon S

    2008-12-01

    The mechanisms of catechol-induced cytotoxicity were studied in cultures of neuroblastoma N2a cells. The minimal cytotoxic concentration after 72 h was 20 micromol x l(-1). The EC50 after 72 h was 38 micromol x l(-1). There was not a correlation between the cytotoxicity and the formation of quinones in the medium. Catechol-induced cytotoxicity was increased significantly when superoxide dismutase (SOD) was added. The addition of catalase did not protect cells, but this enzyme reverted the deleterious effect of SOD. The experimental studies showed a detrimental effect of deferoxamine on catechol-induced cytotoxicity suggesting that cells need iron to maintain its metabolism. NF-kappaB inhibitors increased the cytotoxicity, suggesting that this factor is also important for cell viability. L-cysteine and N-acetyl-L-cysteine protected cells significantly in a dose-dependent manner. The use of monochlorobimane showed that catechol induced reduced glutathione (GSH) depletion after 24 h, prior to cell death. The mode of cell death was studied by flow cytometry after double staining with annexin V and propidium iodide. Catechol induced apoptosis after 72 h. Furthermore, catechol also induced nuclear fragmentation. These data showed that catechol-induced cytotoxicity to N2a cell was not directly a consequence of reactive oxygen species production. Rather, it was due to GSH depletion followed by the induction of apoptosis.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Simultaneous measurement of neural spike recordings and multi-photon calcium imaging in neuroblastoma cells.

    PubMed

    Kim, Suhwan; Jung, Unsang; Baek, Juyeong; Kang, Shinwon; Kim, Jeehyun

    2012-11-08

    This paper proposes the design and implementation of a micro-electrode array (MEA) for neuroblastoma cell culturing. It also explains the implementation of a multi-photon microscope (MPM) customized for neuroblastoma cell excitation and imaging under ambient light. Electrical signal and fluorescence images were simultaneously acquired from the neuroblastoma cells on the MEA. MPM calcium images of the cultured neuroblastoma cell on the MEA are presented and also the neural activity was acquired through the MEA recording. A calcium green-1 (CG-1) dextran conjugate of 10,000 D molecular weight was used in this experiment for calcium imaging. This study also evaluated the calcium oscillations and neural spike recording of neuroblastoma cells in an epileptic condition. Based on our observation of neural spikes in neuroblastoma cells with our proposed imaging modality, we report that neuroblastoma cells can be an important model for epileptic activity studies.

  6. Nano-Bio-Mechanics of Neuroblastoma Cells Using AFM

    NASA Astrophysics Data System (ADS)

    Bastatas, Lyndon; Matthews, James; Kang, Min; Park, Soyeun

    2011-10-01

    We have conducted an in vitro study to determine the elastic moduli of neurobalstoma cell lines using atomic force microscopy. Using a panel of cell lines established from neuroblastoma patients at different stages of disease progress and treatment, we have investigated the differences in elastic moduli during a course of cancer progression and chemotherapy. The cells were grown on the hard substrates that are chemically functionalized to enhance adhesion. We have performed the AFM indentation experiments with different applied forces from the AFM probe. For the purpose of the comparison between cell lines, the indentations were performed only on cell centers. The obtained force-distance curves were analyzed using the Hertz model in order to extract the elastic moduli. We have found that the elastic moduli of human neuroblastoma cells significantly varied during the disease progression. We postulate that the observed difference might be affected by the treatment and chemotherapy.

  7. 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.

  8. Expression of the amplified domain in human neuroblastoma cells.

    PubMed Central

    Michitsch, R W; Montgomery, K T; Melera, P W

    1984-01-01

    Screening of a partial cDNA library prepared from the human neuroblastoma cell line BE(2)-C with genomic DNA probes containing sequences representative of the amplified domain of that cell line allowed us to identify cloned transcripts from an active gene within the domain. The gene BE(2)-C-59 is amplified ca. 150-fold and encodes a 3.0- and a 1.5-kilobase RNA transcript, both of which are overproduced in BE(2)-C cells. A survey of a large variety of human tumor cell types indicated that this gene is amplified to varying degrees in all neuroblastoma cell lines and a retinoblastoma cell line that exhibit obvious cytological manifestations of DNA sequence amplification, i.e., homogeneously staining regions and double-minute chromosomes. The BE(2)-C-59 gene is not amplified, however, in other nonrelated tumor types, even those containing amplified DNA. Although the functional significance of this specific gene amplification in neuroblastoma cells remains unknown, an indication that it may relate to the malignant phenotype of these cells follows from the remainder of our data which show that the amplified BE(2)-C-59 gene shares partial homology with both the second and third exons, but not the first exon, of the human c-myc oncogene. Images PMID:6549047

  9. UBE4B Levels Are Correlated with Clinical Outcomes in Neuroblastoma Patients and with Altered Neuroblastoma Cell Proliferation and Sensitivity to EGFR Inhibitors

    PubMed Central

    Zage, Peter E.; Sirisaengtaksin, Natalie; Liu, Yin; Gireud, Monica; Brown, Brandon S.; Palla, Shana; Richards, Kristen N.; Hughes, Dennis P.M.; Bean, Andrew J.

    2012-01-01

    Background The UBE4B gene, located on chromosome 1p36, encodes a ubiquitin ligase that interacts with Hrs, a protein involved in EGFR trafficking, suggesting a link between EGFR trafficking and neuroblastoma pathogenesis. We have analyzed the roles of UBE4B in the outcomes of neuroblastoma patients and in neuroblastoma tumor cell proliferation, EGFR trafficking, and response to EGFR inhibition. Methods We examined the association of UBE4B expression with neuroblastoma patient survival using available microarray datasets. We measured UBE4B and EGFR protein levels in patient tumor samples and EGFR degradation rates in neuroblastoma cell lines and analyzed the effects of UBE4B on neuroblastoma tumor cell growth. The effects of the EGFR inhibitor cetuximab were examined in neuroblastoma cells expressing wild-type and mutant UBE4B. Results Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma. UBE4B overexpression reduced neuroblastoma tumor cell proliferation, and UBE4B expression was inversely related to EGFR expression in patient tumor samples. EGFR degradation rates correlated with cellular UBE4B levels. Enhanced expression of catalytically active UBE4B resulted in reduced sensitivity to EGFR inhibition. Conclusions We have demonstrated associations between UBE4B expression and neuroblastoma patient outcomes and between UBE4B and EGFR expression in neuroblastoma tumor samples. Moreover, levels of UBE4B influenced neuroblastoma tumor cell proliferation, EGFR degradation, and response to EGFR inhibition. These results suggest UBE4B-mediated GFR trafficking may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions, and that UBE4B expression may be a marker that can predict responses of neuroblastoma tumors to treatment. PMID:22990745

  10. Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

    NASA Astrophysics Data System (ADS)

    Kim, Suhwan; Jung, Unsang; Baek, Juyoung; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-01-01

    Recently, mouse neuroblastoma cells have been considered as an attractive model for the study of human neurological and prion diseases, and they have been intensively used as a model system in different areas. For example, the differentiation of neuro2a (N2A) cells, receptor-mediated ion current, and glutamate-induced physiological responses have been actively investigated with these cells. These mouse neuroblastoma N2A cells are of interest because they grow faster than other cells of neural origin and have a number of other advantages. The calcium oscillations and neural spikes of mouse neuroblastoma N2A cells in epileptic conditions are evaluated. Based on our observations of neural spikes in these cells with our proposed imaging modality, we reported that they can be an important model in epileptic activity studies. We concluded that mouse neuroblastoma N2A cells produce epileptic spikes in vitro in the same way as those produced by neurons or astrocytes. This evidence suggests that increased levels of neurotransmitter release due to the enhancement of free calcium from 4-aminopyridine causes the mouse neuroblastoma N2A cells to produce epileptic spikes and calcium oscillations.

  11. The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

    PubMed

    Briggs, D J; Phillips, R M

    1991-08-01

    Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

  12. Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

    PubMed

    Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

    2017-02-28

    Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

  13. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    PubMed Central

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Background Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. Methods PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Results Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. Conclusion PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration. PMID:19077250

  14. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    SciTech Connect

    Dou, Q.P.; Chen, K.Y.

    1987-05-01

    An 18,000-dalton protein can be metabolically labeled by (TH)putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0.

  15. TRPM7 maintains progenitor-like features of neuroblastoma cells: implications for metastasis formation.

    PubMed

    Middelbeek, Jeroen; Visser, Daan; Henneman, Linda; Kamermans, Alwin; Kuipers, Arthur J; Hoogerbrugge, Peter M; Jalink, Kees; van Leeuwen, Frank N

    2015-04-20

    Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features.

  16. TRPM7 maintains progenitor-like features of neuroblastoma cells: implications for metastasis formation

    PubMed Central

    Middelbeek, Jeroen; Kamermans, Alwin; Kuipers, Arthur J.; Hoogerbrugge, Peter M.; Jalink, Kees; van Leeuwen, Frank N.

    2015-01-01

    Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features. PMID:25797249

  17. Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells.

    PubMed

    Tabata, Keiichi; Nishimura, Yuki; Takeda, Taiji; Kurita, Masahiro; Uchiyama, Taketo; Suzuki, Takashi

    2015-04-01

    Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma.

  18. Ouabain-Induced Signaling and Cell Survival in SK-N-SH Neuroblastoma Cells Differentiated by Retinoic Acid

    PubMed Central

    Akkuratov, Evgeny E.; Wu, Jian; Sowa, David; Shah, Zahoor A.; Liu, Lijun

    2015-01-01

    Ouabain stimulates activation of various signaling cascades such as protein kinase B (Akt) and Extracellular-signaling-regulated kinase 1/2 (ERK 1/2) in various cell lines. Retinoic acid (RA) is commonly used to induce neuroblastoma differentiation in cultures. Upon RA administration, human neuroblastoma cell line, SK-N-SH demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Here we report that ouabain-induced signaling is altered under the action of 1 μM RA in human neuroblastoma SK-N-SH cells. RA increased the expression of p110α subunit of phosphoinositide 3-kinase (PI3K), Akt and β1 subunit of Na+/K+-ATPase. Ouabain activated Akt and ERK 1/2 in differentiated SK-N-SH cells; this effect was not observed in non-differentiated SK-N-SH cells. Long-term incubation of non-differentiated SK-N-SH with 1 μM ouabain led to a decrease in the number of cells; this effect was reduced in differentiated SK-N-SH cells. Taken together, these results suggest that ouabain leads to cell death in neuroblastoma cells rather than neuronal cells due to the different response to ouabain manifested by activation of Akt and ERK 1/2. Highlights • RA increases the expression of p110α subunit of PI3K, Akt and β1 subunit of Na+/K+-ATPase • Ouabain induces activation of Akt and ERK 1/2 in differentiated SK-N-SH cells but not in non-differentiated cells • 1 μM ouabain leads to a decrease in the number of cells in non-differentiated SK-N-SH • Reduction of ouabain-induced cell death in differentiated SK-N-SH

  19. Polysulfide promotes neuroblastoma cell differentiation by accelerating calcium influx.

    PubMed

    Koike, Shin; Shibuya, Norihiro; Kimura, Hideo; Ishii, Kazuyuki; Ogasawara, Yuki

    2015-04-10

    Polysulfides are a typical type of bound sulfur, which is physiologically stable form of sulfur species, derived from the hydrogen sulfide (H2S) that is generated endogenously in cells. We previously reported that bound sulfur protects neuronal cells from oxidative injury. In the present study, we demonstrated that polysulfides inhibited cell growth and promoted neurite outgrowth in mouse neuroblastoma Neuro2A (N2A) cells. However, Na2S showed no effect on neurite outgrowth in N2A cells. Furthermore, 2-APB and SKF96365, which are typical transient receptor potential (TRP) channel inhibitors, suppressed the neurite outgrowth induced by Na2S4. These new findings suggest that bound sulfur could induce neurite outgrowth and cell differentiation of N2A cells by accelerating calcium influx.

  20. What's New in Neuroblastoma Research and Treatment?

    MedlinePlus

    ... Neuroblastoma Research and Treatment? Neuroblastoma About Neuroblastoma What’s New in Neuroblastoma Research and Treatment? Important research into ... cells different from normal cells may lead to new approaches to treating this disease. Newer drugs that ...

  1. TLR3 triggering regulates PD-L1 (CD274) expression in human neuroblastoma cells.

    PubMed

    Boes, Marianne; Meyer-Wentrup, Friederike

    2015-05-28

    Neuroblastoma is the most common extracranial solid tumor in children, causing 12% of all pediatric cancer mortality. Neuroblastoma specific T-cells have been detected in patients, but usually fail to attack and eradicate the tumors. Tumor immune evasion may thus play an important role in neuroblastoma pathogenicity. Recent research in adult cancer patients shows that targeting T-cell check-point molecules PD-1/PD-L1 (or CD279/CD274) may bolster immune reactivity against solid tumors. Also, infections can be associated with spontaneous neuroblastoma regression. In our current study, we therefore investigated if antibody targeting of PD-L1 and triggering of selective pathogen-receptor Toll-like receptors (TLRs) potentiates immunogenicity of neuroblastoma cells. We find this to be the case. TLR3 triggering induced strong upregulation of both MHC class I and PD-L1 on neuroblastoma cells. At the same time TGF-β levels decreased and IL-8 secretion was induced. The combined neuroblastoma cell treatment using PD-L1 blockade and TLR3 triggering using virus analog poly(I:C) moreover induced CD4(+) and CD8(+) T-cell activation. Thus, we propose combined treatment using PD-L1 blockade with synthetic TLR ligands as an avenue toward new immunotherapy against human neuroblastoma.

  2. Identification of nuclear. tau. isoforms in human neuroblastoma cells

    SciTech Connect

    Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I. )

    1990-11-01

    The {tau} proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, {tau} has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire {tau} molecule in the isolated nuclei of neuroblastoma cells. Nuclear {tau} proteins, like the {tau} proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that {tau} may function in processes not directly associated with microtubules and that highly insoluble complexes of {tau} may also play a role in normal cellular physiology.

  3. Differentiation state affects morphine induced cell regulation in neuroblastoma cultured cells.

    PubMed

    Fiore, Giovina; Ghelardini, Carla; Bruni, Giancarlo; Guarna, Massimo; Bianchi, Enrica

    2013-10-25

    Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy. Our purpose was to investigate in vitro how cancer cell survival occurs in presence of morphine in undifferentiated and differentiated SHSY-5Y human neuroblastoma cultured cell line. Exposure of differentiated cells to morphine dose-dependently induced apoptosis in these cells through c-Jun N-terminal kinase (JNK)/caspase pathway. Otherwise, morphine induced activation for mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, caused positive regulation of cell survival in undifferentiated cells. Therefore, cell differentiation state bimodally affects the cellular regulation activity triggered by morphine in isolated cultured neuroblastoma cells raising concerns about the application of morphine to this type of cancer patients.

  4. Stem Cell Transplant-Associated Wernicke Encephalopathy in a Patient with High-Risk Neuroblastoma.

    PubMed

    Darlington, Wendy S; Pinto, Navin; Hecktman, Hillary M; Cohn, Susan L; LaBelle, James L

    2015-12-01

    Children undergoing intense cancer treatment frequently require total parenteral nutrition (TPN). Rarely, vitamins are removed due to hypersensitivity to the carrier vehicle in the formulation. We present the case of a 5-year-old patient with stage 4, high-risk neuroblastoma who developed altered mental status, ataxia, and tachycardia during consolidative autologous stem cell transplantation. Skin findings and brain MRI were consistent with thiamine (vitamin B1) deficiency and Wernicke encephalopathy. Vitamin B1 administration rapidly reversed all skin and neurologic symptoms. This case highlights the importance of close monitoring of micronutrients in pediatric patients receiving prolonged courses of chemotherapy and stem cell transplantation.

  5. Down-regulation of the Lamin A/C in neuroblastoma triggers the expansion of tumor initiating cells.

    PubMed

    Nardella, Marta; Guglielmi, Loredana; Musa, Carla; Iannetti, Ilaria; Maresca, Giovanna; Amendola, Donatella; Porru, Manuela; Carico, Elisabetta; Sessa, Giuseppe; Camerlingo, Rosalba; Dominici, Carlo; Megiorni, Francesca; Milan, Marika; Bearzi, Claudia; Rizzi, Roberto; Pirozzi, Giuseppe; Leonetti, Carlo; Bucci, Barbara; Mercanti, Delio; Felsani, Armando; D'Agnano, Igea

    2015-10-20

    Tumor-initiating cells constitute a population within a tumor mass that shares properties with normal stem cells and is considered responsible for therapy failure in many cancers. We have previously demonstrated that knockdown of the nuclear envelope component Lamin A/C in human neuroblastoma cells inhibits retinoic acid-mediated differentiation and results in a more aggressive phenotype. In addition, Lamin A/C is often lost in advanced tumors and changes in the nuclear envelope composition occur during tumor progression. Based on our previous data and considering that Lamin A/C is expressed in differentiated tissues, we hypothesize that the lack of Lamin A/C could predispose cells toward a stem-like phenotype, thus influencing the development of tumor-initiating cells in neuroblastoma. This paper demonstrates that knockdown of Lamin A/C triggers the development of a tumor-initiating cell population with self-renewing features in human neuroblastoma cells. We also demonstrates that the development of TICs is due to an increased expression of MYCN gene and that in neuroblastoma exists an inverse relationship between LMNA and MYCN expression.

  6. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2013-07-01

    CMV seropositive donors were cocultured with irradiated aAPCs. As shown in Fig 10E, after 10 - 12 days of culture, aAPCs expressing pp65 reactivated CMV ...inferior to the PBMC loaded with pp65 peptides (INF SFU 789 ± 130), aAPCs expressing pp65 were effective in reactivating CMV -specific T cells (INF SFU 83...significantly increased the boosting of CMV -specific T cells (INF SFU 292 ± 56 vs. 502 ± 104 for K562/pp65 and K562/CD40L/pp65, respectively; p=0.034). The

  7. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2012-07-01

    which is the receptor for I-TAC, a chemokine typically released in in- flamed tissues.17 However, because the expression of CXCR3 is moderate in...experiments with 5 mice per group. ***P < 0.001. (C) Mice were pretreated with anti-CD1d blocking or isotype control mAb before transfer of NKT/IL-15 cells...restricted, we repeated the treatment of NB metastases in hu-NSG mice with NKT/IL-15 cells after pretreatment with anti-CD1d blocking or isotype

  8. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2011-07-01

    transfer of Epstein - Barr - virus (EBV)-specific cytotoxic T lymphocytes (EBV- CTLs) genetically modified to express a chimeric antigen receptor (CAR-GD2...4 Introduction. In our recent Phase I study we found that the adoptive transfer of Epstein - Barr - virus (EBV...specific aims. 8 Reference List 1. Pule MA, Savoldo B, Myers GD et al. Virus -specific T cells

  9. Nanopore formation in neuroblastoma cells following ultrashort electric pulse exposure

    NASA Astrophysics Data System (ADS)

    Roth, Caleb C.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    Ultrashort or nanosecond electrical pulses (USEP) cause repairable damage to the plasma membranes of cells through formation of nanopores. These nanopores are able to pass small ions such as sodium, calcium, and potassium, but remain impermeable to larger molecules like trypan blue and propidium iodide. What remains uncertain is whether generation of nanopores by ultrashort electrical pulses can inhibit action potentials in excitable cells. In this paper, we explored the sensitivity of excitable cells to USEP using Calcium Green AM 1 ester fluorescence to measure calcium uptake indicative of nanopore formation in the plasma membrane. We determined the threshold for nanopore formation in neuroblastoma cells for three pulse parameters (amplitude, pulse width, and pulse number). Measurement of such thresholds will guide future studies to determine if USEP can inhibit action potentials without causing irreversible membrane damage.

  10. Biochemical effects of chlorpromazine on mouse neuroblastoma cells.

    PubMed

    Andres, M I; Repetto, G; Sanz, P; Repetto, M

    1999-10-01

    Chlorpromazine and other phenothiazine derivatives are neuroleptic drugs of widespread use for clinical situations beyond the realm of psychiatry, such as to control nausea, vomiting and intractable hiccups. The present study investigated in vitro different cytotoxic effects of chlorpromazine in cultures of mouse neuroblastoma cell line Neuro-2a exposed to different concentrations of this compound. Indicators assessed were cell proliferation by quantification of total protein content of the cell culture, lysosomal function evaluated by the relative uptake of neutral red cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis, mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle, lysosomal beta-galactosidase (GAL) activity, and neuronal acetylcholinesterase activity. Marked inhibitory effects were found for cell proliferation and relative neutral red uptake; PFK, ENL and GAL activities had no significant differences from control. Stimulation was specifically detected on SDH and the Krebs cycle at concentrations up to 30 microM. Chlorpromazine did not have high toxicity for cytotoxic effects on lysosomes.

  11. Effects of YM155 on survivin levels and viability in neuroblastoma cells with acquired drug resistance

    PubMed Central

    Voges, Yvonne; Michaelis, Martin; Rothweiler, Florian; Schaller, Torsten; Schneider, Constanze; Politt, Katharina; Mernberger, Marco; Nist, Andrea; Stiewe, Thorsten; Wass, Mark N; Rödel, Franz; Cinatl, Jindrich

    2016-01-01

    Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action. PMID:27735941

  12. The mechanism of epithelial-mesenchymal transition induced by TGF-β1 in neuroblastoma cells.

    PubMed

    Shao, Jing-Bo; Gao, Zhi-Mei; Huang, Wen-Yan; Lu, Zhi-Bao

    2017-05-01

    Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-β1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of α-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-β1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-β1 indicating that TGF-β1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-β1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-β1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-β1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-β1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-β1-induced EMT independent of Smad signaling.

  13. 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.

  14. Transport of carnitine in neuroblastoma NB-2a cells.

    PubMed

    Nałecz, K A; Korzon, D; Wawrzeńczyk, A; Nałecz, M J

    1995-09-10

    Carnitine accumulation was measured in cultured neuroblastoma NB-2a cells. This process was found partially sodium dependent and its kinetics to be a sum of a saturable transport (Km = 123 +/- 13 microM) and diffusion (D = 63 +/- 7 pmol/mg protein/min/mM). On the contrary to previous reports on neural cells, the accumulation of carnitine was found insensitive to gamma-aminobutyric acid (GABA). Measurements of carnitine accumulation in the presence of different compounds resulted in the conclusion that carnitine transport does not occur through the known systems specific toward choline and/or amino acids. For instance, an observed inhibition of carnitine transport by serine and cysteine, without any effect of alanine, excluded a possible role of ASC amino acid transport system. An involvement of a new transporter is thus postulated, specific toward compounds with a polar group in the beta position with respect to the carboxylic group.

  15. Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival.

    PubMed

    Thole, Theresa M; Lodrini, Marco; Fabian, Johannes; Wuenschel, Jasmin; Pfeil, Sebastian; Hielscher, Thomas; Kopp-Schneider, Annette; Heinicke, Ulrike; Fulda, Simone; Witt, Olaf; Eggert, Angelika; Fischer, Matthias; Deubzer, Hedwig E

    2017-03-02

    The number of long-term survivors of high-risk neuroblastoma remains discouraging, with 10-year survival as low as 20%, despite decades of considerable international efforts to improve outcome. Major obstacles remain and include managing resistance to induction therapy, which causes tumor progression and early death in high-risk patients, and managing chemotherapy-resistant relapses, which can occur years after the initial diagnosis. Identifying and validating novel therapeutic targets is essential to improve treatment. Delineating and deciphering specific functions of single histone deacetylases in neuroblastoma may support development of targeted acetylome-modifying therapeutics for patients with molecularly defined high-risk neuroblastoma profiles. We show here that HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs. Genes necessary for mitotic cell cycle progression and cell division were most prominently enriched in at least two of three time points in whole-genome expression data combined from two cell systems, and all nine genes in these functional categories were strongly repressed, including CENPA, KIF14, KIF23 and RACGAP1. Enforced expression of one selected candidate, RACGAP1, partially rescued the induction of apoptosis caused by HDAC11 depletion. High-level expression of all nine genes in primary neuroblastomas significantly correlated with unfavorable overall and event-free survival in patients, suggesting a role in mediating the more aggressive biological and clinical phenotype of these tumors. Our study identified a group of cell cycle-promoting genes regulated by HDAC11, being both predictors of unfavorable patient outcome and essential for tumor cell viability. The data indicate a significant role of HDAC11 for mitotic cell cycle progression and survival of MYCN-amplified neuroblastoma cells, and suggests that HDAC11 could be a valuable drug target.

  16. 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

  17. 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.

  18. Focal adhesion kinase and p53 synergistically decrease neuroblastoma cell survival.

    PubMed

    Gillory, Lauren A; Stewart, Jerry E; Megison, Michael L; Waters, Alicia M; Beierle, Elizabeth A

    2015-06-15

    Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of neuroblastoma tumor development and progression. The p53 oncogene, although wild type in most neuroblastomas, lacks significant function as a tumor suppressor in these tumors. Recent reports have found that FAK and p53 interact in some tumor types. We have hypothesized FAK and p53 coordinately control each other's expression and also interact in neuroblastoma. In the present study, we showed that not only do FAK and p53 interact but each one controls the expression of the other. In addition, we also examined the effects of FAK inhibition combined with p53 activation in neuroblastoma and showed that these two, in combination, had a synergistic effect on neuroblastoma cell survival. The findings from this present study help to further our understanding of the regulation of neuroblastoma tumorigenesis and may provide novel therapeutic strategies and targets for neuroblastoma and other pediatric solid tumors.

  19. Epidermal growth factor receptor is overexpressed in neuroblastoma tissues and cells.

    PubMed

    Zheng, Chao; Shen, Ruling; Li, Kai; Zheng, Na; Zong, Yuqing; Ye, Danrong; Wang, Qingcheng; Wang, Zuopeng; Chen, Lian; Ma, Yangyang

    2016-08-01

    Neuroblastoma is the most common abdominal malignant tumor in childhood. Immunotoxin (IT) that targets the tumor cell surface receptor is a new supplementary therapeutic treatment approach. The purpose of this study is to detect the expression of epidermal growth factor receptor (EGFR) in neuroblastoma cell lines and tissues, and to explore if IT therapy can be used to treat refractory neuroblastoma. The EGFR expression in human neuroblastoma tissue samples was detected by immunohistochemistry staining. The positive rate of EGFR expression was 81.0% in neuroblastoma tissue and 50.0% in gangliocytoma, respectively, but without statistical significance between them (P > 0.05). The positive rate of EGFR expression in favorable type and unfavorable type was 62.5% and 92.3%, respectively, but they were not statistically different (P > 0.05). Results from pre-chemotherapy and post-chemotherapy samples showed that there was no significant statistical difference (P > 0.05) between them in the EGFR expression. Furthermore, the EGFR expression levels in five neuroblastoma cell lines were measured using cell-based ELISA assay and western blot analysis. The results showed that the expression of EGFR was higher in KP-N-NS and BE(2)-C than those in other cell lines. Our results revealed that there are consistent and widespread expressions of EGFR in neuroblastoma tissues as well as in neuroblastoma cell lines, suggesting that it is possible to develop future treatment strategies of neuroblastoma by targeting at the EGFR.

  20. NeuroD1 promotes neuroblastoma cell growth by inducing the expression of ALK.

    PubMed

    Lu, Fangjin; Kishida, Satoshi; Mu, Ping; Huang, Peng; Cao, Dongliang; Tsubota, Shoma; Kadomatsu, Kenji

    2015-04-01

    Neuroblastoma is derived from the sympathetic neuronal lineage of neural crest cells, and is the most frequently observed of the extracranial pediatric solid tumors. The neuronal differentiation factor, NeuroD1, has previously been shown to promote cell motility in neuroblastoma by suppressing the expression of Slit2. Here we report that NeuroD1 is also involved in the proliferation of neuroblastoma cells, including human cell lines and primary tumorspheres cultured from the tumor tissues of model mice. Interestingly, the growth inhibition of neuroblastoma cells induced by knockdown of NeuroD1 was accompanied by a reduction of ALK expression. ALK is known to be one of the important predisposition genes for neuroblastoma. The phenotype resulting from knockdown of NeuroD1 was suppressed by forced expression of ALK and, therefore, NeuroD1 appears to act mainly through ALK to promote the proliferation of neuroblastoma cells. Furthermore, we showed that NeuroD1 directly bound to the promoter region of ALK gene. In addition, the particular E-box in the promoter was responsible for NeuroD1-mediated ALK expression. These results indicate that ALK should be a direct target gene of NeuroD1. Finally, the expressions of NeuroD1 and ALK in the early tumor lesions of neuroblastoma model mice coincided in vivo. We conclude that the novel mechanism would regulate the expression of ALK in neuroblastoma and that NeuroD1 should be significantly involved in neuroblastoma tumorigenesis.

  1. Primitive neuroectodermal tumor in an ovarian cystic teratoma: natural killer and neuroblastoma cell analysis.

    PubMed

    Tabellini, Giovanna; Benassi, Marzia; Marcenaro, Emanuela; Coltrini, Daniela; Patrizi, Ornella; Ricotta, Doris; Rampinelli, Fabio; Moretta, Alessandro; Parolini, Silvia

    2014-01-01

    In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patient's peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.

  2. Translocation involving 1p and 17q is a recurrent genetic alteration of human neuroblastoma cells

    SciTech Connect

    Savelyeva, L.; Corvi, R.; Schwab, M. )

    1994-08-01

    Human neuroblastoma cells often are monosomic for the distal portion of 1p (1p36). The authors report that the deleted 1p material in cells of neuroblastoma lines is preferentially replaced by material from chromosome 17, resulting from an unbalanced 1;17 translocation. Chromosome 17 often acquires instability, followed by the integration of fragments into various marker chromosomes. As a consequence, 17q material can increase over 17p material. The nonrandom frequency of 1;17 translocations appears to indicate an as-yet-undefined contribution to neuroblastoma development. 35 refs., 4 figs., 1 tab.

  3. Toxicity study of cerium oxide nanoparticles in human neuroblastoma cells.

    PubMed

    Kumari, Monika; Singh, Shailendra Pratap; Chinde, Srinivas; Rahman, Mohammed Fazlur; Mahboob, Mohammed; Grover, Paramjit

    2014-01-01

    The present study consisted of cytotoxic, genotoxic, and oxidative stress responses of human neuroblastoma cell line (IMR32) following exposure to different doses of cerium oxide nanoparticles (CeO2 NPs; nanoceria) and its microparticles (MPs) for 24 hours. Cytotoxicity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays whereas genotoxicity was assessed using the cytokinesis-block micronucleus and comet assays. A battery of assays including lipid peroxidation, reactive oxygen species (ROS), hydrogen peroxide, reduced glutathione, nitric oxide, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase were performed to test the hypothesis that ROS was responsible for the toxicity of nanoceria. The results showed that nanosized CeO2 was more toxic than cerium oxide MPs. Hence, further study on safety evaluation of CeO2 NPs on other models is recommended.

  4. MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

    PubMed Central

    Guglielmi, L; Cinnella, C; Nardella, M; Maresca, G; Valentini, A; Mercanti, D; Felsani, A; D'Agnano, I

    2014-01-01

    Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma

  5. Acute toxicity testing in cultures of mouse neuroblastoma cells.

    PubMed

    Walum, E; Peterson, A

    1983-01-01

    Cultured mouse neuroblastoma cells (C1300) may be used as models for nerve cells since they have a number of properties in common with their normal counterparts in vivo. In order to test the possibility of using C1300 cells as alternative to experimental animals when testing for acute toxicity, cells (clone 41A3) were exposed to a number of common chemicals (CH3HgCl, CdCl2,HgCl2 ppDDT, n-butanol, benzene, dioxan, n-propanol, aceton and t-butanol). The toxic effect was quantified by measuring the degree of cell detachment in the cultures. The concentrations of chemicals that caused 25% of the total cell number to detach (TD25) were used for comparison with LD50 values. In spite of the very simplified situation in culture, where the toxicity of a substance is little or not at all influenced by factors like penetration, storage, metabolism and excretion a good correlation (corr. coeff. 0,98) was obtained between TD25 values and LD50 values. Good correlations between in vitro and in vivo tests have also been reported by others. One possible explanation to these findings could be simplified in vivo toxicokinetics of these substances when tested in high doses for general effects like animal death. If so, simple in vitro tests may be used for predicting acute toxicity of certain groups of substances.

  6. Prostaglandin E2 promotes MYCN non-amplified neuroblastoma cell survival via β-catenin stabilization

    PubMed Central

    Jansen, Sepp R; Holman, Rian; Hedemann, Ilja; Frankes, Ewoud; Elzinga, Carolina R S; Timens, Wim; Gosens, Reinoud; de Bont, Eveline S; Schmidt, Martina

    2015-01-01

    Amplification of MYCN is the most well-known prognostic marker of neuroblastoma risk classification, but still is only observed in 25% of cases. Recent evidence points to the cyclic adenosine monophosphate (cAMP) elevating ligand prostaglandin E2 (PGE2) and β-catenin as two novel players in neuroblastoma. Here, we aimed to define the potential role of PGE2 and cAMP and its potential interplay with β-catenin, both of which may converge on neuroblastoma cell behaviour. Gain and loss of β-catenin function, PGE2, the adenylyl cyclase activator forskolin and pharmacological inhibition of cyclooxygenase-2 (COX-2) were studied in two human neuroblastoma cell lines without MYCN amplification. Our findings show that PGE2 enhanced cell viability through the EP4 receptor and cAMP elevation, whereas COX-2 inhibitors attenuated cell viability. Interestingly, PGE2 and forskolin promoted glycogen synthase kinase 3β inhibition, β-catenin phosphorylation at the protein kinase A target residue ser675, β-catenin nuclear translocation and TCF-dependent gene transcription. Ectopic expression of a degradation-resistant β-catenin mutant enhances neuroblastoma cell viability and inhibition of β-catenin with XAV939 prevented PGE2-induced cell viability. Finally, we show increased β-catenin expression in human high-risk neuroblastoma tissue without MYCN amplification. Our data indicate that PGE2 enhances neuroblastoma cell viability, a process which may involve cAMP-mediated β-catenin stabilization, and suggest that this pathway is of relevance to high-risk neuroblastoma without MYCN amplification. PMID:25266063

  7. T cells targeting NY-ESO-1 demonstrate efficacy against disseminated neuroblastoma.

    PubMed

    Singh, Nathan; Kulikovskaya, Irina; Barrett, David M; Binder-Scholl, Gwendolyn; Jakobsen, Bent; Martinez, Daniel; Pawel, Bruce; June, Carl H; Kalos, Michael D; Grupp, Stephan A

    The cancer-testis antigen NY-ESO-1 is expressed by many solid tumors and has limited expression by mature somatic tissues, making it a highly attractive target for tumor immunotherapy. Targeting NY-ESO-1 using engineered T cells has demonstrated clinical efficacy in the treatment of some adult tumors. Neuroblastoma is a significant cause of cancer mortality in children, and is a tumor type shown to be responsive to immunotherapies. We evaluated a large panel of primarily resected neuroblastoma samples and demonstrated that 23% express NY-ESO-1. After confirming antigen-specific activity of T cells genetically engineered to express an NY-ESO-1 directed high-affinity transgenic T cell receptor in vitro, we performed xenograft mouse studies assessing the efficacy of NY-ESO-1-targeted T cells in both localized and disseminated models of neuroblastoma. Disease responses were monitored by tumor volume measurement and in vivo bioluminescence. After delivery of NY-ESO-1 transgenic TCR T cells, we observed significant delay of tumor progression in mice bearing localized and disseminated neuroblastoma, as well as enhanced animal survival. These data demonstrate that NY-ESO-1 is an antigen target in neuroblastoma and that targeted T cells represent a potential therapeutic option for patients with neuroblastoma.

  8. Culture and characteristics of hormone-responsive neuroblastoma x glioma hybrid cells

    SciTech Connect

    Hamprecht, B.; Glaser, T.; Reiser, G.; Bayer, E.; Propst, F.

    1985-01-01

    Neuroblastoma x glioma hybrid cells were generated by cell fusion of the 6-thioguanine-resistant clonal mouse neuroblastoma cells and the bromodeoxyuridine-resistant rat glioma cells, selection, and cloning. Every characteristics generally ascribed to neurons has been observed with the hybrid cells. The paper explores the morphological differentiation of hybrid cells, procedures for testing the hormonal regulation of intracellular levels of cyclic, (/sup 3/H)AMP in hybrid cells, hormonal regulation of adenylate cyclase in homogenates of hyrbid cells, intracellular levels of cyclic GMP, and uptake of guanidinium ions in hybrid cells.

  9. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  10. Glycolytic inhibitor 2-deoxyglucose simultaneously targets cancer and endothelial cells to suppress neuroblastoma growth in mice.

    PubMed

    Huang, Chao-Cheng; Wang, Shuo-Yu; Lin, Li-Ling; Wang, Pei-Wen; Chen, Ting-Ya; Hsu, Wen-Ming; Lin, Tsu-Kung; Liou, Chia-Wei; Chuang, Jiin-Haur

    2015-10-01

    Neuroblastoma is characterized by a wide range of clinical manifestations and associated with poor prognosis when there is amplification of MYCN oncogene or high expression of Myc oncoproteins. In a previous in vitro study, we found that the glycolytic inhibitor 2-deoxyglucose (2DG) could suppress the growth of neuroblastoma cells, particularly in those with MYCN amplification. In this study, we established a mouse model of neuroblastoma xenografts with SK-N-DZ and SK-N-AS cells treated with 2DG by intraperitoneal injection twice a week for 3 weeks at 100 or 500 mg/kg body weight. We found that 2DG was effective in suppressing the growth of both MYCN-amplified SK-N-DZ and MYCN-non-amplified SK-N-AS neuroblastoma xenografts, which was associated with downregulation of HIF-1α, PDK1 and c-Myc, and a reduction in the number of tumor blood vessels. In vitro study showed that 2DG can suppress proliferation, cause apoptosis and reduce migration of murine endothelial cells, with inhibition of the formation of lamellipodia and filopodia and disorganization of F-actin filaments. The results suggest that 2DG might simultaneously target cancer cells and endothelial cells in the neuroblastoma xenografts in mice regardless of the status of MYCN amplification, providing a potential therapeutic opportunity to use 2DG or other glycolytic inhibitors for the treatment of patients with refractory neuroblastoma.

  11. 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.

  12. Novel proteasome inhibitor ixazomib sensitizes neuroblastoma cells to doxorubicin treatment

    PubMed Central

    Li, Haoyu; Chen, Zhenghu; Hu, Ting; Wang, Long; Yu, Yang; Zhao, Yanling; Sun, Wenijing; Guan, Shan; Pang, Jonathan C.; Woodfield, Sarah E.; Liu, Qing; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial malignant solid tumor seen in children and continues to lead to the death of many pediatric cancer patients. The poor outcome in high risk NB is largely attributed to the development of chemoresistant tumor cells. Doxorubicin (dox) has been widely employed as a potent anti-cancer agent in chemotherapeutic regimens; however, it also leads to chemoresistance in many cancer types including NB. Thus, developing novel small molecules that can overcome dox-induced chemoresistance is a promising strategy in cancer therapy. Here we show that the second generation proteasome inhibitor ixazomib (MLN9708) not only inhibits NB cell proliferation and induces apoptosis in vitro but also enhances dox-induced cytotoxicity in NB cells. Ixazomib inhibits dox-induced NF-κB activity and sensitizes NB cells to dox-induced apoptosis. More importantly, ixazomib demonstrated potent anti-tumor efficacy in vivo by enhancing dox-induced apoptosis in an orthotopic xenograft NB mouse model. Collectively, our study illustrates the anti-tumor efficacy of ixazomib in NB both alone and in combination with dox, suggesting that combination therapy including ixazomib with traditional therapeutic agents such as dox is a viable strategy that may achieve better outcomes for NB patients. PMID:27687684

  13. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation.

    PubMed

    Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E; Pertsemlidis, Alexander; Du, Liqin

    2014-05-15

    Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.

  14. Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells

    NASA Astrophysics Data System (ADS)

    Scalfi-Happ, Claudia; Jauss, Andrea; Hollricher, Olaf; Fulda, Simone; Hauser, Carmen; Steiner, Rudolf; Rück, Angelika

    2007-07-01

    The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the

  15. Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma.

    PubMed

    Song, Liping; Ara, Tasnim; Wu, Hong-Wei; Woo, Chan-Wook; Reynolds, C Patrick; Seeger, Robert C; DeClerck, Yves A; Thiele, Carol J; Sposto, Richard; Metelitsa, Leonid S

    2007-09-01

    Valpha24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.

  16. High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition.

    PubMed

    Bate-Eya, Laurel T; den Hartog, Ilona J M; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E; Westerhout, Ellen M; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J; Dolman, M Emmy M

    2016-05-10

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

  17. High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

    PubMed Central

    Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

    2016-01-01

    The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

  18. IGF2 expression is a marker for paraganglionic/SIF cell differentiation in neuroblastoma.

    PubMed Central

    Hedborg, F.; Ohlsson, R.; Sandstedt, B.; Grimelius, L.; Hoehner, J. C.; Pählman, S.

    1995-01-01

    Neuroblastoma is a childhood tumor of the sympathetic nervous system. Observations in the Beckwith-Wiedemann syndrome suggest that sympathetic embryonal cells with an abundant expression of the insulin-like growth factor 2 gene (IGF2) may be involved in the genesis of low-malignant infant neuroblastomas. We have therefore compared the cell type-specific IGF2 expression of the human sympathetic nervous system during early development with that of neuroblastoma. An abundant expression in normal sympathetic tissue was specific to extra-adrenal chromaffin cells, ie, paraganglia and small intensely fluorescent (SIF) cells, whereas sympathetic neuronal cells were IGF2-negative. A subpopulation of neuroblastomas expressed IGF2, which correlated with an early age at diagnosis, an extra-adrenal tumor origin, and severe hemodynamic signs of catecholamine secretion. Histologically IGF2-expressing tumors displayed a lobular growth pattern, and expression was restricted to the most mature and least proliferative cells. Typically, these cells were morphologically and histochemically similar to paraganglia/SIF cells and formed distinct ring-like zones in the center of the lobules around a core of apoptosis-like tumor cells. The similarities found between IGF2-expressing neuroblastoma cells and paraganglia/SIF cells in terms of histological features, anatomical origin, and age-dependent growth suggest a paraganglionic/SIF cell lineage of most infant tumors and also of extra-adrenal tumors diagnosed after infancy. Furthermore, since paraganglia/SIF cells undergo postnatal involution, the same cellular mechanism may be responsible for spontaneous regression in infant neuroblastoma. Images Figure 2 Figure 3 p839-a Figure 4 PMID:7717451

  19. Antibiotic drug tigecycline reduces neuroblastoma cells proliferation by inhibiting Akt activation in vitro and in vivo.

    PubMed

    Zhong, Xiaoxia; Zhao, Erhu; Tang, Chunling; Zhang, Weibo; Tan, Juan; Dong, Zhen; Ding, Han-Fei; Cui, Hongjuan

    2016-06-01

    As the first member of glycylcycline bacteriostatic agents, tigecycline is approved as a novel expanded-spectrum antibiotic, which is clinically available. However, accumulating evidence indicated that tigecycline was provided with the potential application in cancer therapy. In this paper, tigecycline was shown to exert an anti-proliferative effect on neuroblastoma cell lines. Furthermore, it was found that tigecycline induced G1-phase cell cycle arrest instead of apoptosis by means of Akt pathway inhibition. In neuroblastoma cell lines, the Akt activator insulin-like growth factor-1 (hereafter referred to as IGF-1) reversed tigecycline-induced cell cycle arrest. Besides, tigecycline inhibited colony formation and suppressed neuroblastoma cells xenograft formation and growth. After tigecycline treatment in vivo, the Akt pathway inhibition was confirmed as well. Collectively, our data provided strong evidences that tigecycline inhibited neuroblastoma cells growth and proliferation through the Akt pathway inhibition in vitro and in vivo. In addition, these results were supported by previous studies concerning the application of tigecycline in human tumors treatment, suggesting that tigecycline might act as a potential candidate agent for neuroblastoma treatment.

  20. Disassembly of microtubules and inhibition of neurite outgrowth, neuroblastoma cell proliferation, and MAP kinase tyrosine dephosphorylation by dibenzyl trisulphide.

    PubMed

    Rösner, H; Williams, L A; Jung, A; Kraus, W

    2001-08-22

    Dibenzyl trisulphide (DTS), a main lipophilic compound in Petiveria alliacea L. (Phytolaccaceae), was identified as one of the active immunomodulatory compounds in extracts of the plant. To learn more about its biological activities and molecular mechanisms, we conducted one-dimensional NMR interaction studies with bovine serum albumin (BSA) and tested DTS and related compounds in two well-established neuronal cell-and-tissue culture systems. We found that DTS preferentially binds to an aromatic region of BSA which is rich in tyrosyl residues. In SH-SY5Y neuroblastoma cells, DTS attenuates the dephosphorylation of tyrosyl residues of MAP kinase (erk1/erk2). In the same neuroblastoma cell line and in Wistar 38 human lung fibroblasts, DTS causes a reversible disassembly of microtubules, but it did not affect actin dynamics. Probably due to the disruption of the microtubule dynamics, DTS also inhibits neuroblastoma cell proliferation and neurite outgrowth from spinal cord explants. Related dibenzyl compounds with none, one, or two sulphur atoms were found to be significantly less effective. These data confirmed that the natural compound DTS has a diverse spectrum of biological properties, including cytostatic and neurotoxic actions in addition to immunomodulatory activities.

  1. Induction of thrombospondin 1 by retinoic acid is important during differentiation of neuroblastoma cells.

    PubMed Central

    Castle, V P; Ou, X; O'Shea, S; Dixit, V M

    1992-01-01

    Neuroblastoma, a malignant neoplasm that arises in the adrenal medulla or sympathetic ganglion, is one of the most common solid tumors of childhood. Reports that neuroblastomas spontaneously mature to form benign ganglioneuromas have prompted investigations into the efficacy of using agents that induce neuronal differentiation in the treatment of this malignancy. Retinoic acid is one agent in particular that has been shown to induce growth inhibition and terminal differentiation of neuroblastoma cell lines in vitro. Using the human neuroblastoma cell line SMH-KCNR, we have investigated the role of the extracellular matrix protein thrombospondin in retinoic acid induced neuroblastoma differentiation. Treatment with retinoic acid results in a rapid induction (within 4 h) of thrombospondin (TSP) message which is independent of intervening protein synthesis and superinducible in the presence of cycloheximide. This suggests that TSP functions as a retinoic acid inducible immediate early response gene. A concomitant increase in both cell associated and soluble forms of TSP protein can be detected within 24 h of retinoic acid treatment. A functional role for TSP in SMH-KCNR differentiation was established in experiments which showed that exposure to anti-TSP monoclonal antibodies delay retinoic acid differentiation for 48 h. At the time the cells overcome the effects of TSP inhibition, laminin production becomes maximal. Treatment of the cells with a combination of anti-TSP and antilaminin antibodies results in complete inhibition of differentiation. Images PMID:1430209

  2. In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody

    NASA Astrophysics Data System (ADS)

    Wang, Chung-Hao; Huang, Yao-Jhang; Chang, Chia-Wei; Hsu, Wen-Ming; Peng, Ching-An

    2009-08-01

    Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm-2 for 10 min which was then maintained at 6 W cm-2 for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

  3. Depletion of TFAP2E attenuates adriamycin-mediated apoptosis in human neuroblastoma cells.

    PubMed

    Hoshi, Reina; Watanabe, Yosuke; Ishizuka, Yoshiaki; Hirano, Takayuki; Nagasaki-Maeoka, Eri; Yoshizawa, Shinsuke; Uekusa, Shota; Kawashima, Hiroyuki; Ohashi, Kensuke; Sugito, Kiminobu; Fukuda, Noboru; Nagase, Hiroki; Soma, Masayoshi; Ozaki, Toshinori; Koshinaga, Tsugumichi; Fujiwara, Kyoko

    2017-04-01

    Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system and accounts for approximately 15% of all pediatric cancer-related deaths. To newly identify gene(s) implicated in the progression of neuroblastoma, we investigated aberrantly methylated genomic regions in mouse skin tumors. Previously, we reported that TFAP2E, a member of activator protein-2 transcription factor family, is highly methylated within its intron and its expression is strongly suppressed in mouse skin tumors compared with the normal skin. In the present study, we analyzed public data of neuroblastoma patients and found that lower expression levels of TFAP2E are significantly associated with a shorter survival. The data indicate that TFAP2E acts as a tumor suppressor of neuroblastoma. Consistent with this notion, TFAP2E-depleted neuroblastoma NB1 and NB9 cells displayed a substantial resistance to DNA damage arising from adriamycin (ADR), cisplatin (CDDP) and ionizing radiation (IR). Silencing of TFAP2E caused a reduced ADR-induced proteolytic cleavage of caspase-3 and PARP. Of note, compared with the untransfected control cells, ADR-mediated stimulation of CDK inhibitor p21WAF1 was markedly upregulated in TFAP2E‑knocked down cells. Therefore, our present findings strongly suggest that TFAP2E has a pivotal role in the regulation of DNA damage response in NB cells through the induction of p21WAF1.

  4. Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

    PubMed Central

    Thiele, C J; Cohen, P S; Israel, M A

    1988-01-01

    We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription. Images PMID:3380093

  5. Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model.

    PubMed

    Chakrabarti, Lina; Morgan, Clifford; Sandler, Anthony D

    2015-01-01

    Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.

  6. The Ews/Fli-1 fusion gene changes the status of p53 in neuroblastoma tumor cell lines.

    PubMed

    Rorie, Checo J; Weissman, Bernard E

    2004-10-15

    One hallmark of Ewing's sarcoma/peripheral neuroectodermal tumors is the presence of the Ews/Fli-1 chimeric oncogene. Interestingly, infection of neuroblastoma tumor cell lines with Ews/Fli-1 switches the differentiation program of neuroblastomas to Ewing's sarcoma/peripheral neuroectodermal tumors. Here we examined the status of cytoplasmically sequestered wt-p53 in neuroblastomas after stable expression of Ews/Fli-1. Immunofluorescence revealed that in the neuroblastoma-Ews/Fli-1 infectant cell lines, p53 went from a punctate-pattern of cytoplasmic sequestration to increased nuclear localization. Western blot analysis revealed that PARC was down-regulated in one neuroblastoma cell line but not expressed in the second. Therefore, decreased PARC expression could not fully account for relieving p53 sequestration in the neuroblastoma tumor cells. Neuroblastoma-Ews/Fli-1 infectant cell lines showed marked increases in p53 protein expression without transcriptional up-regulation. Interestingly, p53 was primarily phosphorylated, without activation of its downstream target p21(WAF1). Western blot analysis revealed that whereas MDM2 gene expression does not change, p14(ARF), a negative protein regulator of MDM2, increases. These observations suggest that the downstream p53 pathway may be inactivated as a result of abnormal p53. We also found that p53 has an extended half-life in the neuroblastoma-Ews/Fli-1 infectants despite the retention of a wild-type sequence in neuroblastoma-Ews/Fli-1 infectant cell lines. We then tested the p53 response pathway and observed that the neuroblastoma parent cells responded to genotoxic stress, whereas the neuroblastoma-Ews/Fli-1 infectants did not. These results suggest that Ews/Fli-1 can directly abrogate the p53 pathway to promote tumorigenesis. These studies also provide additional insight into the relationship among the p53 pathway proteins.

  7. P-selectin mediates adhesion of platelets to neuroblastoma and small cell lung cancer.

    PubMed Central

    Stone, J P; Wagner, D D

    1993-01-01

    Activated platelets and stimulated endothelial cells express P-selectin, an integral membrane protein receptor that binds monocytes and neutrophils. P-selectin mediates adhesion to glycoproteins with carbohydrate structures containing sialyl-Lewis X. Since many carcinoma cells also express these carbohydrate structures and are known to interact with platelets, we asked whether P-selectin may mediate this interaction. Both small cell lung cancer and neuroblastoma cell lines bound to activated platelets, and this interaction was blocked with inhibitory anti-P-selectin antibodies and by pretreatment of these cancer cells with neuraminidase or trypsin. Platelet binding to the small cell lung cancer cells was not inhibited with anti-GP IIb-IIIa antibody or Arg-Gly-Asp-Ser peptide. Pretreatment of the neuroblastoma cells with inhibitors of N-linked carbohydrate biosynthesis had little effect on binding to P-selectin, indicating that relevant carbohydrate ligand(s) may be O-linked. In addition, lipospheres containing P-selectin specifically bound to cryostat sections derived from a small cell lung tumor and two neuroblastoma tumors, but not to sections of normal lung. These observations demonstrate that P-selectin mediates binding of platelets to small cell lung cancer and to neuroblastoma and suggest a possible role for this lectin in metastasis. Images PMID:7688763

  8. Standardization of the Immunocytochemical Detection of Neuroblastoma Cells in Bone Marrow

    PubMed Central

    Swerts, Katrien; Ambros, Peter F.; Brouzes, Chantal; Navarro, José M. Fernandez; Gross, Nicole; Rampling, Dyanne; Schumacher-Kuckelkorn, Roswitha; Sementa, Angela R.; Ladenstein, Ruth; Beiske, Klaus

    2005-01-01

    Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 × 106 cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 × 106 mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 × 106. This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma. PMID:15956022

  9. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  10. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    SciTech Connect

    Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  11. Synapse formation between clonal neuroblastoma X glioma hybrid cells and striated muscle cells.

    PubMed Central

    Nelson, P; Christian, C; Nirenberg, M

    1976-01-01

    Clonal neuroblastoma X glioma hybrid cells were shown to form synapses with cultured, striated muscle cells. The properties of the synapses between hybrid and muscle cells were similar to those of the normal, neuromuscular synapse at an early stage of development. The number of synapses formed and the efficiency of transmission across synapses were found to be regulated, apparently independently, by components in the culture medium. Under appropriate conditions synapses were found with 20% of the hybrid-muscle cell pairs examined; thus, the hybrid cells form synapses with relatively high frequency. Images PMID:1061105

  12. Stimulation of phosphatidic acid of calcium influx and cyclic GMP synthesis in neuroblastoma cells.

    PubMed

    Ohsako, S; Deguchi, T

    1981-11-10

    Phosphatidic acid added to the medium markedly elevated intracellular cyclic GMP content in cultured neuroblastoma N1E 115 cells. There was a significant elevation of cyclic GMP with 1 micrograms/ml and a maximum (70-fold) elevation with 100 micrograms/ml of phosphatidic acid. Other natural phospholipids did not increase, or increased only slightly, the cyclic GMP content in the cells. The elevation of cyclic GMP content by phosphatidic acid was absolutely dependent on extracellular calcium. Phosphatidic acid stimulated the influx of calcium into neuroblastoma cells 2- to 5-fold. The pattern of the calcium influx induced by phosphatidic acid was comparable to that of cyclic GMP elevation. The stimulation of calcium influx by phosphatidic acid was also observed in cultured heart cells, indicating that phosphatidic acid acts as a calcium ionophore or opens a specific calcium-gate in a variety of cell membranes. Treatment of neuroblastoma cells with phospholipase C increased 32Pi labeling of phosphatidic acid, stimulated the influx of calcium, and elevated the cyclic GMP content in the cells. Thus exogenous as well as endogenous phosphatidic acid stimulates the translocation of calcium across cell membranes and, as a consequence, induces the synthesis of cyclic GMP in the neuroblastoma cells.

  13. Comparison of the Microtubule Proteins of Neuroblastoma Cells, Brain, and Chlamydomonas Flagella

    PubMed Central

    Olmsted, J. B.; Witman, G. B.; Carlson, K.; Rosenbaum, Joel L.

    1971-01-01

    Intact A microtubules isolated from outer doublet microtubules of Chlamydomonas flagella contain two separable proteins (tubulins) that differ in molecular weight and in amino-acid composition. The microtubule protein isolated from brain or neuroblastoma cells also has two electrophoretically distinct tubulins. Although the two tubulins of brain and neuroblastoma cells are electrophoretically similar to each other, only one of these tubulins migrates with the flagellar tubulins. This is the first evidence that (a) isolated, morphologically intact, single microtubules from flagella contain at least two different tubulins, and (b) at least one of these tubulins differs from tubulins that are isolated from other sources. Images PMID:5289385

  14. Synergistic effect of choline and carnitine on acetylcholine synthesis in neuroblastoma NB-2a cells.

    PubMed

    Wawrzeńczyk, A; Nałecz, K A; Nałecz, M J

    1994-07-15

    An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 microM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.

  15. Methyl jasmonate downregulates expression of proliferating cell nuclear antigen and induces apoptosis in human neuroblastoma cell lines.

    PubMed

    Tong, Qiang-Song; Jiang, Guo-Song; Zheng, Li-Duan; Tang, Shao-Tao; Cai, Jia-Bin; Liu, Yuan; Zeng, Fu-Qing; Dong, Ji-Hua

    2008-07-01

    Recent evidence indicates that methyl jasmonate, a plant stress hormone, exhibits anticancer activity on human cancer cells. Whether methyl jasmonate could inhibit the growth of human neuroblastoma cells still, however, remains largely unknown. In this study, administration of methyl jasmonate to cultured neuroblastoma cell lines, SK-N-SH and BE(2)-C, resulted in a decrease of cell viability in a dose-dependent and time-dependent manner as demonstrated by MTT colorimetry and colony formation assay. The results from RT-PCR indicated that the expression of proliferating cell nuclear antigen, but not of cyclin D1, was downregulated by methyl jasmonate. Accordingly, the cell cycle of methyl jasmonate-treated neuroblastoma cells was arrested at the G0/G1 phase. Moreover, incubation of SK-N-SH and BE(2)-C cells with methyl jasmonate resulted in characteristic changes of apoptosis, as demonstrated by acridine orange-ethidium bromide (AO/EB) staining, Hoechst 33258 staining and flow cytometry. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of the inhibitors of apoptosis protein family, in neuroblastoma cells. These findings indicate that methyl jasmonate suppresses the growth of cultured human neuroblastoma cells associated with downregulation of proliferating cell nuclear antigen, and induces apoptosis accompanied by downregulation of the X-linked inhibitor of apoptosis protein and survivin, which lays the groundwork for further investigation into the mechanisms of methyl jasmonate-mediated anticancer activities.

  16. Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

    PubMed Central

    Martínez-Soto, Soledad; Legarra, Sheila; Pata-Merci, Noémie; Guegan, Justine; Danglot, Giselle; Bernheim, Alain; Meléndez, Bárbara; Rey, Juan A.; Castresana, Javier S.

    2014-01-01

    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells. PMID:25392930

  17. Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells

    PubMed Central

    Condorelli, F; Gnemmi, I; Vallario, A; Genazzani, A A; Canonico, P L

    2007-01-01

    Background and purpose: Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti-cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability. Experimental approach: Effects of HDAC inhibitors on survival of the I-type SK-N-BE and the N-type NB SH-SY5Y neuroblastoma cell lines were assessed by the MTT assay. Molecular pathways leading to this were examined by western blot, confocal microscopy and cytofluorometry. The mRNA levels of apoptotic mediators were assessed semi-quantitatively by RT-PCR. Tumour-suppressor p53 trans activity was assessed in EMSA experiments. HDAC inhibitors were also studied in cells subjected to plasmid-based p53 interference (p53i). Key results: HDAC inhibitors induced cell death via the mitochondrial pathway of apoptosis with recruitment of Bcl-2 family members. Bcl-2 overexpression rendered neuroblastoma cells resistant to HDAC inhibitor treatment. Low concentrations of HDAC inhibitors (0.9 mM) caused a G2 cell-cycle arrest and a marked upregulation of the p21/Waf1/Cip1 protein. HDAC inhibitors also activate the p53 protein via hyper-acetylation and nuclear re-localization, without affecting its protein expression. Accordingly, HDAC inhibitor-induced cell-killing and p21/Waf1/Cip1 upregulation is impaired in p53i-cells. Conclusions and implications: In neuroblastoma cells, HDAC inhibitors may overcome the resistance to classical chemotherapeutic drugs by restoring the p53 tumour-repressor function via its hyper-acetylation and nuclear migration, events usually impaired in such tumours. In neuroblastoma cells, HDAC inhibitors are not able to induce p21/Waf1/Cip1 in the absence of a functional p53. PMID:18059320

  18. Matrix metalloproteinase-9 and cell division in neuroblastoma cells and bone marrow macrophages.

    PubMed

    Sans-Fons, M Gloria; Sole, Sonia; Sanfeliu, Coral; Planas, Anna M

    2010-12-01

    Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages.

  19. A pilot study of tandem high-dose chemotherapy with stem cell rescue as consolidation for high-risk neuroblastoma: Children's Oncology Group study ANBL00P1.

    PubMed

    Seif, A E; Naranjo, A; Baker, D L; Bunin, N J; Kletzel, M; Kretschmar, C S; Maris, J M; McGrady, P W; von Allmen, D; Cohn, S L; London, W B; Park, J R; Diller, L R; Grupp, S A

    2013-07-01

    Increasing treatment intensity has improved outcomes for children with neuroblastoma. We performed a pilot study in the Children's Oncology Group to assess the feasibility and toxicity of a tandem myeloablative regimen without TBI supported by autologous CD34-selected peripheral blood stem cells. Forty-one patients with high-risk neuroblastoma were enrolled; eight patients did not receive any myeloablative consolidation procedure and seven received only one. Two patients out of 41 (4.9%) experienced transplant-related mortality. CD34 selection was discontinued after subjects were enrolled due to serious viral illness. From the time of study enrollment, the overall 3-year EFS and OS were 44.8 ± 9.6% and 59.2 ± 9.2% (N=41). These results demonstrate that tandem transplantation in the cooperative group setting is feasible and support a randomized comparison of single vs tandem myeloablative consolidation with PBSC support for high-risk neuroblastoma.

  20. Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse

    PubMed Central

    Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L.; Bown, Nick; Tweddle, Deborah A.

    2016-01-01

    Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ∼85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression. PMID:27888620

  1. Antibody-dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells.

    PubMed

    Mise, Naoko; Takami, Mariko; Suzuki, Akane; Kamata, Toshiko; Harada, Kazuaki; Hishiki, Tomoro; Saito, Takeshi; Terui, Keita; Mitsunaga, Tetsuya; Nakata, Mitsuyuki; Ikeuchi, Takayuki; Nakayama, Toshinori; Yoshida, Hideo; Motohashi, Shinichiro

    2016-03-01

    Anti-ganglioside GD2 antibodies mainly work through antibody-dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high-risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti-GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti-tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti-GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co-cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti-GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK-NKT cell contact or NK cell-dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell-based immunotherapy could be an appropriate candidate for anti-GD2 antibody therapy for neuroblastoma.

  2. Neuroblastoma: A neurochemical approach

    SciTech Connect

    Schor, N.F. )

    1991-07-01

    Neuroblastoma is among the most common malignancies of childhood. Despite greatly improved therapy for some pediatric tumors, the prognosis for children with metastatic neuroblastoma has not changed significantly in the past 10 years. With conventional chemotherapy, radiation therapy, and surgery, children with metastatic neuroblastoma have a 20% long-term survival rate. The authors describe here approaches to neuroblastoma that target its neuronal characteristics. On the one hand, the neurotransmitter receptors on the surface of the neuroblastoma cells and, on the other hand, specific isozymes that distinguish neuroblastoma cells from their normal counterparts are the focus of these experimental therapies. In the former case, specificity for tumor cells is effected by (1) selective protection of normal neuronal elements from toxicity, or (2) selective potentiation of toxicity for neural tumor cells. It is hoped that these strategies will be generalizable to other neural crest-derived tumors. 32 references.

  3. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    PubMed

    Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  4. NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

    SciTech Connect

    Shoji, Wataru; Suenaga, Yusuke; Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer; Yokoi, Sana; Nio, Masaki; Nakagawara, Akira

    2015-06-05

    NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase.

  5. Cell lines from MYCN transgenic murine tumours reflect the molecular and biological characteristics of human neuroblastoma.

    PubMed

    Cheng, Andy J; Cheng, Ngan Ching; Ford, Jette; Smith, Janice; Murray, Jayne E; Flemming, Claudia; Lastowska, Maria; Jackson, Michael S; Hackett, Christopher S; Weiss, William A; Marshall, Glenn M; Kees, Ursula R; Norris, Murray D; Haber, Michelle

    2007-06-01

    Overexpression of the human MYCN oncogene driven by a tyrosine hydroxylase promoter causes tumours in transgenic mice that recapitulate the childhood cancer neuroblastoma. To establish an in vitro model to study this process, a series of isogenic cell lines were developed from these MYCN-driven murine tumours. Lines were established from tumours arising in homozygous and hemizygous MYCN transgenic mice. Hemizygous tumours gave rise to cell lines growing only in suspension. Homozygous tumours gave rise to similar suspension lines as well as morphologically distinct substrate-adherent lines characteristic of human S-type neuroblastoma cells. FISH analysis demonstrated selective MYCN transgene amplification in cell lines derived from hemizygous mice. Comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) analysis confirmed a range of neuroblastoma-associated genetic changes in the various lines, in particular, gain of regions syntenic with human 17q. These isogenic lines together with the transgenic mice thus represent valuable models for investigating the biological characteristics of aggressive neuroblastoma.

  6. PrP-dependent cell adhesion in N2a neuroblastoma cells.

    PubMed

    Mangé, Alain; Milhavet, Ollivier; Umlauf, David; Harris, David; Lehmann, Sylvain

    2002-03-13

    The cellular isoform of prion protein (PrP(C)) is a ubiquitous glycoprotein expressed by most tissues and with a biological function yet to be determined. Here, we have used a neuroblastoma cell model to investigate the involvement of PrP in cell adhesion. Incubation of single cell suspension induced cell-cell adhesion and formation of cell aggregates. Interestingly, cells overexpressing PrP exhibit increased cation-independent aggregation. Aggregation was reduced after phosphatidylinositol-specific phospholipase C release of the protein and by pre-incubation of cells with an antibody raised against the N-terminal part of PrP(C). Our paradigm allows the study of the function of PrP as an intercellular adhesion molecule and a cell surface ligand or receptor.

  7. SILENCING GASTRIN-RELEASING PEPTIDE RECEPTOR SUPPRESSES KEY REGULATORS OF AEROBIC GLYCOLYSIS IN NEUROBLASTOMA CELLS

    PubMed Central

    Rellinger, Eric J.; Romain, Carmelle; Choi, SunPhil; Qiao, Jingbo; Chung, Dai H.

    2015-01-01

    Background Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin-releasing peptide receptor (GRP-R) signaling regulates glucose metabolism. Procedure Neuroblastoma cell lines, BE(2)-C and SK-N-AS, were used. PCR microararay for glucose metabolism was performed on GRP-R silenced cells. Target protein expression was validated using Western blotting and VEGF ELISA. Cobalt chloride (CoCl2) was used to induce chemical hypoxia. Efficacy of targeting PDK regulation in neuroblastoma was assessed using dichloroacetate (DCA) by conducting cell viability assays and Western blotting for apoptotic markers. Results Silencing GRP-R decreased HIF-1α expression and blocked VEGF expression and secretion in both normoxic and CoCl2 induced hypoxia. PCR array analysis identified that GRP-R silencing reduced PDK4 and increased PDP2 mRNA expression. These findings were validated by Western blotting. CoCl2 induced hypoxia increased VEGF secretion, HIF-1α, and PDK4 expression. PDK4 silencing decreased HIF-1α expression and VEGF expression and secretion. DCA treatment decreased BE(2)-C and SK-N-AS proliferation while promoting cell death. GRP-R silencing and DCA treatment synergistically halted BE(2)-C proliferation. Conclusions We report that GRP-R regulates glucose metabolism in neuroblastoma by modulating HIF-1α, PDK4 and PDP2. PDK4 regulates glucose metabolism, in part, via regulation of HIF-1α. Synergistic consequences of GRP-R inhibition and DCA treatment may suggest a novel therapeutic strategy for the treatment of aggressive neuroblastoma. PMID:25630799

  8. In vitro differentiation of human neuroblastoma cells induced by sodium phenylacetate.

    PubMed

    Cinatl, J; Cinatl, J; Mainke, M; Weissflog, A; Rabenau, H; Kornhuber, B; Doerr, H W

    1993-06-15

    Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.

  9. 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.

  10. Exposure to low level GSM 935 MHZ radiofrequency fields does not induce apoptosis in proliferating or differentiated murine neuroblastoma cells.

    PubMed

    Moquet, J; Ainsbury, E; Bouffler, S; Lloyd, D

    2008-01-01

    The aim of this study was to investigate whether radiofrequency (RF) fields characteristic of mobile phones at non-thermal levels can induce apoptosis in murine neuroblastoma (N2a) cells in both proliferating and differentiated states. Cells were exposed continuously for 24 h to one of the three 935-MHz RF signals: global system for mobile communication (GSM) basic, GSM talk and a continuous wave, unmodulated signal; all at a specific energy absorption rate of 2 W kg(-1). The measured increase in temperature of the cells due to the RF fields was around 0.06 degrees C. At a number of time points between 0 and 48 h post-exposure, the cells were assessed for apoptosis under a fluorescence microscope using three independent assays: Annexin V, caspase activation and in situ end-labelling. No statistically significant differences in apoptosis levels were observed between the exposed and sham-exposed cells using the three assays at any time point post-exposure. These data suggest that RF exposures, characteristic of GSM mobile phones, do not significantly affect the apoptosis levels in proliferating and differentiated murine neuroblastoma cell line N2a.

  11. Phenylacetate synergizes with retinoic acid in inducing the differentiation of human neuroblastoma cells.

    PubMed

    Sidell, N; Wada, R; Han, G; Chang, B; Shack, S; Moore, T; Samid, D

    1995-02-08

    Phenylacetate, a natural metabolite of phenylalanine which was originally described as a plant growth hormone, has recently gained attention as a possible differentiation inducer for a variety of human tumor cell types. This interest prompted us to assess the ability of sodium phenylacetate (NaPA) to promote the differentiation of human neuroblastoma cells, both alone and in combination with retinoic acid (RA), a known inducer of neuroblastoma differentiation and maturation. Using the LA-N-5 cell line, we have determined that NaPA can stimulate the differentiation of neuroblastoma cells, as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity and reduction of N-myc expression. Furthermore, NaPA and RA synergized in inducing differentiation, in that combination treatment resulted in cessation of cell growth along with morphologic and biochemical changes indicative of the loss of malignant properties. We have determined that NaPA can markedly enhance mRNA levels of the nuclear RA receptor-beta (RAR beta) in LA-N-5 cells prior to morphologic or other phenotypic changes induced by this compound. This effect appeared to be distinct from the ability of NaPA to alter tumor cell lipid metabolism via inhibition of protein isoprenylation. Thus among its varied effects on LA-N-5 cells, NaPA appears to interact with the RA pathway at the nuclear level by up-regulating RAR beta expression.

  12. Role of CXCL13-CXCR5 crosstalk between malignant neuroblastoma cells and Schwannian stromal cells in neuroblastic tumors.

    PubMed

    Del Grosso, Federica; Coco, Simona; Scaruffi, Paola; Stigliani, Sara; Valdora, Francesca; Benelli, Roberto; Salvi, Sandra; Boccardo, Simona; Truini, Mauro; Croce, Michela; Ferrini, Silvano; Longo, Luca; Tonini, Gian Paolo

    2011-07-01

    Neuroblastoma is a stroma-poor (SP) aggressive pediatric cancer belonging to neuroblastic tumors, also including ganglioneuroblastoma and ganglioneuroma, two stroma-rich (SR) less aggressive tumors. Our previous gene-expression profiling analysis showed a different CXCL13 mRNA expression between SP and SR tumors. Therefore, we studied 13 SP and 13 SR tumors by reverse transcription quantitative real-time PCR (RT-qPCR) and we found that CXCR5b was more expressed in SP than in SR and CXCL13 was predominantly expressed in SR tumors. Then, we isolated neuroblastic and Schwannian stromal cells by laser capture microdissection and we found that malignant neuroblasts express CXCR5b mRNA, whereas Schwannian stromal cells express CXCL13. Immunohistochemistry confirmed that stroma expresses CXCL13 but not CXCR5. To better understand the role of CXCL13 and CXCR5 in neuroblastic tumors we studied 11 neuroblastoma cell lines and we detected a heterogeneous expression of CXCL13 and CXCR5b. Interestingly, we found that only CXCR5b splice variant was expressed in both tumors and neuroblastoma lines, whereas CXCR5a was never detected. Moreover, we found that neuroblastoma cells expressing CXCR5 receptor migrate toward a source of recombinant CXCL13. Lastly, neuroblastoma cells induced to glial cell differentiation expressed CXCL13 mRNA and protein. The chemokine released in the culture medium was able to stimulate chemotaxis of LA1-5S neuroblastoma cells. Collectively, our data suggest that CXCL13 produced by stromal cells may contribute to the generation of an environment in which the malignant neuroblasts are retained, thus limiting the possible development of metastases in patients with SR tumor.

  13. Mouse neuroblastoma cell based model and the effect of epileptic events on calcium oscillations and neural spikes

    NASA Astrophysics Data System (ADS)

    Kim, Suhwan; Baek, Juyeong; Jung, Unsang; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-05-01

    Recently, Mouse neuroblastoma cells are considered as an attractive model for the study of human neurological and prion diseases, and intensively used as a model system in different areas. Among those areas, differentiation of neuro2a (N2A) cells, receptor mediated ion current, and glutamate induced physiological response are actively investigated. The reason for the interest to mouse neuroblastoma N2A cells is that they have a fast growing rate than other cells in neural origin with a few another advantages. This study evaluated the calcium oscillations and neural spikes recording of mouse neuroblastoma N2A cells in an epileptic condition. Based on our observation of neural spikes in mouse N2A cell with our proposed imaging modality, we report that mouse neuroblastoma N2A cells can be an important model related to epileptic activity studies. It is concluded that the mouse neuroblastoma N2A cells produce the epileptic spikes in vitro in the same way as produced by the neurons or the astrocytes. This evidence advocates the increased and strong level of neurotransmitters release by enhancement in free calcium using the 4-aminopyridine which causes the mouse neuroblastoma N2A cells to produce the epileptic spikes and calcium oscillation.

  14. Advances in neuroblastoma research

    SciTech Connect

    Evans, A.E.; D'Angio, G.J.; Seeger, R.C.

    1985-01-01

    This book contains over 50 papers. Some of the titles are: Studies on the expression of the amplified domain in human neuroblastoma cells; Comparison studies of oncogenes in retinoblastoma and neuroblastoma; Chromosome abnormalities, gene amplification and tumor progression; and Peripheral neuroepithelioma: Genetic analysis of tumor derived cell lines.

  15. Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation.

    PubMed Central

    Abemayor, E; Sidell, N

    1989-01-01

    Neuroblastoma is a childhood solid tumor composed of primitive cells derived from precursors of the autonomic nervous system. This neoplasm has the highest rate of spontaneous regression of all cancer types and has been noted to undergo spontaneous and chemically induced differentiation into elements resembling mature nervous tissue. As such, neuroblastoma has been a prime model system for the study of neuronal differentiation and the process of cancer cell maturation. In this paper we review those agents that have been described to induce the differentiation of neuroblastoma, with an emphasis on the effects and possible mechanisms of action of a group of related compounds, the retinoids. With this model system and the availability of subclones that are both responsive and resistant to chemically induced differentiation, fundamental questions regarding the mechanisms and processes underlying cell maturation have become more amenable to in vitro study. Images FIGURE 1. A FIGURE 1. B FIGURE 1. C FIGURE 2. A FIGURE 2. B PMID:2538324

  16. 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

  17. 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

  18. SGO1 is involved in the DNA damage response in MYCN-amplified neuroblastoma cells

    PubMed Central

    Murakami-Tonami, Yuko; Ikeda, Haruna; Yamagishi, Ryota; Inayoshi, Mao; Inagaki, Shiho; Kishida, Satoshi; Komata, Yosuke; Jan Koster, J K; Takeuchi, Ichiro; Kondo, Yutaka; Maeda, Tohru; Sekido, Yoshitaka; Murakami, Hiroshi; Kadomatsu, Kenji

    2016-01-01

    Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of SGO1 impaired proliferation and induced DNA damage followed by a senescence-like phenotype only in MYCN-overexpressing neuroblastoma cells. In these cells, SGO1 knockdown induced DNA damage, even during interphase, and this effect was independent of cohesin. Furthermore, MYCN-promoted SGO1 transcription and SGO1 expression tended to be higher in MYCN- or MYC-overexpressing cancers. Together, these findings indicate that SGO1 plays a role in the DNA damage response in interphase. Therefore, we propose that SGO1 represents a potential molecular target for treatment of MYCN-amplified neuroblastoma. PMID:27539729

  19. 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

  20. Valproic Acid Increases CD133 Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma

    PubMed Central

    Khalil, Mohamed Ashraf; Hraběta, Jan; Groh, Tomáš; Procházka, Pavel; Doktorová, Helena; Eckschlager, Tomáš

    2016-01-01

    Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through its action as a histone deacetylase inhibitor. CD133 is considered to be a cancer stem cell marker in several tumors including neuroblastoma. CD133 transcription is strictly regulated by epigenetic modifications. We evaluated the epigenetic effects of treatment with 1mM VPA and its influence on the expression of CD133 in four human neuroblastoma cell lines. Chemoresistance and cell cycle of CD133+ and CD133− populations were examined by flow cytometry. We performed bisulfite conversion followed by methylation-sensitive high resolution melting analysis to assess the methylation status of CD133 promoters P1 and P3. Our results revealed that VPA induced CD133 expression that was associated with increased acetylation of histones H3 and H4. On treatment with VPA and cytostatics, CD133+ cells were mainly detected in the S and G2/M phases of the cell cycle and they showed less activated caspase-3 compared to CD133− cells. UKF-NB-3 neuroblastoma cells which express CD133 displayed higher colony and neurosphere formation capacities when treated with VPA, unlike IMR-32 which lacks for CD133 protein. Induction of CD133 in UKF-NB-3 was associated with increased expression of phosphorylated Akt and pluripotency transcription factors Nanog, Oct-4 and Sox2. VPA did not induce CD133 expression in cell lines with methylated P1 and P3 promoters, where the CD133 protein was not detected. Applying the demethylating agent 5-aza-2’-deoxycytidine to the cell lines with methylated promoters resulted in CD133 re-expression that was associated with a drop in P1 and P3 methylation level. In conclusion, CD133 expression in neuroblastoma can be regulated by histone acetylation and/or methylation of its CpG promoters. VPA can induce CD133+ cells which display high proliferation potential and low sensitivity to cytostatics in neuroblastoma. These results give new insight into the possible

  1. Synthesis and turnover of plasma-membrane proteins and glycoproteins in a neuroblastoma cell line.

    PubMed Central

    Mathews, R A; Johnson, T C; Hudson, J E

    1976-01-01

    A kinetic analysis of the appearance of 14C-labelled proteins in the surface membranes isolated from exponentially growing neuroblastoma cells (N2a) showed that the total membrane proteins reached a steady-state specific radioactivity in 18-20 h. However, examination of individual protein bands resolved by sodium dodecyl sulphate-urea-polyacrylamide-gel electrophoresis illustrated that differences in the kinetics of specific surface-membrane proteins could be detected. Although most of the protein bands reached a steady-state specific radioactivity at a time similar to that for total membrane proteins, at least two bands (mol. wt. 180000 and 130000) attained the steady-state within 8-10 h. It was shown by the use of dual-labelling techniques that these two protein bands turned over in the surface membranes of neuroblastoma N2a cells at least 180 and 150% faster than the total membrane protein. These two proteins were glycosylated and located on the outer surface of the cells, since they were labelled with radioactive carbohydrates and readily removed by treatment of the intact neuroblastoma cell with proteinases. PMID:1275913

  2. Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells.

    PubMed

    Celia, Christian; Trapasso, Elena; Locatelli, Marcello; Navarra, Michele; Ventura, Cinzia Anna; Wolfram, Joy; Carafa, Maria; Morittu, Valeria Maria; Britti, Domenico; Di Marzio, Luisa; Paolino, Donatella

    2013-12-01

    Citrus extracts, particularly bergamot essential oil (BEO) and its fractions, have been found to exhibit anticancer efficacy. However, the poor water solubility, low stability and limited bioavailability have prevented the use of BEO in cancer therapy. To overcome such drawbacks, we formulated BEO liposomes that improved the water solubility of the phytocomponents and increased their anticancer activity in vitro against human SH-SY5Y neuroblastoma cells. The results warrant further investigation of BEO liposomes for in vivo applications.

  3. Inducing trauma into neuroblastoma cells and synthetic neural networks using optical tweezers

    NASA Astrophysics Data System (ADS)

    Schneider, Patrick William

    The laser tweezers have become a very useful tool in the fields of physics, chemistry, and biology. My intent is to use the laser tweezers to induce trauma into neuroblastoma cells, cells that resemble neural cells when treated with retinoic acid, to try to surmise what happens when neural cells and networks are disrupted or destroyed. The issues presented will deal with the obtaining, maintenance, and differentiation of the cells, as well as the inner operations of the laser tweezers themselves, and what kind of applications it has been applied to, as well as to my work in this project.

  4. Activated β-catenin forces N2A cell-derived neurons back to tumor-like neuroblasts and positively correlates with a risk for human neuroblastoma.

    PubMed

    Zhi, Feng; Gong, Guangming; Xu, Yan; Zhu, Yan; Hu, Die; Yang, Yilin; Hu, Yiqiao

    2012-01-01

    Neuroblastoma is an embryonic malignancy arising from neuroblasts. The mechanisms that regulate the origination of neuroblastoma are still not very clear. In this study, we revealed that 6-bromoindirubin 3'-oxime (BIO), a specific GSK-3β inhibitor, promoted N2A cells-derived neurons to become tumor-like neuroblasts. Moreover, constitutively activated β-catenin (S33Y) also promoted this process, whereas, silencing endogenous expression of β-catenin abolished BIO-induced effects. These results implicated the potential relationship between the Wnt/β-catenin signaling and neuroblastoma formation. Indeed, we found that the amount of β-catenin in nucleus, which indicated the activation of Wnt/β-catnin signaling, was accumulated in human neuroblastoma specimens and positively correlated with clinical risk of neuroblastoma. These results give us a new sight into the neuroblastoma initiation and progression, and provide a potential drug target for neuroblastoma treatment.

  5. 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

  6. How Is Neuroblastoma Diagnosed?

    MedlinePlus

    ... Neuroblastoma Early Detection, Diagnosis, and Staging How Is Neuroblastoma Diagnosed? Neuroblastomas are usually found when a child ... Ask Your Child’s Doctor About Neuroblastoma? More In Neuroblastoma About Neuroblastoma Causes, Risk Factors, and Prevention Early ...

  7. Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL

    PubMed Central

    1979-01-01

    Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus- endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1. PMID:457774

  8. Transcriptional upregulation of retinoic acid receptor beta (RAR beta) expression by phenylacetate in human neuroblastoma cells.

    PubMed

    Sidell, N; Chang, B; Yamashiro, J M; Wada, R K

    1998-02-25

    Sodium phenylacetate (NaPA) has been shown to synergize with retinoic acid (RA) in inducing the differentiation of human neuroblastoma cells. Our studies indicated that NaPA can impact on the RA differentiation program by upregulating nuclear retinoic acid receptor-beta (RAR beta) expression. We have found that NaPA does not alter the half-life of RAR beta mRNA; thus, increased stability of mRNA levels does not contribute to NaPA induction. In contrast, NaPA was able to specifically activate a reporter gene construct (delta SV beta RE-CAT) which contains a retinoic acid response element (RARE beta) that is located in the RAR beta promoter. Activation of delta SV beta RE-CAT by NaPA also occurred in neuroblastoma cells cotransfected with a nuclear retinoic acid receptor expression vector, demonstrating the independence of this activation on cellular RAR levels. Taken together, our findings suggest that induction of RAR beta by NaPA is regulated at the level of transcription and mediated through the retinoic acid response element, RARE beta. This effect may account, at least in part, for the strong synergy between NaPA and RA in promoting neuroblastoma differentiation.

  9. Microarray expression analysis of MYCN-amplified neuroblastoma cells after inhibition of CDK2.

    PubMed

    Song, H; Wu, F; Li, S; Wang, Z; Liu, X; Cui, Y; Lin, C

    2017-03-03

    The study was aimed to explore the underlying molecular mechanisms of CDK2 inhibition in neuroblastoma by bioinformatics analysis. Gene expression profile GSE16480 was downloaded from the Gene Expression Omnibus. The differentially expressed genes (DEGs) were identified from IMR32 between each time point and average expression of all time points. Gene significance was calculated using dSVDsig algorithm of dnet package. Protein-protein interaction (PPI) network was built. Then, integrated with gene significance, a core PPI network was detected by dNetPipeline algorithm in dnet package. Finally, pathway enrichment analysis was performed for genes in network. Total 1524 DEGs were identified. CCNA2 (cyclin A2), EXO1 (exonuclease 1), RAD51AP1 (RAD51 associated protein 1), TOP2A (topoisomerase (DNA) II alpha) and CDK1 (cyclin-dependent kinase 1) were selected as DEGs with higher connectivity after PPI network analysis. In the network, CCNA2, CDK1, BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B) and CCNB1 (cyclin B1) were involved in cell cycle pathway. Additionally, CCNB1, CDK1, CCNE2 (Cyclin E2), and RRM2B (ribonucleotide reductase subunit M2B) were involved in p53 signaling pathway. Cell cycle and p53 signaling pathway were closely associated with neuroblastoma after CDK2 inhibition. The DEGs, such as CCNA2, CCNB1, CDK1 and RRM2B may be the potential targets for neuroblastoma.

  10. 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.

  11. Evidence of asymmetric cell division and centrosome inheritance in human neuroblastoma cells.

    PubMed

    Izumi, Hideki; Kaneko, Yasuhiko

    2012-10-30

    Asymmetric cell division (ACD) is believed to be a physiological event that occurs during development and tissue homeostasis in a large variety of organisms. ACD produces two unequal daughter cells, one of which resembles a multipotent stem and/or progenitor cell, whereas the other has potential for differentiation. Although recent studies have shown that the balance between self-renewal and differentiation potentials is precisely controlled and that alterations in the balance may lead to tumorigenesis in Drosophila neuroblasts, it is largely unknown whether human cancer cells directly show ACD in an evolutionarily conserved manner. Here, we show that the conserved polarity/spindle protein NuMA is preferentially localized to one side of the cell cortex during cell division, generating unequal inheritance of fate-altering molecules in human neuroblastoma cell lines. We also show that the cells with a single copy of MYCN showed significantly higher percentages of ACD than those with MYCN amplification. Moreover, suppression of MYCN in MYCN-amplified cells caused ACD, whereas expression of MYCN in MYCN-nonamplified cells enhanced symmetric cell division. Furthermore, we demonstrate that centrosome inheritance follows a definite rule in ACD: The daughter centrosome with younger mother centriole is inherited to the daughter cell with NuMA preferentially localized to the cell cortex, whereas the mother centrosome with the older mother centriole migrates to the other daughter cell. Thus, the mechanisms of cell division of ACD or symmetric cell division and centrosome inheritance are recapitulated in human cancer cells, and these findings may facilitate studies on cancer stem cells.

  12. A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.

    PubMed

    Prapa, Malvina; Caldrer, Sara; Spano, Carlotta; Bestagno, Marco; Golinelli, Giulia; Grisendi, Giulia; Petrachi, Tiziana; Conte, Pierfranco; Horwitz, Edwin M; Campana, Dario; Paolucci, Paolo; Dominici, Massimo

    2015-09-22

    Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

  13. MEIS homeobox genes in neuroblastoma.

    PubMed

    Geerts, Dirk; Revet, Ingrid; Jorritsma, Gerda; Schilderink, Nathalie; Versteeg, Rogier

    2005-10-18

    The common pediatric tumor neuroblastoma originates from primitive neural crest-derived precursor cells of the peripheral nervous system. Neuroblastoma especially affects very young children, and can already be present at birth. Its early onset and cellular origin predict the involvement of developmental control genes in neuroblastoma etiology. These genes are indispensable for the tight regulation of normal embryonic development but as a consequence cause cancer and congenital diseases upon mutation or aberrant expression. To date however, the connotation of these genes in neuroblastoma pathogenesis is scant. This review recapitulates data on the MEIS homeobox control genes in cancer and focuses on neuroblastoma.

  14. β-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α.

    PubMed

    Kim, Yoo-Sun; Lee, Hyun-Ah; Lim, Ji Ye; Kim, Yang; Jung, Chang-Hwan; Yoo, Sang-Ho; Kim, Yuri

    2014-06-01

    Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While β-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of β-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of β-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with β-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following β-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered β-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following β-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following β-carotene treatment. In conclusion, the present study provides the first evidence that β-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.

  15. Next generation sequencing of microRNAs from isogenic neuroblastoma cell lines isolated before and after treatment.

    PubMed

    Roth, Sarah Andrea; Knutsen, Erik; Fiskaa, Tonje; Utnes, Peter; Bhavsar, Swapnil; Hald, Øyvind H; Løkke, Cecilie; Mestdagh, Pieter; Johansen, Steinar D; Flægstad, Trond; Einvik, Christer

    2016-03-01

    Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma.

  16. SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR-410 in human neuroblastoma cells

    PubMed Central

    Boyineni, Jerusha; Tanpure, Smita; Gnanamony, Manu; Antony, Reuben; Fernández, Karen S.; Lin, Julian; Pinson, David; Gondi, Christopher S.

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N-BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the over-expression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410. PMID:27498840

  17. Generation of a Functional Human Neural Network by NDM29 Overexpression in Neuroblastoma Cancer Cells.

    PubMed

    Alloisio, Susanna; Garbati, Patrizia; Viti, Federica; Dante, Silvia; Barbieri, Raffaella; Arnaldi, Giovanni; Petrelli, Alessia; Gigoni, Arianna; Giannoni, Paolo; Quarto, Rodolfo; Nobile, Mario; Vassalli, Massimo; Pagano, Aldo

    2016-10-03

    Recent advances in life sciences suggest that human and rodent cell responses to stimuli might differ significantly. In this context, the results achieved in neurotoxicology and biomedical research practices using neural networks obtained from mouse or rat primary culture of neurons would benefit of the parallel evaluation of the same parameters using fully differentiated neurons with a human genetic background, thus emphasizing the current need of neuronal cells with human origin. In this work, we developed a human functionally active neural network derived by human neuroblastoma cancer cells genetically engineered to overexpress NDM29, a non-coding RNA whose increased synthesis causes the differentiation toward a neuronal phenotype. These cells are here analyzed accurately showing functional and morphological traits of neurons such as the expression of neuron-specific proteins and the possibility to generate the expected neuronal current traces and action potentials. Their morphometrical analysis is carried out by quantitative phase microscopy showing soma and axon sizes compatible with those of functional neurons. The ability of these cells to connect autonomously forming physical junctions recapitulates that of hippocampal neurons, as resulting by connect-ability test. Lastly, these cells self-organize in neural networks able to produce spontaneous firing, in which spikes can be clustered in bursts. Altogether, these results show that the neural network obtained by NDM29-dependent differentiation of neuroblastoma cells is a suitable tool for biomedical research practices.

  18. Differentiation characteristics of human neuroblastoma cells in the presence of growth modulators and antimitotic drugs.

    PubMed

    Gupta, M; Notter, M F; Felten, S; Gash, D M

    1985-03-01

    Morphological characteristics of undifferentiated and differentiated human neuroblastoma cells were studied. Monolayer cultures of a human neuroblastoma, IMR-32 clone, were grown in Eagle's minimum essential medium with fetal calf serum in tissue culture dishes with polystyrene film liners. After 48 h, cultures were treated with either mitomycin C and 5-bromodeoxyuridine or prostaglandin E1 (PGE1) and dibutyryl adenosine 3',5'-cyclophosphate (cAMP). A third dish was untreated to study as an undifferentiated control. Three days later, all cultures were processed for acetylcholinesterase staining, scanning and transmission electron microscopy and high performance liquid chromatography. Treatment with mitomycin/5-bromodeoxyuridine and PGE1/cAMP inhibited growth as seen by the growth curves and caused morphological differentiation as seen by the extension of long neurites. The treated cells showed increased acetylcholinesterase staining compared to the controls. With the scanning electron microscope, the differentiated cells showed long neurites, processes with beaded varicosities and growth cones. By transmission electron microscopy, these cells contained a large number of neurosecretory granules in their cytoplasm and neurites. Specialized cell contacts were also observed between the treated cells. This is the first study demonstrating that both the treated and control cells of IMR-32 clone contain large quantities of serotonin and comparatively small amounts of norepinephrine and dopamine.

  19. Microarray of neuroblastoma cells on the selectively functionalized nanocrystalline diamond thin film surface

    NASA Astrophysics Data System (ADS)

    Park, Young-Sang; Son, Hyeong-Guk; Kim, Dae-Hoon; Oh, Hong-Gi; Lee, Da-Som; Kim, Min-Hye; Lim, Ki-Moo; Song, Kwang-Soup

    2016-01-01

    Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O2 or C3F8 gas environment in order to induce wettability. The oxygenated-NCD (O-NCD) film surface was hydrophilic and the fluorinated-NCD (F-NCD) surface was hydrophobic. The efficiency of early cell adhesion, which is dependent on the wettability of the cell culture plate and necessary for the growth and proliferation of cells, was 89.62 ± 3.92% on the O-NCD film and 7.78 ± 0.77% on the F-NCD film surface after 3 h of cell culture. The wettability of the NCD film surface was artificially modified using a metal mask and plasma treatment to fabricate a micro-pattern. Four types of micro-patterns were fabricated (line, circle, mesh, and word) on the NCD film surface. We precisely arrayed the neuroblastoma cells on the micro-patterned NCD film surfaces by controlling the surface wettability and cell seeding density. The neuroblastoma cells adhered and proliferated along the O-NCD film surface.

  20. Mechanisms of Neuroblastoma Cell Growth Inhibition by CARP-1 Functional Mimetics

    PubMed Central

    Muthu, Magesh; Cheriyan, Vino T.; Munie, Sara; Levi, Edi; Frank, John; Ashour, Abdelkader E.; Singh, Mandip; Rishi, Arun K.

    2014-01-01

    Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and β proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents. PMID:25033461

  1. Cytotoxicity of alpha-particle-emitting m-[211At]astatobenzylguanidine on human neuroblastoma cells.

    PubMed

    Strickland, D K; Vaidyanathan, G; Zalutsky, M R

    1994-10-15

    Radioiodinated m-iodobenzylguanidine (MIBG) has been used with only limited success for the treatment of neural crest tumors including neuroblastoma. Use of an MIBG analogue labeled with 211At could be advantageous because of the shorter range and higher linear energy transfer of its alpha-particle emissions compared with the beta-particles emitted by 131I. The potential utility of m-[211At]astatobenzylguanidine for the treatment of neuroblastoma was investigated in vitro using 3 human neuroblastoma cell lines known to take up MIBG [SK-N-SH, SK-N-BE(2C), and SK-SY5Y] and a control line lacking MIBG uptake (SK-N-MC). Maximum binding of m-[211At]astatobenzylguanidine ([211At] MABG) to 5 x 10(5) cells after a 2-h incubation ranged from 61% for SK-N-SH to 1% for SK-N-MC. Using a limiting dilution clonogenic assay, the cytotoxicity for SK-N-SH cells of [211At]MABG was compared with [211At]astatide and no-carrier-added [131I]MIBG. A D0 of 5.8 nCi/ml was calculated for [211At]MABG compared with 482 nCi/ml for [211At] astatide, indicating a more than 80-fold enhanced cytotoxicity for the specifically targeted alpha-particles of [211At]MABG. For [211At]MABG, the D0 corresponded to only 6.4 211At atoms bound/cell compared with 9000 atoms/cell for no-carrier-added [131I]MIBG. The D0 values measured for [211At]MABG treatment of SK-SY5Y, SK-N-BE(2C), and SK-N-MC cells were 50, 5.8, and 11,043 nCi/ml, respectively, corresponding to 7.04, 6.46, and 171.79 211At atoms bound/cell. In conclusion, these results have demonstrated that [211At]MABG is considerably more cytotoxic than [131I]MIBG and that [211At]MABG could have great potential as a radiotherapeutic agent for the treatment of neuroblastoma.

  2. Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

    SciTech Connect

    Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki . E-mail: mikeda.emb@tmd.ac.jp

    2006-02-03

    The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

  3. Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells

    PubMed Central

    Cordeau, Martine; Belounis, Assila; Lelaidier, Martin; Cordeiro, Paulo; Sartelet, Hervé; Duval, Michel

    2016-01-01

    High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB. PMID:27716850

  4. Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model.

    PubMed

    Giménez-Xavier, Pol; Francisco, Roser; Santidrián, Antonio F; Gil, Joan; Ambrosio, Santiago

    2009-07-01

    Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.

  5. Trim32 facilitates degradation of MYCN on spindle poles and induces asymmetric cell division in human neuroblastoma cells.

    PubMed

    Izumi, Hideki; Kaneko, Yasuhiko

    2014-10-01

    Asymmetric cell division (ACD) is a physiologic process during development and tissue homeostasis. ACD produces two unequal daughter cells: one has stem/progenitor cell activity and the other has potential for differentiation. Recent studies showed that misregulation of the balance between self-renewal and differentiation by ACD may lead to tumorigenesis in Drosophila neuroblasts. However, it is still largely unknown whether human cancer stem-like cells exhibit ACD or not. Here, using human neuroblastoma cells as an ACD model, we found that MYCN accumulates at spindle poles by GSK-3β phosphorylation during mitosis. In parallel, the ACD-related ubiquitin ligase Trim32 was recruited to spindle poles by CDK1/cyclin B-mediated phosphorylation. Trim32 interacted with MYCN at spindle poles during mitosis, facilitating proteasomal degradation of MYCN at spindle poles and inducing ACD. Trim32 also suppressed sphere formation of neuroblastoma-initiating cells, suggesting that the mechanisms of ACD produce differentiated neuroblastoma cells that will eventually die. Thus, Trim32 is a positive regulator of ACD that acts against MYCN and should be considered as a tumor-suppressor candidate. Our findings offer novel insights into the mechanisms of ACD and clarify its contributions to human tumorigenesis.

  6. NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

    SciTech Connect

    Ogbomo, Henry; Hahn, Anke; Geiler, Janina; Michaelis, Martin; Doerr, Hans Wilhelm; Cinatl, Jindrich . E-mail: Cinatl@em.uni-frankfurt.de

    2006-01-06

    The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.

  7. Morroniside protects SK-N-SH human neuroblastoma cells against H2O2-induced damage

    PubMed Central

    Zhang, Jing-Xing; Wang, Rui; Xi, Jin; Shen, Lin; Zhu, An-You; Qi, Qi; Wang, Qi-Yi; Zhang, Lun-Jun; Wang, Feng-Chao; Lü, He-Zuo; Hu, Jian-Guo

    2017-01-01

    Oxidative stress-induced cell injury has been linked to the pathogenesis of neurodegenerative disorders such as spinal cord injury, Parkinson's disease, and multiple sclerosis. Morroniside is an antioxidant derived from the Chinese herb Shan-Zhu-Yu. The present study investigated the neuroprotective effect of morroniside against hydrogen peroxide (H2O2)-induced cell death in SK-N-SH human neuroblastoma cells. H2O2 increased cell apoptosis, as determined by flow cytometry and Hoechst 33342 staining. This effect was reversed by pretreatment with morroniside at concentrations of 1–100 µM. The increase in intracellular reactive oxygen species (ROS) generation and lipid peroxidation induced by H2O2 was also abrogated by morroniside. H2O2 induced a reduction in mitochondrial membrane potential, increased caspase-3 activity, and caused downregulation of B cell lymphoma-2 (Bcl-2) and upregulation of Bcl-2-associated X protein (Bax) expression. These effects were blocked by morroniside pretreatment. Thus, morroniside protects human neuroblastoma cells against oxidative damage by inhibiting ROS production while suppressing Bax and stimulating Bcl-2 expression, thereby blocking mitochondrial-mediated apoptosis. These results indicate that morroniside has therapeutic potential for the prevention and treatment of neurodegenerative diseases. PMID:28204825

  8. Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

    SciTech Connect

    Ponzoni, M.; Lanciotti, M.; Melodia, A.; Casalaro, A.; Cornaglia-Ferraris, P. )

    1989-03-01

    The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 {mu}g/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in ({sup 3}H)thymidine incorporation was also observed within 24 hours and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

  9. Morroniside protects SK-N-SH human neuroblastoma cells against H2O2-induced damage.

    PubMed

    Zhang, Jing-Xing; Wang, Rui; Xi, Jin; Shen, Lin; Zhu, An-You; Qi, Qi; Wang, Qi-Yi; Zhang, Lun-Jun; Wang, Feng-Chao; Lü, He-Zuo; Hu, Jian-Guo

    2017-03-01

    Oxidative stress-induced cell injury has been linked to the pathogenesis of neurodegenerative disorders such as spinal cord injury, Parkinson's disease, and multiple sclerosis. Morroniside is an antioxidant derived from the Chinese herb Shan-Zhu-Yu. The present study investigated the neuroprotective effect of morroniside against hydrogen peroxide (H2O2)-induced cell death in SK-N-SH human neuroblastoma cells. H2O2 increased cell apoptosis, as determined by flow cytometry and Hoechst 33342 staining. This effect was reversed by pretreatment with morroniside at concentrations of 1-100 µM. The increase in intracellular reactive oxygen species (ROS) generation and lipid peroxidation induced by H2O2 was also abrogated by morroniside. H2O2 induced a reduction in mitochondrial membrane potential, increased caspase-3 activity, and caused downregulation of B cell lymphoma-2 (Bcl-2) and upregulation of Bcl-2-associated X protein (Bax) expression. These effects were blocked by morroniside pretreatment. Thus, morroniside protects human neuroblastoma cells against oxidative damage by inhibiting ROS production while suppressing Bax and stimulating Bcl-2 expression, thereby blocking mitochondrial-mediated apoptosis. These results indicate that morroniside has therapeutic potential for the prevention and treatment of neurodegenerative diseases.

  10. Effects of inorganic lead on the differentiation and growth of cultured hippocampal and neuroblastoma cells.

    PubMed

    Audesirk, T; Audesirk, G; Ferguson, C; Shugarts, D

    1991-01-01

    Lead exposure has devastating effects on the developing nervous system, and has been implicated in variety of behavioral and cognitive deficits as well as neural morphological abnormalities. Since lead impacts many calcium-dependent processes, one likely mechanism of lead toxicity is its disruption of calcium dependent processes, among which is neuronal differentiation. We investigated the effects of inorganic lead on survival and several parameters of differentiation of cultured neurons. Three different cell types were used: Rat hippocampal neurons (a primary CNS cell type), B50 rat neuroblastoma cells (a transformed CNS-derived cell line), and N1E-115 mouse neuroblastoma cells (a transformed peripherally-derived cell line). Lead concentrations ranged from low nM to 1 mM. Lead effects differed considerably among the three cell types, with B50 cells least affected. Lead effects were generally multimodal, with fewest effects observed at intermediate concentrations. Lead inhibited neurite initiation in hippocampal neurons, but stimulated initiation in N1E-115 cells. In those cells that differentiated, lead increased dendrite numbers in hippocampal neurons and neurite numbers in N1E-115 cells. Lead exposure increased both the length and the degree of branching of axons in hippocampal neurons and the length of neurites in N1E-115 cells. We hypothesize that lead impacts multiple regulatory processes that influence neuron survival and differentiation, and that its effects show differing dose-dependencies. The differing responses of the different cell types to lead suggests that differentiation may be regulated in different ways by the three types of cells. Alternatively, or additionally, the cell types may differ in their ability to compensate for, sequester, or expel lead.

  11. 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

  12. 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.

  13. Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

    PubMed

    Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

    2014-06-01

    Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer.

  14. The CacyBP/SIP protein is sumoylated in neuroblastoma NB2a cells.

    PubMed

    Wasik, Urszula; Filipek, Anna

    2013-11-01

    The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

  15. Immune Therapies for Neuroblastoma

    PubMed Central

    Navid, Fariba; Armstrong, Michael; Barfield, Raymond C.

    2009-01-01

    Neuroblastoma, a solid tumor arising from developing cells of the sympathetic nervous system, is the most common extracranial tumor in children. The prognosis for high-risk neuroblastoma remains poor with conventional treatment, and new approaches are therefore being explored to treat this disease. One such alternative therapy that holds promise is immune therapy. We review here the recent advances in 4 types of immune therapy – cytokine, vaccine, antibody, and cellular therapy – to treat neuroblastoma. We present preclinical research and clinical trials on several promising candidates such as IL-12, dendritic cell vaccines, anti-GD2 antibodies, and allogeneic hematopoietic stem cell transplant. An optimal treatment plan for neuroblastoma will most likely involve multimodal approaches and combinations of immune therapies. PMID:19342881

  16. Early thyroid hormone-induced gene expression changes in N2a-β neuroblastoma cells.

    PubMed

    Bedó, Gabriela; Pascual, Angel; Aranda, Ana

    2011-10-01

    Thyroid hormone has long been known to regulate neural development. Hypothyroidism during pregnancy and early postnatal period has severe neurological consequences including even mental retardation. The purpose of this study was to characterize gene expression pattern during thyroid hormone-induced differentiation of neuro-2a β cells in order to select "direct response genes" for further analysis. In this neuroblastoma cell line, thyroid hormone blocks proliferation and induces differentiation. Changes in gene expression level were examined after a T3 treatment of 3 and 24 h using cDNA arrays. Sixteen genes were significantly up-regulated and 79 down-regulated by T3 treatment. Five up-regulated genes not previously described as regulated by thyroid hormone and selected for their putative significance to understand T3 action on cell differentiation, were verified by RT-PCR analysis. The transcription factors Phox2a and basic helix-loop-helix domain containing, class B2 mRNAs exhibited a clear increase after 3- and 24-h treatment. The guanine-nucleotide exchange factor RalGDS was greatly up-regulated after 3-h treatment but not 24 h after. The results suggest an early involvement of these genes in T3 action during neuroblastoma cell differentiation probably mediating later changes in gene expression pattern.

  17. 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

  18. Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells

    PubMed Central

    Nobile, Mario; Vercellino, Paolo

    1997-01-01

    The action of the anticonvulsant drug phenytoin on K+ currents was investigated in neuroblastoma cells by whole-cell voltage-clamp recording.Neuroblastoma cells expressed an outward K+ current with a voltage- and time-dependence which resembled the delayed-rectifier K+ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n=65). Inhibition was concentration-dependent with a half-maximal inhibitory concentration of 30.9±0.8 μM.The K+ current inhibition by phenytoin was voltage-dependent with block by phenytoin being relieved by depolarization.The times taken to reach steady-state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K+ current nor the K+ channel availability were significantly altered by the blocking drug. A use-dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.These results suggest that phenytoin affects K+ currents and that this effect might lead to a reduction in neuronal excitability. PMID:9051303

  19. 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.

  20. Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

    PubMed Central

    Arsenault, Jason; Cuijpers, Sabine A G; Ferrari, Enrico; Niranjan, Dhevahi; Rust, Aleksander; Leese, Charlotte; O'Brien, John A; Binz, Thomas; Davletov, Bazbek

    2014-01-01

    Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal-associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations. PMID:24372287

  1. Neuroblastoma stem cells - mechanisms of chemoresistance and histone deacetylase inhibitors.

    PubMed

    Khalil, M A; Hrabeta, J; Cipro, S; Stiborova, M; Vicha, A; Eckschlager, T

    2012-01-01

    Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133- ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs.

  2. Viologen-phosphorus dendrimers exhibit minor toxicity against a murine neuroblastoma cell line.

    PubMed

    Lazniewska, Joanna; Milowska, Katarzyna; Katir, Nadia; El Kadib, Abdelkim; Bryszewska, Maria; Majoral, Jean-Pierre; Gabryelak, Teresa

    2013-09-01

    Dendrimers containing viologen (derivatives of 4,4'-bipyridyl) units in their structure have been demonstrated to exhibit antiviral activity against human immunodeficiency virus (HIV-1). It has also recently been revealed that novel dendrimers with both viologen units and phosphorus groups in their structure show different antimicrobial, cytotoxic and hemotoxic properties, and have the ability to influence the activity of cholinesterases and to inhibit α-synuclein fibrillation. Since the influence of viologen-phosphorus structures on basic cellular processes had not been investigated, we examined the impact of such macromolecules on the murine neuroblastoma cell line (N2a). We selected three water-soluble viologen-phosphorus (VPD) dendrimers, which differ in their core structure, number of viologen units and number and type of surface groups, and analyzed several aspects of the cellular response. These included cell viability, generation of reactive oxygen species (ROS), alterations in mitochondrial activity, morphological modifications, and the induction of apoptosis and necrosis. The MTT assay results suggest that all of the tested dendrimers are only slightly cytotoxic. Although some changes in ROS formation and mitochondrial function were detected, the three compounds did not induce apoptosis or necrosis. In light of these results, we can assume that the tested VPD are relatively safe for mouse neuroblastoma cells. Although more research on their safety is needed, VPD seem to be promising nanoparticles for further biomedical investigation.

  3. Presence of fucosyl residues on the oligosaccharide antennae of membrane glycopeptides of human neuroblastoma cells

    SciTech Connect

    Santer, U.V.; Glick, M.C.

    1983-09-01

    Fucosyl residues linked alpha 1 leads to 3 or 4 to N-acetylglucosamine were found in large amounts on glycopeptides from the membranes of human tumor cells of neurectodermal origin but not on membrane glycopeptides from human fibroblasts. The fucosyl residues were detected by release of radioactive fucose from the glycopeptides with an almond alpha-L-fucosidase specific for fucosyl alpha 1 leads to 3(4)-N-acetylglucosamine. In other studies, the linkage was shown to be alpha 1 leads to 3 by nuclear magnetic resonance analysis. Glycopeptides containing these fucosyl residues from four human neuroblastoma cell lines were defined by binding to immobilized lectins. In addition, the glycopeptides from one human neuroblastoma cell line, CHP-134, were further characterized by enzyme degradation and columns calibrated for size and charge. The antennary position of fucosyl alpha 1 leads to 3-N-acetylglucosamine on the glycopeptides was demonstrated by the use of exoglycosidases and endoglycosidase D, since complete degradation to yield fucosyl-N-acetylglucosaminylasparagine was obtained only after treatment with almond alpha-L-fucosidase prior to the sequential degradation. Fucosyl alpha 1 leads to 3-N-acetylglucosamine was present on most size and charge classes of membrane glycopeptides and therefore was not limited to a few glycoproteins. Since the almond alpha-L-fucosidase cleaves fucosyl residues from glycoproteins, the physiological effects of the increased specific fucosylation on human tumors of neurectodermal origin can be examined.

  4. Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells.

    PubMed

    Rhein, V; Baysang, G; Rao, S; Meier, F; Bonert, A; Müller-Spahn, F; Eckert, A

    2009-09-01

    Evidence suggests that amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Here we investigated the specific effects of Abeta on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Abeta protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

  5. Effect of graphene on growth of neuroblastoma cells.

    PubMed

    Park, Hye-Bin; Nam, Hyo-Geun; Oh, Hong-Gi; Kim, Jung-Hyun; Kim, Chang-Man; Song, Kwang-Soup; Jhee, Kwang-Hwan

    2013-02-01

    The unique properties of graphene have earned much interest in the fields of materials science and condensed-matter physics in recent years. However, the biological applications of graphene remain largely unexplored. In this study, we investigated the conditions and viability of a cell culture exposed to graphene onto glass and SiO2/Si, using a human nerve cell line, SH-SY5Y. Cell viability was 84% when cultured on glass and SiO2/Si coated with graphene as compared with culturing on polystyrene surface. Fluorescence data showed that the presence of graphene did not influence cell morphology. These findings suggest that graphene may be used for biological applications.

  6. Calcium protects differentiating neuroblastoma cells during 50 Hz electromagnetic radiation.

    PubMed

    Tonini, R; Baroni, M D; Masala, E; Micheletti, M; Ferroni, A; Mazzanti, M

    2001-11-01

    Despite growing concern about electromagnetic radiation, the interaction between 50- to 60-Hz fields and biological structures remains obscure. Epidemiological studies have failed to prove a significantly correlation between exposure to radiation fields and particular pathologies. We demonstrate that a 50- to 60-Hz magnetic field interacts with cell differentiation through two opposing mechanisms: it antagonizes the shift in cell membrane surface charges that occur during the early phases of differentiation and it modulates hyperpolarizing K channels by increasing intracellular Ca. The simultaneous onset of both mechanisms prevents alterations in cell differentiation. We propose that cells are normally protected against electromagnetic insult. Pathologies may arise, however, if intracellular Ca regulation or K channel activation malfunctions.

  7. Dynamic and selective HERV RNA expression in neuroblastoma cells subjected to variation in oxygen tension and demethylation.

    PubMed

    Hu, Lijuan; Uzhameckis, Dmitrijs; Hedborg, Fredrik; Blomberg, Jonas

    2016-01-01

    We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups (HERVs) (HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cell lines SH-SY5Y, SK-N-DZ, and SK-N-AS were studied using reverse transcription and real-time quantitative PCR (QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N-DZ. In the SK-N-DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli.

  8. Norcantharidin Inhibits SK-N-SH Neuroblastoma Cell Growth by Induction of Autophagy and Apoptosis.

    PubMed

    Han, Zeping; Li, Baoxia; Wang, Juanjuan; Zhang, Xiangqiang; Li, Zhenhua; Dai, Liting; Cao, Mingrong; Jiang, Jianwei

    2017-02-01

    Norcantharidin, a low-toxic analog of the active anticancer compound cantharidin in Mylabris, can inhibit proliferation and induce apoptosis of multiple types of cancer cells. However, the anticancer activities of norcantharidin with respect to neuroblastoma, and its underlying mechanisms, have not been investigated. Therefore, our study was designed to determine the efficacy of norcantharidin on SK-N-SH neuroblastoma cell death and to elucidate detailed mechanisms of activity. In the present study, norcantharidin suppressed the proliferation and cloning ability of SK-N-SH cells in a dose-dependent manner, apparently by reducing the mitochondrial membrane potential and arresting SK-N-SH cells at the G2/M stage, accompanied by elevated expressions of p21 and decreased expressions of cyclin B1 and cell division control 2. Treatment by norcantharidin induced significant mitophagy and autophagy, as demonstrated by a decrease in Translocase Of Outer Mitochondrial Membrane 20 (TOM20), increased beclin1 and LC3-II protein expression, reduced protein SQSTM1/p62 expression, and accumulation of punctate LC3 in the cytoplasm of SK-N-SH cells. In addition, norcantharidin induced apoptosis through regulating the expression of B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 and B-cell lymphoma 2-associated X protein/myeloid cell leukemia 1 and activating caspase-3 and caspase-9-dependent endogenous mitochondrial pathways. We also observed an increase in phosphor-AMP-activated protein kinase accompanied with a decrease in phosphor-protein kinase B and mammalian target of rapamycin expression after treatment with norcantharidin. Subsequent studies indicated that norcantharidin participates in cellular autophagy and apoptosis via activation of the c-Jun NH2-terminal kinases/c-Jun pathway. In conclusion, our results demonstrate that norcantharidin can reduce the mitochondrial membrane potential, induce mitophagy, and subsequently arouse cellular autophagy and apoptosis; the

  9. Novel multiple tyrosine kinase inhibitor ponatinib inhibits bFGF-activated signaling in neuroblastoma cells and suppresses neuroblastoma growth in vivo

    PubMed Central

    Lu, Jiaxiong; Pan, Jessie; Yu, Yang; Zhao, Yanling; Zhang, Huiyuan; Hu, Ting; Liu, Qing; Yang, Jianhua

    2017-01-01

    Neuroblastoma (NB) is one of the most common pediatric malignancies in children. Abnormal activation of receptor tyrosine kinases contributes to the pathological development of NB. Therefore, targeting tyrosine kinase receptors to cure NB is a promising strategy. Here, we report that a multi-targeted tyrosine kinase inhibitor ponatinib inhibited NB cell proliferation and induced NB cell apoptosis in a dose-dependent manner. In addition, ponatinib suppressed the colony formation ability of NB cells. Mechanistically, ponatinib effectively inhibited the FGFR1-activated signaling pathway. Ponatinib also enhanced the cytotoxic effects of doxorubicin on NB cells. Furthermore, ponatinib demonstrated anti-tumor efficacy in vivo by inhibiting tumor growth in an orthotopic xenograft NB mouse model. In summary, our results showed that ponatinib inhibited NB growth both in vitro and in vivo. PMID:27564113

  10. 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.

  11. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    PubMed

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  12. 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.

  13. Radiofrequency radiation-induced calcium ion efflux enhancement from human and other neuroblastoma cells in culture

    SciTech Connect

    Dutta, S.K.; Ghosh, B.; Blackman, C.F.

    1989-01-01

    To test the generality of radiofrequency radiation-induced changes in /sup 45/Ca2+ efflux from avian and feline brain tissues, human neuroblastoma cells were exposed to electromagnetic radiation at 147 MHz, amplitude-modulated (AM) at 16 Hz, at specific absorption rates (SAR) of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005 W/kg. Significant /sup 45/Ca2+ efflux was obtained at SAR values of 0.05 and 0.005 W/kg. Enhanced efflux at 0.05 W/kg peaked at the 13-16 Hz and at the 57.5-60 Hz modulation ranges. A Chinese hamster-mouse hybrid neuroblastoma was also shown to exhibit enhanced radiation-induced /sup 45/Ca2+ efflux at an SAR of 0.05 W/kg, using 147 MHz, AM at 16 Hz. These results confirm that amplitude-modulated radiofrequency radiation can induce responses in cells of nervous tissue origin from widely different animal species, including humans. The results are also consistent with the reports of similar findings in avian and feline brain tissues and indicate the general nature of the phenomenon.

  14. Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells.

    PubMed Central

    Di Marzo, V; De Petrocellis, L; Sepe, N; Buono, A

    1996-01-01

    Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role

  15. TNF-α Contributes to Caspase-3 Independent Apoptosis in Neuroblastoma Cells: Role of NFAT

    PubMed Central

    Álvarez, Susana; Blanco, Almudena; Fresno, Manuel; Muñoz-Fernández, Ma Ángeles

    2011-01-01

    There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation. PMID:21298033

  16. In Vitro Anti-Neuroblastoma Activity of Thymoquinone Against Neuro-2a Cells via Cell-cycle Arrest.

    PubMed

    Paramasivam, Arumugam; Raghunandhakumar, Subramanian; Priyadharsini, Jayaseelan Vijayashree; Jayaraman, Gopalswamy

    2015-01-01

    We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose- dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

  17. Nitro-oxidative Stress Is Involved in Anticancer Activity of 17β-Estradiol Derivative in Neuroblastoma Cells.

    PubMed

    Gorska, Magdalena; Kuban-Jankowska, Alicja; Milczarek, Ryszard; Wozniak, Michal

    2016-04-01

    Neuroblastoma is one of the most common childhood malignancies and the primary cause of death from pediatric cancer. Derivatives of 17β-estradiol, 2-methoxyestradiol, as well as selective estrogen receptor modulators, such as fulvestrant, are novel potentially active anticancer agents. In particular, 2-methoxyestradiol is effective in treatment of numerous malignancies, including breast and prostate cancer, Ewing sarcoma, and osteosarcoma. Herein, we treated neuroblastoma SH-SY5Y cells with physiologically and pharmacologically relevant concentrations of 2-methoxyestradiol. We used flow cytometry in order to determine cell viability, cell death, level of nitric oxide and mitochondrial membrane potential. We demonstrated that at pharmacologically relevant concentrations, 2-methoxyestradiol results in induction of apoptosis of neuroblastoma SH-SY5Y cells via nitric oxide generation and reduction of mitochondrial membrane potential. Based on the obtained data, we propose that 2-methoxyestradiol may be a natural modulator of cancer cell death and survival through nitro-oxidative stress-dependent mechanisms. Moreover, the results confirm the efficiency of 2-methoxyestradiol in treatment of neuroblastoma.

  18. MiR-128 up-regulation inhibits Reelin and DCX expression and reduces neuroblastoma cell motility and invasiveness.

    PubMed

    Evangelisti, Cristina; Florian, Maria Carolina; Massimi, Isabella; Dominici, Carlo; Giannini, Giuseppe; Galardi, Silvia; Buè, Maria Cristina; Massalini, Simone; McDowell, Heather P; Messi, Elio; Gulino, Alberto; Farace, Maria Giulia; Ciafrè, Silvia Anna

    2009-12-01

    MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.

  19. 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.

  20. Hydrochloric acid alters the effect of L-glutamic acid on cell viability in human neuroblastoma cell cultures.

    PubMed

    Croce, Nicoletta; Bernardini, Sergio; Di Cecca, Stefano; Caltagirone, Carlo; Angelucci, Francesco

    2013-07-15

    l-Glutamic acid (l-glutamate) is used to induce excitotoxicity and test neuroprotective compounds in cell cultures. However, because l-glutamate powder is nearly insoluble in water, many manufacturers recommend reconstituting l-glutamate in hydrochloric acid (HCl) prior to successive dilutions. Nevertheless, HCl, even at low concentrations, may alter the pH of the cell culture medium and interfere with cell activity. Thus, the aim of this study was to evaluate whether the reconstitution of l-glutamate powder in HCl alters its capacity to induce neurotoxicity in different human neuroblastoma cell lines. SH-SY5Y, IMR-32 and SK-N-BE(2) cells were exposed to various concentrations of l-glutamate, which was either reconstituted in HCl (1M) or post re-equilibrated to the pH of the culture medium (7.5). After 24 and 48h of incubation, changes in the cell viability of treated versus untreated cells were evaluated. The effect of an identical amount of HCl present in the l-glutamate dilutions on neuroblastoma cell survival was also investigated. Our data showed that the neurotoxicity of glutamate reconstituted in HCl was comparable to that of HCl alone. Moreover, the pH variations induced by glutamate or HCl in the culture medium were similar. When the pH of the glutamate stock solution was re-equilibrated, l-glutamate induced variation in cell viability to a lower extent and after a longer incubation time. This study demonstrated that HCl used to reconstitute l-glutamate powder might alter the effect of glutamate itself in neuroblastoma cell cultures. Thus, this information might be useful to scientists who use l-glutamate to induce excitotoxicity or to test neuroprotective agents.

  1. Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

    SciTech Connect

    Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

    1987-01-01

    Muscarinic receptor-linked increases in intracellular free Ca/sup 2 +/ as measured with quin-2 and Ca/sup 2 +/ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca/sup 2 +/ and stimulation of Ca/sup 2 +/ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, /sup 3/H-NMS, by a non-labelled agonist was studied.

  2. Membrane raft disruption promotes axonogenesis in n2a neuroblastoma cells.

    PubMed

    Petro, Kimberly A; Schengrund, Cara-Lynne

    2009-01-01

    Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

  3. Manipulation of PrPres production in scrapie-infected neuroblastoma cells.

    PubMed

    Bate, Clive; Langeveld, Jan; Williams, Alun

    2004-09-30

    In the present study the accumulation of protease resistant prion protein (PrPres) in scrapie-infected neuroblastoma cells (ScN2a cells) was shown to be dependent on culture conditions. The highest levels of PrPres were found in slow growing cells. Further increases in PrPres accumulation were observed in ScN2a cells treated with retinoic acid, a compound that is associated with neuronal differentiation. The effects of retinoic acid were dose-dependent with a maximal effect at 200 ng/ml. A similar increase in PrPres was observed in another prion-infected cell line, scrapie-mouse brain (SMB) cells, treated with retinoic acid while retinoic acid increased the amount of PrPC in non-infected cells. Other drugs reported to cause neuronal differentiation, such as phorbol esters, did not increase the PrPres content of ScN2a cells. The survival of retinoic acid-treated ScN2a cells co-cultured with microglia was significantly reduced when compared to untreated ScN2a cells and an inverse correlation was demonstrated between the PrPres content of cells and their survival when co-cultured with microglia. The production of interleukin-6 by microglia cultured with retinoic acid-treated ScN2a cells was significantly higher than that of microglia cultured with untreated ScN2a cells.

  4. 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

  5. Transplantation of Human Neuroblastoma Cells, Catecholaminergic and Non-Catecholaminergic: Effects on Rotational Behavoir in Parkinson's Rat Model

    PubMed Central

    Manaster, Jacob S.; Feuerman, Tony; Reynolds, C. Patrick; Markham, Charles H.

    1992-01-01

    Cultured human catecholaminergic and noncatecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model. PMID:1355366

  6. Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells.

    PubMed

    Tiewcharoen, Supathra; Malainual, Nat; Junnu, Virach; Chetanachan, Pruksawan; Rabablert, Jundee

    2008-04-01

    The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.

  7. N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

    PubMed Central

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

    2013-01-01

    Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. PMID:23941992

  8. Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.

    PubMed

    Kita, Masaki; Black, David StC; Ohno, Osamu; Yamada, Kaoru; Kigoshi, Hideo; Uemura, Daisuke

    2009-12-23

    The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

  9. 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

  10. Relationship between interphasic nucleolar organizer regions and growth rate in two neuroblastoma cell lines.

    PubMed Central

    Derenzini, M.; Pession, A.; Farabegoli, F.; Trerè, D.; Badiali, M.; Dehan, P.

    1989-01-01

    The relationship between the quantity of silver-stained interphasic nucleolar organizer regions (NORs) and nuclear synthetic activity, caryotype, and growth rate was studied in two established neuroblastoma cell lines (CHP 212 and HTB 10). Statistical analysis of silver-stained NORs revealed four times as many in CHP 212 cells compared with HTB 10 cells. No difference was observed in the ribosomal RNA synthesis between the two cell lines. The caryotype index was 1.2 for CHP 212 and 1.0 for HTB 10 cells. The number of chromosomes carrying NORs and the quantity of ribosomal genes was found to be the same for the two cell lines. Doubling time of CHP 212 cells was 20 hours compared with 54 hours for HTB 10 cells. In CHP 212 cells bindering of cell duplication by serum deprivation induced a progressive lowering (calculated at 48, 72, and 96 hours) of the quantity of silver-stained interphasic NORs. Recovery of duplication by new serum addition induced, after 24 hours, an increase of the quantity of silver-stained interphasic NORs up to control levels. In the light of available data, these results indicate that the quantity of interphasic NORs is strictly correlated only to the growth rate of the cell. Images Figure 2 Figure 3 Figure 4 PMID:2705511

  11. Functional Sphere Profiling Reveals the Complexity of Neuroblastoma Tumor-Initiating Cell Model12

    PubMed Central

    Coulon, Aurélie; Flahaut, Marjorie; Mühlethaler-Mottet, Annick; Meier, Roland; Liberman, Julie; Balmas-Bourloud, Katia; Nardou, Katya; Yan, Pu; Tercier, Stéphane; Joseph, Jean-Marc; Sommer, Lukas; Gross, Nicole

    2011-01-01

    Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically “profile” the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB. PMID:22028624

  12. In vivo expansion and activation of γδ T cells as immunotherapy for refractory neuroblastoma

    PubMed Central

    Pressey, Joseph G.; Adams, Julia; Harkins, Lualhati; Kelly, David; You, Zhiying; Lamb, Lawrence S.

    2016-01-01

    Abstract Introduction: CD3+ γδ+ T cells comprise 2% to 5% of circulating T cells with Vγ9Vδ2+ cells the dominant circulating subtype. Vγ9Vδ2+ cells recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of γδ T cells represent a promising immunotherapeutic strategy for treatment of solid malignancies including neuroblastoma (NB). In this prospective, non-randomized Phase I trial, we assessed whether circulating Vγ9Vδ2+ cells could be safely expanded using intravenous ZOL (Zoledronate [Zometa®]) and subcutaneous Interleukin-2 (IL-2) in patients with refractory NB. Methods: Patients 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options received ZOL on day 1, and IL-2 on days 1 to 5 and 15 to 19 of each 28-day cycle (n = 4). Lymphocyte immunophenotyping was assessed weekly. Immunophenotyping studies from the treatment group were compared with healthy pediatric controls (n = 16; range, 5y–15y) and of untreated NB disease controls (n = 9; range, 4m–18y). Results: Treatment was well tolerated with no unexpected grade 3 and 4 toxicities. Lymphocyte subset counts did not differ significantly between volunteers and disease controls with the exception of γδ+ T cell counts that were significantly higher in healthy volunteers (212 + 93 vs. 89 + 42, P = 0.05). Study patients showed increases in circulating γδ+ T cell count (3–10 fold) after the first week, increasing into the range seen in healthy volunteers (125 + 37, P = 0.1940). Interestingly, all ZOL + IL-2 treated patients showed significant increases in CD3+CD4+CD27hiCD127dim T cells that rose weekly in 2 patients throughout the 4 weeks of observation (maximum 41% and 24% of total CD3+CD4+ T cells, respectively). Conclusions: In summary, combined ZOL and IL-2 is well tolerated and restored γδ+ T cell counts to the normal range with a moderate

  13. 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.

  14. A c-Myc-regulated stem cell-like signature in high-risk neuroblastoma: A systematic discovery (Target neuroblastoma ESC-like signature).

    PubMed

    Yang, Xinan Holly; Tang, Fangming; Shin, Jisu; Cunningham, John M

    2017-12-01

    c-Myc dysregulation is hypothesized to account for the 'stemness' - self-renewal and pluripotency - shared between embryonic stem cells (ESCs) and adult aggressive tumours. High-risk neuroblastoma (HR-NB) is the most frequent, aggressive, extracranial solid tumour in childhood. Using HR-NB as a platform, we performed a network analysis of transcriptome data and presented a c-Myc subnetwork enriched for genes previously reported as ESC-like cancer signatures. A subsequent drug-gene interaction analysis identified a pharmacogenomic agent that preferentially interacted with this HR-NB-specific, ESC-like signature. This agent, Roniciclib (BAY 1000394), inhibited neuroblastoma cell growth and induced apoptosis in vitro. It also repressed the expression of the oncogene c-Myc and the neural ESC marker CDK2 in vitro, which was accompanied by altered expression of the c-Myc-targeted cell cycle regulators CCND1, CDKN1A and CDKN2D in a time-dependent manner. Further investigation into this HR-NB-specific ESC-like signature in 295 and 243 independent patients revealed and validated the general prognostic index of CDK2 and CDKN3 compared with CDKN2D and CDKN1B. These findings highlight the very potent therapeutic benefits of Roniciclib in HR-NB through the targeting of c-Myc-regulated, ESC-like tumorigenesis. This work provides a hypothesis-driven systems computational model that facilitates the translation of genomic and transcriptomic signatures to molecular mechanisms underlying high-risk tumours.

  15. Scanning electron microscopic study of human neuroblastoma cells affected with Naegleria fowleri Thai strains.

    PubMed

    Tiewcharoen, Supathra; Rabablert, Jundee; Chetanachan, Pruksawan; Junnu, Virach; Worawirounwong, Dusit; Malainual, Nat

    2008-10-01

    In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24 h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson's medium at 37 degrees C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1 day) was shorter than the Vero cells (12 days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.

  16. Differentiation of human neuroblastoma cells toward the osteogenic lineage by mTOR inhibitor

    PubMed Central

    Carpentieri, A; Cozzoli, E; Scimeca, M; Bonanno, E; Sardanelli, A M; Gambacurta, A

    2015-01-01

    Current hypothesis suggest that tumors can originate from adult cells after a process of 'reprogramming' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic 'Warburg' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that 'in vitro' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate 'stem' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate. PMID:26561783

  17. In vitro cytocompatibility assessment of amorphous carbon structures using neuroblastoma and Schwann cells.

    PubMed

    Jain, Shilpee; Sharma, Ashutosh; Basu, Bikramjit

    2013-05-01

    The development of scaffolds for neural tissue engineering application requires an understanding of cell adhesion, proliferation, and migration of neuronal cells. Considering the potential application of carbon as scaffold materials and the lack of understanding of compatibility of amorphous carbon with neuronal cells, the carbon-based materials in the forms of carbon films and continuous electrospun carbon nanofibers having average diameter of ~200 nm are being investigated with or without ultraviolet (UV) and oxy-plasma (OP) treatments for cytocompatibility property using mouse Neuroblastoma (N2a) and rat Schwann cells (RT4-D6P2T). The use of Raman spectroscopy in combination with Fourier transform infrared (FTIR) and X-ray diffraction establishes the amorphous nature and surface-bonding characteristics of the studied carbon materials. Although both UV and OP treatments make carbon surfaces more hydrophilic, the cell viability of N2a cells is statistically more significant on OP treated fibers/films compared to UV fiber/film substrates after 4 days in culture. The electrospun carbon fibrous substrate provides the physical guidance to the cultured Schwann cells. Overall, the experimental results of this study demonstrate that the electrospun amorphous carbon nanofibrous scaffolds can be used as a suitable biomaterial substrate for supporting cell adhesion and proliferation of neuronal cells in the context of their applications as artificial nerve implants.

  18. Specific binding of nerve growth factor (NGF) by murine C 1300 neuroblastoma cells.

    PubMed

    Revoltella, R; Bertolini, L; Pediconi, M; Vigneti, E

    1974-08-01

    Murine C 1300 neuroblastoma cells bind with high avidity on their membrane surface the nerve growth factor (NGF), a protein capable of inducing differentiation of sympathetic nerve cells. The total binding capacity of NGF by the cells was quantitatively measured by a radioimmunoassay technique, using (125)I-labeled NGF. An average number of about 10(6) molecules of NGF could be bound, at saturation, by each cell with an average relative association constant of about 10(7) liters/mol. Using synchronized cells, it was found, however, that either the number of molecules of ligand bound or the avidity of the binding interaction between NGF and cells varied depending upon their growth cycle, the maximal-binding occurring during the G(1) and early S phase. Binding of [(125)I]NGF was suppressed by trypsin treatment of the cells, however new receptor sites were rapidly replaced onto the membrane surface within 1-2 h. Cells exposed to 3 M KCl released into the supernate a protein product exhibiting similar high avidity for NGF. Acrylamide gel electrophoresis suggested a restricted molecular heterogeneity of this product, with a major component in the 52,000 mol wt region. Antibodies made specific to this protein were capable, in the absence of the complement, of inhibiting the binding of [(125)I]NGF by the cells and in the presence of the complement they killed them.

  19. Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells

    PubMed Central

    1983-01-01

    Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells. PMID:6833390

  20. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

    PubMed

    Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

    2017-04-01

    Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein.

  1. 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.

  2. Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents.

    PubMed

    Lentz, T L; Fu, Y; Lewis, P

    1997-06-01

    IMR-32 human neuroblastoma cells are a continuous nerve cell line expressing neuronal nicotinic acetylcholine receptors. These cells were found to be susceptible to infection by rabies virus (CVS strain). After infection, viral antigen accumulated in the cell body in puncta and larger masses and spread out into the processes until at 3-4 days the entire cell was filled with antigen and lysed. A variety of chemical agents including cholinergic agonists and antagonists were tested for ability to inhibit infection of IMR-32 cells in a fluorescent focus assay. Agents found to inhibit infection were antibodies against the viral glycoprotein, gangliosides, a synthetic peptide of the neurotoxin-binding site of Torpedo acetylcholine receptor alpha1 subunit, alpha-bungarotoxin, and lysosomotropic agents. All other agents tested including other cholinergic ligands and synthetic peptides were not effective. Except for lysosomotropic agents, the agents which inhibited infection also inhibited attachment of virus to the cell surface. These results indicate that IMR-32 cells are a useful model in studying the interaction of a neurotropic virus with human neurons. The ability of alpha-bungarotoxin to inhibit infection suggests that neuronal alpha-bungarotoxin-binding receptors might serve as central nervous system receptors for rabies virus.

  3. Protective effect of edaravone against Alzheimer's disease-relevant insults in neuroblastoma N2a cells.

    PubMed

    Yan, Yufang; Gong, Kai; Ma, Tuo; Zhang, Lihai; Zhao, Nanming; Zhang, Xiufang; Tang, Peifu; Gong, Yandao

    2012-12-07

    Oxidative stress has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). Thus, antioxidant therapy may represent a promising avenue for the treatment of AD. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent free radical scavenger and has been shown to provide neuroprotection in both animal models of cerebral ischemia and stroke patients. In the present study, we investigated the protective effect of edaravone against AD-relevant insults in neuroblastoma N2a cells and explored the potential mechanisms involved. N2a/Swe.Δ9 cells were used as the AD model cells, which exhibited reduced cell viability, increased apoptosis and oxidative stress as well as decreased mitochondrial membrane potential compared with N2a/Wt cells. All of these phenotypes were significantly reversed by edaravone treatment. Edaravone treatment significantly elevated cell viability, reduced apoptotic rate, attenuated oxidative stress and improved mitochondrial membrane potential in N2a/Swe.Δ9 cells. Furthermore, edaravone treatment inhibited mitochondria-dependent apoptosis pathways in N2a/Swe.Δ9 cells through decreasing the Bax/Bcl-2 ratio, attenuating cytochrome c release and suppressing the activation of caspase-3. These results demonstrate that edaravone provides neuroprotection in an AD-related in vitro model and therefore, may be a potential complement for AD therapy.

  4. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

  5. Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

    PubMed Central

    Becchetti, A; Arcangeli, A; Del Bene, M R; Olivotto, M; Wanke, E

    1992-01-01

    The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models. PMID:1330041

  6. γ-secretase inhibitor I inhibits neuroblastoma cells, with NOTCH and the proteasome among its targets

    PubMed Central

    Dorneburg, Carmen; Goß, Annika V.; Fischer, Matthias; Roels, Frederik; Barth, Thomas F.E.; Berthold, Frank; Kappler, Roland; Oswald, Franz; Siveke, Jens T.; Molenaar, Jan J.; Debatin, Klaus-Michael; Beltinger, Christian

    2016-01-01

    As high-risk neuroblastoma (NB) has a poor prognosis, new therapeutic modalities are needed. We therefore investigated the susceptibility of NB cells to γ-secretase inhibitor I (GSI-I). NOTCH signaling activity, the cellular effects of GSI-I and its mechanisms of cytotoxicity were evaluated in NB cells in vitro and in vivo. The results show that NOTCH signaling is relevant for human NB cells. Of the GSIs screened in vitro GSI-I was the most effective inhibitor of NB cells. Both MYCN-amplified and non-amplified NB cells were susceptible to GSI-I. Among the targets of GSI-I in NB cells were NOTCH and the proteasome. GSI-I caused G2/M arrest that was enhanced by acute activation of MYCN and led to mitotic dysfunction. GSI-I also induced proapoptotic NOXA. Survival of mice bearing an MYCN non-amplified orthotopic patient-derived NB xenograft was significantly prolonged by systemic GSI-I, associated with mitotic catastrophe and reduced angiogenesis, and without evidence of intestinal toxicity. In conclusion, the activity of GSI-I on multiple targets in NB cells and the lack of gastrointestinal toxicity in mice are advantageous and merit further investigations of GSI-I in NB. PMID:27588497

  7. Retinoic acid inhibits the cytoproliferative response to weak 50-Hz magnetic fields in neuroblastoma cells

    PubMed Central

    TRILLO, MARÍA ÁNGELES; MARTÍNEZ, MARÍA ANTONIA; CID, MARÍA ANTONIA; ÚBEDA, ALEJANDRO

    2012-01-01

    We previously reported that intermittent exposure to a 50-Hz magnetic field (MF) at 100 μT stimulates cell proliferation in the human neuroblastoma cell line NB69. The present study aimed to investigate whether the magnetic field-induced growth promotion also occurs at a lower magnetic flux density of 10 μT. To this purpose, NB69 cells were subjected for 42 h to intermittent exposure, 3 h on/3 h off, to a 50-Hz MF at a 10 or 100 μT magnetic flux density. The field exposure took place either in the presence or in the absence of the antiproliferative agent retinoic acid. At the end of the treatment and/or incubation period, the cell growth was estimated by hemocytometric counting and spectrophotometric analysis of total protein and DNA contents. Potential changes in DNA synthesis were also assessed through proliferating cell nuclear antigen (PCNA) immunolabeling. The results confirmed previously reported data that a 42-h exposure to a 50-Hz sine wave MF at 100 μT promotes cell growth in the NB69 cell line, and showed that 10 μT induces a similar proliferative response. This effect, which was significantly associated and linearly correlated with PCNA expression, was abolished by the presence of retinoic acid in the culture medium. PMID:23292364

  8. Comparative analysis of some aspects of mitochondrial metabolism in differentiated and undifferentiated neuroblastoma cells.

    PubMed

    Klepinin, Aleksandr; Chekulayev, Vladimir; Timohhina, Natalja; Shevchuk, Igor; Tepp, Kersti; Kaldma, Andrus; Koit, Andre; Saks, Valdur; Kaambre, Tuuli

    2014-02-01

    The aim of the present study is to clarify some aspects of the mechanisms of regulation of mitochondrial metabolism in neuroblastoma (NB) cells. Experiments were performed on murine Neuro-2a (N2a) cell line, and the same cells differentiated by all-trans-retinoic acid (dN2a) served as in vitro model of normal neurons. Oxygraphy and Metabolic Control Analysis (MCA) were applied to characterize the function of mitochondrial oxidative phosphorylation (OXPHOS) in NB cells. Flux control coefficients (FCCs) for components of the OXPHOS system were determined using titration studies with specific non-competitive inhibitors in the presence of exogenously added ADP. Respiration rates of undifferentiated Neuro-2a cells (uN2a) and the FCC of Complex-II in these cells were found to be considerably lower than those in dN2a cells. Our results show that NB is not an exclusively glycolytic tumor and could produce a considerable part of ATP via OXPHOS. Two important enzymes - hexokinase-2 and adenylate kinase-2 can play a role in the generation of ATP in NB cells. MCA has shown that in uN2a cells the key sites in the regulation of OXPHOS are complexes I, II and IV, whereas in dN2a cells complexes II and IV. Results obtained for the phosphate and adenine nucleotide carriers showed that in dN2a cells these carriers exerted lower control over the OXPHOS than in undifferentiated cells. The sum of FCCs for both types of NB cells was found to exceed significantly that for normal cells suggesting that in these cells the respiratory chain was somehow reorganized or assembled into large supercomplexes.

  9. Second-generation proteasome inhibitor carfilzomib sensitizes neuroblastoma cells to doxorubicin-induced apoptosis

    PubMed Central

    Guan, Shan; Zhao, Yanling; Lu, Jiaxiong; Yu, Yang; Sun, Wenjing; Mao, Xinfang; Chen, Zhenghu; Xu, Xin; Pan, Jessie; Sun, Surong; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common extracranial malignant neoplasm in children. Elevated level of proteasome activity promotes cancer development and the inhibition of proteasome activity is a promising strategy for cancer treatment. Therefore, targeting proteasome by small molecule inhibitors may be a viable option for NB therapy. Here in this study, we show that a novel proteasome inhibitor Carfilzomib (CFZ) exerts anti-tumor effect on NB. CFZ caused decreased cell viability and attenuated colony formation ability of a subset of NB cell lines. CFZ induced cell apoptosis in NB cells. Moreover, CFZ enhanced the cytotoxic effect of doxorubicin (Dox) on NB cells and Dox-induced p38 and JNK phosphorylation. In addition, CFZ inhibited Dox-induced NF-κB activation by stabilizing the protein level of IκBα. Furthermore, CFZ induced apoptosis and augmented Dox-induced apoptosis in NB tumor cells in orthotopic xenograft mouse models. In summary, our study suggests that proteasome is a therapeutic target in NB and proteasome inhibition by CFZ is a potential therapeutic strategy for treating NB patients. PMID:27713150

  10. Diazinon oxon affects the differentiation of mouse N2a neuroblastoma cells.

    PubMed

    Sidiropoulou, Erasmia; Sachana, Magdalini; Flaskos, John; Harris, Wayne; Hargreaves, Alan J; Woldehiwet, Zerai

    2009-04-01

    The aim of this study was to assess the neurotoxicity of diazinon oxon (DZO), a major in vivo metabolite of the phosphorothionate insecticide diazinon (DZ), on differentiating mouse N2a neuroblastoma cells. When used at concentrations of 1, 5 and 10 microM, DZO did not cause cell death but it impaired the outgrowth of axon-like processes after 24 h. Densitometric scanning of Western blots of lysates of N2a cells revealed that exposure to 5 or 10 microM DZO for 24 h increased the expression of phosphorylated neurofilament heavy chain (NFH) compared to controls, while there was no significant change in total NFH. By contrast, treatment of N2a cells with 1-10 microM DZO resulted in marked reductions in the expression of the axon growth-associated protein GAP-43. DZO-treated cells also showed an increased expression of the heat shock protein HSP-70 compared to controls. The above biochemical changes were not temporally related to inhibition of acetylcholinesterase (AChE). These data suggest that biologically relevant, subcytotoxic levels of DZO may exert neurotoxic effects on differentiating cells and that the mechanisms involved are different from those attributed to its parent compound.

  11. 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.

  12. Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells

    PubMed Central

    Subramaniam, Vinod; Canters, Gerard W.; Schmidt, Thomas; Aartsma, Thijs J.

    2016-01-01

    Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson’s disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments. PMID:27105068

  13. 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.

  14. An improved sensitive assay for the detection of PSP toxins with neuroblastoma cell-based impedance biosensor.

    PubMed

    Zou, Ling; Wu, Chunsheng; Wang, Qin; Zhou, Jie; Su, Kaiqi; Li, Hongbo; Hu, Ning; Wang, Ping

    2015-05-15

    Paralytic shellfish poisoning (PSP) toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of neurological disorders symptoms. However, PSP toxins can inhibit the cytotoxicity effect of compounds (e.g., ouabain and veratridine). Under the treatment of ouabain and veratridine, neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na(+) and ouabain inhibits the activity of Na(+)/K(+)-ATPases. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na(+). Based on the antagonism effect of PSP toxins, we designed an improved cell-based assay to detect PSP toxins using a neuroblastoma cell-based impedance biosensor. The results demonstrated that this biosensor showed high sensitivity and good specificity for saxitoxins detection. The detection limit of this biosensor was as low as 0.03 ng/ml, which was lower than previous reported cell-based assays and mouse bioassays. With the improvement of biosensor performance, the neuroblastoma cell-based impedance biosensor has great potential to be a universal PSP screening method.

  15. Neuroblastoma Screening

    MedlinePlus

    ... is the most common type of cancer in infants. The number of new cases of neuroblastoma is ... credited as the source. Please note that blog posts that are written by individuals from outside the ...

  16. Acetyl-CoA metabolism in amprolium-evoked thiamine pyrophosphate deficits in cholinergic SN56 neuroblastoma cells.

    PubMed

    Bizon-Zygmańska, D; Jankowska-Kulawy, A; Bielarczyk, H; Pawełczyk, T; Ronowska, A; Marszałł, M; Szutowicz, A

    2011-08-01

    Inhibition of pyruvate (PDHC) and ketoglutarate (KDHC) dehydrogenase complexes induced by thiamine pyrophosphate deficits is known cause of disturbances of cholinergic transmission in the brain, yielding clinical symptoms of cognitive, vegetative and motor deficits. However, particular alterations in distribution of key acetylcholine precursor, acetyl-CoA, in the cholinergic neuron compartment of thiamine pyrophosphate-deficient brain remain unknown. Therefore, the aim of our work was to find out how amprolium-induced thiamine pyrophosphate deficits (TD) affect distribution of acetyl-CoA in the compartment of pure cholinergic neuroblastoma SN56 cells originating from murine septum. Amprolium caused similar concentration-dependent decreases in thiamine pyrophosphate levels in nondifferentiated (NC) and differentiated (DC) cells cultured in low thiamine medium. In such conditions DC displayed significantly greater loss of viability than the NC ones, despite of lesser suppressions of PDHC activities and tetrazolium salt reduction rates in the former. On the other hand, intramitochondrial acetyl-CoA levels in DC were 73% lower than in NC, which explains their greater susceptibility to TD. Choline acetyltransferase activity and acetylcholine content in DC were two times higher than in NC. TD caused 50% decrease of cytoplasmic acetyl-CoA levels that correlated with losses of acetylcholine pool in DC but not in NC. These data indicate that particular sensitivity of DC to TD may result from relative shortage of acetyl-CoA due to its higher utilization in acetylcholine synthesis.

  17. 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

  18. 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.

  19. Nutrient deprivation in neuroblastoma cells alters 4-hydroxynonenal-induced stress response.

    PubMed

    Zimmermann, Lars; Moldzio, Rudolf; Vazdar, Katarina; Krewenka, Christopher; Pohl, Elena E

    2017-01-31

    4-hydroxy-2-nonenal (HNE), a toxic lipid peroxidation product, is associated with oxidative damage in cells and involved in various diseases including the initiation and progression of cancer. Cancer cells have a high, adaptable metabolism with a shift from oxidative phosphorylation to glycolysis and rely on high levels of glucose and glutamine as essential nutrients for cell growth. Here we investigated whether the toxic effects of HNE on the mitochondrial membrane potential (MMP) of cancer cells depends on their metabolic state by deprivation of glucose and/or glutamine. The addition of 16 μM HNE to N18TG2 neuroblastoma cells incubated in glucose medium led to a severe reduction of MMP, which was similar to the MMP of cells fed with both glucose and glutamine. In contrast, HNE addition to cells starved in glutamine medium increased their MMP slightly for a prolonged time period and this was accompanied by increased cellular survival. We found that ß-oxidation of HNE did not cause the increased MMP, since the aldehyde dehydrogenase was distinctly more active in cells with glucose medium. However, after blocking fatty acid ß-oxidation in cells starved in glutamine medium with etomoxir, which inhibits carnitine palmitoyltransferase 1, HNE addition induced a strong reduction of MMP similar to cells in glucose medium. Surprisingly, the effect of more toxic 4-oxo-2-nonenal was less pronounced. Our results suggest that in contrast to cells fed with glucose, glutamine-fed cancer cells are capable of ß-oxidizing fatty acids to maintain their MMP to combat the toxic effects of HNE.

  20. Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

    PubMed Central

    2005-01-01

    We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal. PMID:15673285

  1. The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration.

    PubMed

    Villanueva, Andrea A; Falcón, Paulina; Espinoza, Natalie; R, Luis Solano; Milla, Luis A; Hernandez-SanMiguel, Esther; Torres, Vicente A; Sanchez-Gomez, Pilar; Palma, Verónica

    2017-02-07

    Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.

  2. Quantitative Nuclear Proteomics Identifies that miR-137-mediated EZH2 Reduction Regulates Resveratrol-induced Apoptosis of Neuroblastoma Cells*

    PubMed Central

    Ren, Xiaoqing; Bai, Xue; Zhang, Xuefei; Li, Zheyi; Tang, Lingfang; Zhao, Xuyang; Li, Zeyang; Ren, Yanfei; Wei, Shicheng; Wang, Qingsong; Liu, Cong; Ji, Jianguo

    2015-01-01

    Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4′-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment. PMID:25505154

  3. Networks of neuroblastoma cells on porous silicon substrates reveal a small world topology.

    PubMed

    Marinaro, Giovanni; La Rocca, Rosanna; Toma, Andrea; Barberio, Marianna; Cancedda, Laura; Di Fabrizio, Enzo; Decuzzi, Paolo; Gentile, Francesco

    2015-02-01

    The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized.

  4. The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells.

    PubMed

    Sachana, M; Flaskos, J; Alexaki, E; Glynn, P; Hargreaves, A J

    2001-01-01

    The aim of this work was to study the effects of chlorpyrifos (CPF) on the outgrowth of axons by differentiating mouse N2a neuroblastoma cells. This was achieved by morphological, Western blotting and enzymatic analyses of cells induced to differentiate in the presence and absence of CPF added either at the same time (co-differentiation) or 16 h after (post-differentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 h exposure to CPF in both co- and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8 h of exposure but no significant effect at 4 h under both co- and post-differentiation conditions. By contrast, levels of the heat shock protein HSP-70 were raised at both time points, but only in co-differentiation experiments. Neuropathy target esterase (NTE) activity was lower than controls following 4 or 8 h of exposure under co-differentiation conditions, but not under any post-differentiation conditions. The results suggest that the inhibition of axon production and maintenance by CPF in differentiating N2a cells may involve multiple targets, which are different under co- and post-differentiation conditions.

  5. Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells.

    PubMed

    Sun, Ying-Jian; Long, Ding-Xin; Li, Wei; Hou, Wei-Yuan; Wu, Yi-Jun; Shen, Jian-Zhong

    2010-02-01

    Avermectins (AVMs) are macrocyclic lactone compounds that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is associated with the efflux transport of AVMs and other drugs across the blood-brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivatives: abamectin (ABM) and doramectin (DOR). We found that both these compounds caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addition, Western blotting analysis showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, beta-actin and beta-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compounds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.

  6. Protective effects of ginsenoside Rg1 against hydrogen peroxide-induced injury in human neuroblastoma cells

    PubMed Central

    Sun, Zhi-gao; Chen, Li-ping; Wang, Fa-wei; Xu, Cheng-yong; Geng, Miao

    2016-01-01

    The active ingredient of ginseng, ginsenosides Rg1, has been shown to scavenge free radicals and improve antioxidant capacity. This study hypothesized that ginsenosides Rg1 has a protective role in human neuroblastoma cells injured by H2O2. Ginsenosides Rg1 at different concentrations (50 and 100 μM) was used to treat H2O2 (150 μM)-injured SH-SY5Y cells. Results demonstrated that ginsenoside Rg1 elevated the survival rate of SH-SY5Y cells injured by H2O2, diminished the amount of leaked lactate dehydrogenase, and increased superoxide dismutase activity. Ginsenoside Rg1 effectively suppressed caspase-3 immunoreactivity, and contributed to heat shock protein 70 gene expression, in a dose-dependent manner. These results indicate that ginsenoside Rg1 has protective effects on SH-SY5Y cells injured by H2O2 and that its mechanism of action is associated with anti-oxidation and the inhibition of apoptosis. PMID:27630703

  7. Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells

    PubMed Central

    LANGE, INGO; MOSCHNY, JULIA; TAMANYAN, KAMILLA; KHUTSISHVILI, MANANA; ATHA, DANIEL; BORRIS, ROBERT P.; KOOMOA, DANA-LYNN

    2016-01-01

    Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB. PMID:26848085

  8. Neurotoxic effects of local anesthetics on the mouse neuroblastoma NB2a cell line.

    PubMed

    Mete, M; Aydemir, I; Tuglu, I M; Selcuki, M

    2015-04-01

    Local anesthetics are used clinically for peripheral nerve blocks, epidural anesthesia, spinal anesthesia and pain management; large concentrations, continuous application and long exposure time can cause neurotoxicity. The mechanism of neurotoxicity caused by local anesthetics is unclear. Neurite outgrowth and apoptosis can be used to evaluate neurotoxic effects. Mouse neuroblastoma cells were induced to differentiate and generate neurites in the presence of local anesthetics. The culture medium was removed and replaced with serum-free medium plus 20 μl combinations of epidermal growth factor and fibroblast growth factor containing tetracaine, prilocaine, lidocaine or procaine at concentrations of 1, 10, 25, or 100 μl prior to neurite measurement. Cell viability, iNOS, eNOS and apoptosis were evaluated. Local anesthetics produced toxic effects by neurite inhibition at low concentrations and by apoptosis at high concentrations. There was an inverse relation between local anesthetic concentrations and cell viability. Comparison of different local anesthetics showed toxicity, as assessed by cell viability and apoptotic potency, in the following order: tetracaine > prilocaine > lidocaine > procaine. Procaine was the least neurotoxic local anesthetic and because it is short-acting, may be preferred for pain prevention during short procedures.

  9. Identification of novel targets for PGC-1{alpha} and histone deacetylase inhibitors in neuroblastoma cells

    SciTech Connect

    Cowell, Rita M. Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-02-06

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1{alpha} express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1{alpha} in neuronal cells and whether there are ways to pharmacologically target PGC-1{alpha} in neurons. Here, PGC-1{alpha} overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1{alpha} and glucose transporter 4 (GLUT4). These results suggest that PGC-1{alpha} regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1{alpha} and GLUT4 in HD and other neurological disorders.

  10. Ethanol activates midkine and anaplastic lymphoma kinase signaling in neuroblastoma cells and in the brain.

    PubMed

    He, Donghong; Chen, Hu; Muramatsu, Hisako; Lasek, Amy W

    2015-11-01

    Alcohol engages signaling pathways in the brain. Midkine (MDK) is a neurotrophic factor that is over-expressed in the prefrontal cortex of alcoholics. MDK and one of its receptors, anaplastic lymphoma kinase (ALK), also regulate behavioral responses to ethanol in mice. The goal of this study was to determine whether MDK and ALK expression and signaling are activated by ethanol. We found that ethanol treatment of neuroblastoma cells increased MDK and ALK expression. We also assessed activation of ALK by ethanol in cells and found that ALK and ALK-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation increased rapidly with ethanol exposure. Similarly, treatment of cells with recombinant MDK protein increased ALK, ERK and STAT3 phosphorylation, suggesting that ethanol may utilize MDK to activate ALK signaling. In support of this, transfection of cells with MDK siRNAs attenuated ALK signaling in response to ethanol. Ethanol also activates ERK signaling in the brain. We found that inhibition of ALK or knockout of MDK attenuated ethanol-induced ERK phosphorylation in mouse amygdala. These results demonstrate that ethanol engages MDK and ALK signaling, which has important consequences for alcohol-induced neurotoxicity and the regulation of behaviors related to alcohol abuse.

  11. Neuroprotection from diazinon-induced toxicity in differentiating murine N2a neuroblastoma cells.

    PubMed

    Harris, Wayne; Sachana, Magda; Flaskos, John; Hargreaves, Alan J

    2009-11-01

    In previous work, the outgrowth of axon-like processes by differentiating mouse N2a neuroblastoma cells was shown to be inhibited by exposure to 10 microM diazinon. In the present work, N2a cells were induced to differentiate for 24 h in the presence and absence of 10 microM diazinon and 20% (v/v) conditioned medium derived from differentiating rat C6 glioma cells. Cells were then stained or lysed for morphological and biochemical analyses, respectively. The data showed that co-treatment with conditioned medium prevented the neurite inhibitory effect of diazinon. Furthermore, a significant recovery was also observed in the reduced levels of neurofilament heavy chain (NFH), heat shock protein-70 (HSP-70) and growth-associated protein-43 (GAP-43) observed as a result of diazinon treatment in the absence of conditioned medium, as seen by densitometric analysis of Western blots of cell lysates probed with monoclonal antibodies N52, BRM-22 and GAP-7B10. By contrast, no significant change was noted in the reactivity of cell lysates with antibodies against alpha- and beta-tubulin under any condition tested. After pre-incubation with a polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) antibody, conditioned medium derived from rat C6 glioma cells lost its ability to protect N2a cells against the neurite inhibitory effects of diazinon. In conclusion, these data demonstrate that C6 conditioned medium protects N2a cells from the neurite inhibitory effects of diazinon by blocking molecular events leading to axon damage and that GDNF is implicated in these effects.

  12. Fenofibrate suppressed proliferation and migration of human neuroblastoma cells via oxidative stress dependent of TXNIP upregulation

    SciTech Connect

    Su, Cunjin; Shi, Aiming; Cao, Guowen; Tao, Tao; Chen, Ruidong; Hu, Zhanhong; Shen, Zhu; Tao, Hong; Cao, Bin; Hu, Duanmin; Bao, Junjie

    2015-05-15

    There are no appropriate drugs for metastatic neuroblastoma (NB), which is the most common extra-cranial solid tumor for childhood. Thioredoxin binding protein (TXNIP), the endogenous inhibitor of ROS elimination, has been identified as a tumor suppressor in various solid tumors. It reported that fenofibrate exerts anti-tumor effects in several human cancer cell lines. However, its detail mechanisms remain unclear. The present study assessed the effects of fenofibrate on NB cells and investigated TXNIP role in its anti-tumor mechanisms. We used MTT assay to detect cells proliferation, starch wound test to investigate cells migration, H{sub 2}DCF-DA to detect intracellular ROS, siRNA to interfere TXNIP and peroxisome proliferator-androgen receptor-alpha (PPAR-α) expression, western blot to determine protein levels, flow cytometry to analyze apoptosis. Fenofibrate suppressed proliferation and migration of NB cells, remarkably increased intracellular ROS, upregulated TXNIP expression, promoted cell apoptosis. Furthermore, inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. Our results indicated the anti-tumor role of fenofibrate on NB cells by exacerbating oxidative stress and inducing apoptosis was dependent on the upregulation of TXNIP. - Highlights: • We found that fenofibrate suppressed proliferation and migration of NB cells. • We found that fenofibrate remarkably increased intracellular ROS, upregulated TXNIP expression, and promoted cell apoptosis. • Inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. • Our results indicated the anti-tumor role of fenofibrate on NB cells was dependent on the upregulation of TXNIP.

  13. 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

  14. Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells.

    PubMed

    De Girolamo, L A; Hargreaves, A J; Billett, E E

    2001-02-01

    We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.

  15. Potential anticancer activity of carvone in N2a neuroblastoma cell line.

    PubMed

    Aydın, Elanur; Türkez, Hasan; Keleş, Mevlüt Sait

    2015-08-01

    Carvone (CVN) is a monocyclic monoterpene found in the essential oils of Mentha spicata var. crispa (Lamiaceae) and Carum carvi L. (Apiaceae) plants and has been reported to have antioxidant, antimicrobial, anticonvulsant, and antitumor activities. The beneficial health properties of CVN have encouraged us to look into its anticancer activity. To the best of our knowledge, reports are not available on the anticancer activity of CVN in cultured primary rat neuron and N2a neuroblastoma (NB) cells. Therefore, the present study is an attempt toward exploring the potential anticancer activity of CVN, if any, in cultured primary rat neuron and N2a NB cells. Our results indicated that CVN (only at 25 mg/L) treatment led to an increase in the total antioxidant capacity levels in cultured primary rat neuron cells compared with control cells. Also, CVN (at concentrations higher than 100 mg/L) treatment led to an increase in the total oxidative stress levels in both cell types. The mean values of the total scores of cells showing DNA damage (for comet assay) were not found to be significantly different from the control values in both cells (p > 0.05). On the other hand, after 24 h treatment with CVN, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay showed that CVN application significantly reduced the cell viability rates in both cell types at concentrations higher than 100 mg/L. Summarizing, our data suggest that CVN represents little potential for promising anticancer agent to improve brain tumors therapy.

  16. miR-362-5p inhibits proliferation and migration of neuroblastoma cells by targeting phosphatidylinositol 3-kinase-C2β.

    PubMed

    Wu, Kai; Yang, Liucheng; Chen, Jianfeng; Zhao, Haijun; Wang, Jianjun; Xu, Shuai; Huang, Zonghai

    2015-07-08

    miR-362-5p is down-regulated in high-risk neuroblastoma and can function as a tumor suppressor. However, its role remains poorly understood. We show that miR-362-5p is down-regulated in metastatic neuroblastoma compared with primary neuroblastoma. Overexpression of miR-362-5p inhibits cell proliferation, migration and invasion of neuroblastoma cells in vitro and suppresses tumor growth of neuroblastoma in vivo. Phosphatidylinositol 3-kinase (PI3K)-C2β is a target of miR-362-5p. Knockdown of PI3K-C2β by siRNA had a similar effect to overexpression of miR-362-5p on SH-SY5Y cells. Overexpression of PI3K-C2β partially reversed tumor-suppressive effects of miR-362-5p. We suggest that miR-362-5p suppresses neuroblastoma cell growth and motility, partially by targeting PI3K-C2β.

  17. 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

  18. Fipronil interferes with the differentiation of mouse N2a neuroblastoma cells.

    PubMed

    Sidiropoulou, Erasmia; Sachana, Magdalini; Flaskos, John; Harris, Wayne; Hargreaves, Alan J; Woldehiwet, Zerai

    2011-02-25

    The purpose of this study was to evaluate the neurotoxic potential of the pesticide fipronil (FIP) towards the differentiation of mouse N2a neuroblastoma cells. At concentrations of 1, 5 and 10 μM that were not cytotoxic, as shown by two different cell viability assays, FIP impaired potently after 24h the development of axon-like processes, with a concentration of 1 μM causing 50% inhibition. Densitometric analysis of immunoblots of extracts of N2a cells exposed to FIP demonstrated that the axon-inhibitory action of the pesticide was not accompanied by significant changes in the levels of total and phosphorylated neurofilament heavy chain (NFH). FIP also induced no alteration in the levels of total and tyrosinated α-tubulin. On the other hand, this pesticide caused severe disruption of the developmentally important ERK 1/2-MAP kinase signal transduction pathway, as evidenced by significant reductions in the activation state of MAPK kinase (MEK 1/2) and, particularly, ERK 1/2. The above data seem to justify very recent concerns that FIP has the capacity to induce developmental neurotoxicity in mammals.

  19. [Effect of estrogen on nucleotide excision repair of N2a neuroblastoma cells].

    PubMed

    Yoshioka, Akira; Yamamoto, Aya; Mori, Toshio; Nakamura, Yu; Morikawa, Masayuki; Yoshino, Hiroki; Kiuchi, Kuniaki; Makinodan, Manabu; Kishimoto, Toshifumi

    2007-04-01

    Until now reduced estrogen level has been considered to affect some psychiatric symptoms, because there are sex differences in onset of Schizophrenia and Alzheimer's disease. Estrogen is associated with cognitive functions, and it has been reported to protect oxidative damage of DNA related to base excision repair (BER). Some patients with Xeroderma Pigmentosum, who have normal BER and impaired nucleotide excision repair (NER), are known to be suffering from mental retardation. Therefore we hypothesized that impaired NER was partly associated with pathology of mental disorder and investigated the effects of estrogen on NER for ultraviolet-induced DNA damage. The N2a neuroblastoma cell line was used as a representative of neuronal cells and 17p-estradiol was selected as one of the most active estrogen derivatives. There were no significant effects of 17p-estradiol on prevention of DNA damage, promotion of DNA repair, or cell survival at the concentration of 0-0.1 microM 17p-estradiol (below cytotoxicity level). These results described that estrogen might not directly affect NER except through another DNA repair system.

  20. Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis

    PubMed Central

    Bouzas-Rodriguez, Jimena; Cabrera, Jorge Ruben; Delloye-Bourgeois, Céline; Ichim, Gabriel; Delcros, Jean-Guy; Raquin, Marie-Anne; Rousseau, Raphaël; Combaret, Valérie; Bénard, Jean; Tauszig-Delamasure, Servane; Mehlen, Patrick

    2010-01-01

    Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor with tyrosine kinase activity that was expected to be oncogenic. However, it has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis; and it was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand. Here we show that the TrkC ligand neurotrophin-3 (NT-3) is upregulated in a large fraction of aggressive human neuroblastomas (NBs) and that it blocks TrkC-induced apoptosis of human NB cell lines, consistent with the idea that TrkC is a dependence receptor. Functionally, both siRNA knockdown of NT-3 expression and incubation with a TrkC-specific blocking antibody triggered apoptosis in human NB cell lines. Importantly, disruption of the NT-3 autocrine loop in malignant human neuroblasts triggered in vitro NB cell death and inhibited tumor growth and metastasis in both a chick and a mouse xenograft model. Thus, we believe that our data suggest that NT-3/TrkC disruption is a putative alternative targeted therapeutic strategy for the treatment of NB. PMID:20160348

  1. Aberrant regulation of choline metabolism by mitochondrial electron transport system inhibition in neuroblastoma cells

    PubMed Central

    Baykal, Ahmet T.; Jain, Mohit R.

    2009-01-01

    Anomalous choline metabolic patterns have been consistently observed in vivo using Magnetic Resonance Spectroscopy (MRS) analysis of patients with neurodegenerative diseases and tissues from cancer patient. It remains unclear; however, what signaling events may have triggered these choline metabolic aberrancies. This study investigates how changes in choline and phospholipid metabolism are regulated by distinct changes in the mitochondrial electron transport system (ETS). We used specific inhibitors to down regulate the function of individual protein complexes in the ETS of SH-SY5Y neuroblastoma cells. Interestingly, we found that dramatic elevation in the levels of phosphatidylcholine metabolites could be induced by the inhibition of individual ETS complexes, similar to in vivo observations. Such interferences produced divergent metabolic patterns, which were distinguishable via principal component analysis of the cellular metabolomes. Functional impairments in ETS components have been reported in several central nervous system (CNS) diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases. PMID:19774105

  2. MicroRNA-23a promotes neuroblastoma cell metastasis by targeting CDH1.

    PubMed

    Cheng, Lin; Yang, Tao; Kuang, Yongqin; Kong, Bin; Yu, Sixun; Shu, Haifeng; Zhou, Hutian; Gu, Jianwen

    2014-03-01

    CDH1 inactivation is important in tumor metastasis. In the present study, it was suggested that the mRNA and protein levels of CDH1 decreased in metastatic neuroblastoma (NB) tissues compared with those in primary NB tissues. The aim of the study was to explore the regulatory mechanisms of CDH1 downregulation in metastatic NB. MicroRNAs are small non-coding RNAs (~22 nt in length) that negatively regulate target mRNAs and are involved in various cancer-related processes, including metastasis. In the current study, miR-23a was shown to be upregulated in human metastatic NB tissues compared with primary NB tissues. Inhibition of miR-23a may significantly suppress NB cell migration and invasion. In vitro reporter assay suggested that CDH1 is a direct target gene of miR-23a. Furthermore, blocking the expression of miR-23a partly restored the expression of CDH1 in NB cells. These findings provide evidence that miR-23a is key in promoting NB cell migration and invasion through targeting CDH1, and suggest that exogenous miR-23a may have therapeutic value in treating NB metastasis.

  3. 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.

  4. The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells.

    PubMed

    Chen, JinYan; Sun, MengRu; Wang, XingHua; Lu, Jing; Wei, Yan; Tan, Yan; Liu, Ying; Götz, Jürgen; He, RongQiao; Hua, Qian

    2014-04-01

    The herbal medicine Tong Luo Jiu Nao (TLJN) contains geniposide (GP) and ginsenoside Rg1 at a molar ratio of 10:1. Rg1 is the major component of another herbal medicine, panax notoginseng saponin (PNS). TLJN has been shown to strengthen brain function in humans, and in animals it improves learning and memory. We have previously shown that TLJN reduces amyloidogenic processing in Alzheimer's disease (AD) mouse models. Together this suggests TLJN may be a potential treatment for patients with dementia. Because chronic damage of the central nervous system by formaldehyde (FA) has been presented as a risk factor for age-associated cognitive dysfunction, in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA. FA-exposed murine N2a neuroblastoma cells were incubated with TLJN, its main ingredient GP, as well as PNS, to measure cell viability and morphology, the rate of apoptosis and expression of genes encoding Akt, FOXO3, Bcl2 and p53. The CCK-8 assay, cytoskeletal staining and flow cytometry were used to test cell viability, morphology and apoptosis, respectively. Fluorescent quantitative real-time PCR (qRT-PCR) was used to monitor changes in gene expression, and HPLC to determine the rate of FA clearance. Treatment of N2a cells with 0.09 mmol L(-1) FA for 24 h significantly reduced cell viability, changed cell morphology and promoted apoptosis. Both TLJN and GP conferred neuroprotection to FA-treated N2a cells, whereas PNS, which had to be used at lower concentrations because of its toxicity, did not. Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient, GP, has a major role in this efficacy. This presents purified GP as a drug or lead compound for the treatment of AD.

  5. Natural killer cells and neuroblastoma: tumor recognition, escape mechanisms, and possible novel immunotherapeutic approaches.

    PubMed

    Bottino, Cristina; Dondero, Alessandra; Bellora, Francesca; Moretta, Lorenzo; Locatelli, Franco; Pistoia, Vito; Moretta, Alessandro; Castriconi, Roberta

    2014-01-01

    Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and arises from developing sympathetic nervous system. Most primary tumors localize in the abdomen, the adrenal gland, or lumbar sympathetic ganglia. Amplification in tumor cells of MYCN, the major oncogenic driver, patients' age over 18 months, and the presence at diagnosis of a metastatic disease (stage IV, M) identify NB at high risk of treatment failure. Conventional therapies did not significantly improve the overall survival of these patients. Moreover, the limited landscape of somatic mutations detected in NB is hampering the development of novel pharmacological approaches. Major efforts aim to identify novel NB-associated surface molecules that activate immune responses and/or direct drugs to tumor cells and tumor-associated vessels. PVR (Poliovirus Receptor) and B7-H3 are promising targets, since they are expressed by most high-risk NB, are upregulated in tumor vasculature and are essential for tumor survival/invasiveness. PVR is a ligand of DNAM-1 activating receptor that triggers the cytolytic activity of natural killer (NK) cells against NB. In animal models, targeting of PVR with an attenuated oncolytic poliovirus induced tumor regression and elimination. Also B7-H3 was successfully targeted in preclinical studies and is now being tested in phase I/II clinical trials. B7-H3 down-regulates NK cytotoxicity, providing NB with a mechanism of escape from immune response. The immunosuppressive potential of NB can be enhanced by the release of soluble factors that impair NK cell function and/or recruitment. Among these, TGF-β1 modulates the cytotoxicity receptors and the chemokine receptor repertoire of NK cells. Here, we summarize the current knowledge on the main cell surface molecules and soluble mediators that modulate the function of NK cells in NB, considering the pros and cons that must be taken into account in the design of novel NK cell-based immunotherapeutic approaches.

  6. Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

    SciTech Connect

    Khung, Y.L.; Barritt, G.; Voelcker, N.H.

    2008-02-15

    The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce sample numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For example, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell-cell interactions crosslinked the cell layer to the substratum surface, while direct substrate-cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of < 20 nm.

  7. Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

    PubMed Central

    Zhang, J; Loew, L M; Davidson, R M

    1996-01-01

    Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

  8. 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).

  9. Identification of Multiple Hypoxia Signatures in Neuroblastoma Cell Lines by l1-l2 Regularization and Data Reduction

    PubMed Central

    Fardin, Paolo; Cornero, Andrea; Barla, Annalisa; Mosci, Sofia; Acquaviva, Massimo; Rosasco, Lorenzo; Gambini, Claudio; Verri, Alessandro; Varesio, Luigi

    2010-01-01

    Hypoxia is a condition of low oxygen tension occurring in the tumor and negatively correlated with the progression of the disease. We studied the gene expression profiles of nine neuroblastoma cell lines grown under hypoxic conditions to define gene signatures that characterize hypoxic neuroblastoma. The l1-l2 regularization applied to the entire transcriptome identified a single signature of 11 probesets discriminating the hypoxic state. We demonstrate that new hypoxia signatures, with similar discriminatory power, can be generated by a prior knowledge-based filtering in which a much smaller number of probesets, characterizing hypoxia-related biochemical pathways, are analyzed. l1-l2 regularization identified novel and robust hypoxia signatures within apoptosis, glycolysis, and oxidative phosphorylation Gene Ontology classes. We conclude that the filtering approach overcomes the noisy nature of the microarray data and allows generating robust signatures suitable for biomarker discovery and patients risk assessment in a fraction of computer time. PMID:20652058

  10. Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma.

    PubMed

    Gutova, Margarita; Goldstein, Leanne; Metz, Marianne; Hovsepyan, Anahit; Tsurkan, Lyudmila G; Tirughana, Revathiswari; Tsaturyan, Lusine; Annala, Alexander J; Synold, Timothy W; Wan, Zesheng; Seeger, Robert; Anderson, Clarke; Moats, Rex A; Potter, Philip M; Aboody, Karen S

    2017-03-17

    Despite improved survival for children with newly diagnosed neuroblastoma (NB), recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs) expressing a modified rabbit carboxylesterase (rCE) can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11) to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093) and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

  11. 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.

  12. A discriminant block among K+ channel types by phenytoin in neuroblastoma cells

    PubMed Central

    Nobile, M; Lagostena, L

    1998-01-01

    The action of the anticonvulsant drug phenytoin on K+ channels was investigated in neuroblastoma cells (N2A) by using the single-channel patch-clamp technique. N2A cells expressed three types of delayed rectifier K+ channels, which were found to have a conductance of 10–20 pS in a `physiological' K+ gradient. When added to the external solution at concentrations ranging between 1 and 200 μM, phenytoin decreased single channel activity, whereas the unitary current amplitude was unaffected in all three types of channels. The open probability of the biggest channel decreased, according to an exponential distribution of open and closed times, from 40% in control conditions to 10% in the presence of 50 μM phenytoin (Vm=40 mv). The reduction in the open-channel probability was concentration-dependent with a IC50=27.2±0.9 μM. A transient type of K+ channel was identified that was affected by cumulative inactivation and had a conductance of a mean value equal to 26 pS. Finally, a voltage-and Ca2+-dependent K+ channel with a unitary conductance of 95 pS was recorded. Both the channel's amplitude and kinetics were unaffected by phenytoin. These results confirm the phenytoin effect on K+ currents and suggest that the drug may be considered a selective blocker of delayed rectifier K+ channels. PMID:9756386

  13. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    PubMed

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

  14. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  15. Seladin-1/DHCR24 protects neuroblastoma cells against Aβ toxicity by increasing membrane cholesterol content

    PubMed Central

    Cecchi, C; Rosati, F; Pensalfini, A; Formigli, L; Nosi, D; Liguri, G; Dichiara, F; Morello, M; Danza, G; Pieraccini, G; Peri, A; Serio, M; Stefani, M

    2008-01-01

    The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-β-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD. PMID:18194465

  16. JARID1B Expression Plays a Critical Role in Chemoresistance and Stem Cell-Like Phenotype of Neuroblastoma Cells

    PubMed Central

    Adebayo, Bamodu Oluwaseun; Shih, Ping-Hsiao; Lee, Wei-Hwa; Wang, Liang-Shun; Liao, Yung-Feng; Hsu, Wen-Ming; Yeh, Chi-Tai; Lin, Chien-Min

    2015-01-01

    Neuroblastoma (NB) is a common neural crest-derived extracranial solid cancer in children. Among all childhood cancers, NB causes devastating loss of young lives as it accounts for 15% of childhood cancer mortality. Neuroblastoma, especially high-risk stage 4 NB with MYCN amplification has limited treatment options and associated with poor prognosis. This necessitates the need for novel effective therapeutic strategy. JARID1B, also known as KDM5B, is a histone lysine demethylase, identified as an oncogene in many cancer types. Clinical data obtained from freely-accessible databases show a negative correlation between JARID1B expression and survival rates. Here, we demonstrated for the first time the role of JARID1B in the enhancement of stem cell-like activities and drug resistance in NB cells. We showed that JARID1B may be overexpressed in either MYCN amplification (SK-N-BE(2)) or MYCN-non-amplified (SK-N-SH and SK-N-FI) cell lines. JARID1B expression was found enriched in tumor spheres of SK-N-BE(2) and SK-N-DZ. Moreover, SK-N-BE(2) spheroids were more resistant to chemotherapeutics as compared to parental cells. In addition, we demonstrated that JARID1B-silenced cells acquired a decreased propensity for tumor invasion and tumorsphere formation, but increased sensitivity to cisplatin treatment. Mechanistically, reduced JARID1B expression led to the downregulation of Notch/Jagged signaling. Collectively, we provided evidence that JARID1B via modulation of stemness-related signaling is a putative novel therapeutic target for treating malignant NB. PMID:25951238

  17. Morus alba Accumulates Reactive Oxygen Species to Initiate Apoptosis via FOXO-Caspase 3-Dependent Pathway in Neuroblastoma Cells

    PubMed Central

    Kwon, Young Hwi; Bishayee, Kausik; Rahman, Ataur; Hong, Jae Seung; Lim, Soon-Sung; Huh, Sung-Oh

    2015-01-01

    Morus alba root extract (MARE) has been used to treat hyperglycaemic conditions in oriental medicine. Here, we studied whether MARE possesses a cytotoxic effect on neuroblastoma. To check the cytotoxicity generated by MARE was whether relatively higher against the cancer cells rather than normal cells, we chose a neuroblastoma cell line (B103) and a normal cell line (Rat-2). A CCK assay revealed that MARE (10 μg/ml) reduced cell viability to approximately 60% compared to an untreated control in B103 cells. But in Rat-2 cells, MARE induced relatively lower cytotoxicity. To investigate the mechanisms underlying the cytotoxic effect of MARE, we used flow cytometry combined with immunoblot analyses. We found that MARE-treatment could accumulate ROS and depolarize mitochondria membrane potential of B103 cells. Further treatment with MARE in B103 cells also could damage DNA and induce apoptosis. An expression study of p-Akt also suggested that there was a reduction in cellular proliferation and transcription along with the process of apoptosis, which was further evidenced by an increase in Bax and cleaved-caspase 3 activity. Together, our findings suggest that MARE produces more cytotoxicity in cancer cells while having a relatively attenuated effect on normal cells. As such, MARE may be a safer option in cancer therapeutics, and it also shows potential for the patients with symptoms of hyperglycemia and cancer. PMID:25921607

  18. Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1

    PubMed Central

    Ma, Qi; Hu, Qing-song; Xu, Ran-jie; Zhen, Xue-chu; Wang, Guang-hui

    2015-01-01

    Aim: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process. Methods: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 μmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software. Results: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1. Conclusion: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi. PMID:26238290

  19. Investigating the neuroglial differentiation effect of neuroblastoma conditioned medium in human endometrial stem cells cultured on 3D nanofibrous scaffold.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Norouzi Javidan, Abbas; Ai, Jafar

    2015-08-01

    Neural tissue engineering is an important area of research in the field of tissue-engineering especially for neurodegenerative disease such as spinal cord injury. The differentiation capacity of human endometrial stem cells (hEnSCs) into neuronal cells has yet to be elucidated. Here, the major aim of the present study was to investigate the differentiation ability of hEnSCs cultured on polylactic acid/chitosan (PLA/CS) nanofibrous scaffold into neuroglial cells in response to conditioned medium of BE(2)-C human neuroblastoma cells and growth factors. Here we investigated the use PLA/CS scaffold as a three dimensional (3D) system that increased neuro-glial cells differentiation. Human EnSCs after three passages were differentiated in neuro-glial like cells under neuroblastoma conditioned medium with FGF2/PDGF-AA on PLA/CS scaffold. By day 18, differentiated cells were analyzed for expression of neuroglial markers by qRT-PCR and immunofluorescence. The results revealed that hEnSCs attach, grow and differentiation on the nanofibrous PLA/CS scaffold. Additionally, our study showed the expression of neural and glial lineage markers such as Nestin, NF-L, MAP2, PDGFRa, CNP, Olig2, MBP, and GFAP in the level of mRNA and MAP2, Tuj-1, and NF-L in the protein level after 18 days. Our results demonstrate that hEnSCs cultured on PLA/CS nanofibrous scaffold have the potential to differentiate in neuronal and glial cells in presence of neuroblastoma conditioned medium on PLA/CS scaffold. The result of this study may have impact in tissue engineering and cells-base therapy of neurodegenerative diseases and have a great potential for wide application.

  20. Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

    PubMed Central

    Yeung, Pan; Sin, Hoi Shun; Chan, Shing; Chan, Godfrey Chi Fung; Chan, Barbara Pui

    2015-01-01

    There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies. PMID:26657086

  1. CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells.

    PubMed

    Zhou, D; Song, Z H

    2001-08-15

    The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain.

  2. TNF-α stimulates efficient JC virus replication in neuroblastoma cells.

    PubMed

    Nukuzuma, Souichi; Nakamichi, Kazuo; Kameoka, Masanori; Sugiura, Shigeki; Nukuzuma, Chiyoko; Tasaki, Takafumi; Takegami, Tsutomu

    2014-12-01

    JC polyomavirus (JCV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS) in immunocompromised patients, and particularly in the severe immunosuppression associated with acquired immunodeficiency syndrome (AIDS). HIV-1 can lead to the production of tumor necrosis factor-alpha (TNF-α) in the CNS. Our aim was to examine the effects of TNF-α on JCV gene expression and replication using a human neuroblastoma cell line, IMR-32, transfected with JCV DNA, M1-IMRb. Quantitative RT-PCR analysis of JCV large T antigen and VP1 mRNA, the viral DNA replication assay, and the DNase protection assay were carried out. TNF-α treatment of IMR-32 cells transfected with JCV DNA induced large T antigen mRNA and JCV DNA replication, while other effects on VP1 mRNA expression and virus production were marginal. In addition, ELISA analysis of the nuclear p65 subunit of nuclear factor κB (NF-κB), which is a hallmark of NF-κB pathway activation, of IMR-32 cells upon TNF-α treatment showed that TNF-α treatment activated the NF-κB pathway in IMR-32 cells. Taken together, our results suggest that TNF-α stimulation could induce JCV replication associated with the induction of JCV large T antigen mRNA through the NF-κB pathway in IMR-32 cells transfected with JCV DNA. Our findings may contribute to further understanding of the pathogenesis of AIDS-related PML.

  3. IKAP/hELP1 downregulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

    PubMed Central

    Cohen-Kupiec, Rachel; Weinstein, Shiri; Kantor, Gal; Peer, Dan

    2010-01-01

    A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP-downregulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development. PMID:20671422

  4. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

    PubMed Central

    1993-01-01

    Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by

  5. ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells.

    PubMed

    Liu, Zhiying; Qiao, Luxin; Zhang, Yulin; Zang, Yunjing; Shi, Ying; Liu, Kai; Zhang, Xin; Lu, Xiaofan; Yuan, Lin; Su, Bin; Zhang, Tong; Wu, Hao; Chen, Dexi

    2017-01-01

    HIV invasion of the central nervous system (CNS) in the majority of patients infected with HIV-1, leads to dysfunction and injury within the CNS, showing a variety of neurological symptoms which was broadly termed HIV-associated neurocognitive disorder (HAND). But the molecular mechanisms are not completely understood. It has been suggested that apoptosis and autophagic dysfunction in neurons may play an important role in the development of HAND. Previous studies have indicated that p53 may be involved in the onset of neurological disorder in AIDS. Apoptosis-stimulating protein of p53-2 (ASPP2), a p53-binding protein with specific function of inducing p53, has been reported to modulate autophagy. In the present study, we observed that gp120 induces autophagy and apoptosis in SH-SY5Y neuroblastoma cells. Adenovirus-mediated overexpression of ASPP2 significantly inhibited autophagy and apoptosis induced by low dose of gp120 protein (50 ng/mL), but induced autophagy and apoptosis when treated by high dose of gp120 protein (200 ng/mL). Further, ASPP2 knockdown attenuated autophagy and apoptosis induced by gp120. Conclusion: ASPP2 had different effects on the autophagy and apoptosis of neurons induced by different concentration of gp120 protein. It may be a potential therapeutic agent for HAND through modulating autophagy and apoptosis in CNS.

  6. ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells

    PubMed Central

    Liu, Zhiying; Qiao, Luxin; Zhang, Yulin; Zang, Yunjing; Shi, Ying; Liu, Kai; Zhang, Xin; Lu, Xiaofan; Yuan, Lin; Su, Bin; Zhang, Tong; Wu, Hao; Chen, Dexi

    2017-01-01

    HIV invasion of the central nervous system (CNS) in the majority of patients infected with HIV-1, leads to dysfunction and injury within the CNS, showing a variety of neurological symptoms which was broadly termed HIV-associated neurocognitive disorder (HAND). But the molecular mechanisms are not completely understood. It has been suggested that apoptosis and autophagic dysfunction in neurons may play an important role in the development of HAND. Previous studies have indicated that p53 may be involved in the onset of neurological disorder in AIDS. Apoptosis-stimulating protein of p53-2 (ASPP2), a p53-binding protein with specific function of inducing p53, has been reported to modulate autophagy. In the present study, we observed that gp120 induces autophagy and apoptosis in SH-SY5Y neuroblastoma cells. Adenovirus-mediated overexpression of ASPP2 significantly inhibited autophagy and apoptosis induced by low dose of gp120 protein (50 ng/mL), but induced autophagy and apoptosis when treated by high dose of gp120 protein (200 ng/mL). Further, ASPP2 knockdown attenuated autophagy and apoptosis induced by gp120. Conclusion: ASPP2 had different effects on the autophagy and apoptosis of neurons induced by different concentration of gp120 protein. It may be a potential therapeutic agent for HAND through modulating autophagy and apoptosis in CNS. PMID:28392757

  7. Rab 11 regulates constitutive dopamine transporter trafficking and function in N2A neuroblastoma cells.

    PubMed

    Furman, Cheryse A; Lo, Charles B; Stokes, Stephanie; Esteban, Jose A; Gnegy, Margaret E

    2009-09-29

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission which undergoes constitutive and substrate-mediated trafficking to and from the membrane. Although, considerable research has been done to elucidate the regulation of substrate-stimulated DAT trafficking, less is known about which trafficking proteins are involved in constitutive DAT trafficking. Rab proteins are GTPases known to regulate the trafficking of proteins to and from specific endocytic compartments. Rabs 8 and 11, in particular, are involved in trafficking proteins from intracellular compartments to the plasma membrane. In this study, we sought to determine whether Rabs 8 and 11 would modulate DAT activity and trafficking in N2A neuroblastoma cells. We used Rab mutations known to confer constitutively active or dominant negative activity of these proteins to investigate the role of Rab activity in constitutive DAT trafficking and function. We found that constitutively active Rab 11 upregulates DAT function and surface expression while neither the constitutively active nor the dominant negative mutant of Rab 8 had any effect on DA uptake. Furthermore, immunofluorescence experiments revealed that dominant negative Rab 11 overexpression results in decreased surface DAT indicating a necessary function of Rab 11 in DAT trafficking to the plasma membrane. These data show for the first time a functional role of Rab proteins in the constitutive recycling of DAT to the plasma membrane.

  8. Antidepressants modulate intracellular amyloid peptide species in N2a neuroblastoma cells.

    PubMed

    Aboukhatwa, Marwa; Luo, Yuan

    2011-01-01

    It is estimated that 30%-50% of Alzheimer's disease (AD) patients are diagnosed with major or minor depression. Research that addresses the relationship between these two diseases will benefit patients who suffer from depression comorbid with AD and allow further understanding of the neuroanatomy of depression. A clinical study showed that the use of the antidepressant fluoxetin concomitantly with the FDA-approved AD drug rivastigmine provided an improvement in the daily activities and the overall functioning in the patients with cognitive impairment. In an attempt to understand the underlying mechanism for the antidepressant's beneficial effect in AD patients, we evaluated the effects of different classes of antidepressants on the amyloid-β peptide (Aβ) species in N2a neuroblastoma cells overexpressing amyloid-β protein precursor. The effect of increasing antidepressant concentrations on the intracellular and secreted Aβ species is investigated by Western blotting. The tested antidepressants include fluoxetine, paroxetine, maprotiline, and imipramine. Fluoxetine and paroxetine at 10 μM significantly decreased the intracellular level of Aβ oligomers and increased the level of Aβ monomers. However, imipramine and maprotiline increased the intracellular amount of Aβ monomers without affecting Aβ oligomers. Based on these results, it is possible that fluoxetine and paroxetine could be beneficial to AD patients via reducing the level of the cytotoxic oligomers and keeping the Aβ peptide in the monomeric form. These data could explain some of the beneficial effects of antidepressants in AD patients observed in clinical studies.

  9. Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study.

    PubMed

    Kintzios, S; Yiakoumetis, I; Moschopoulou, G; Mangana, O; Nomikou, K; Simonian, A

    2007-11-30

    In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.

  10. Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

    SciTech Connect

    Suwalsky, Mario; Gonzalez, Raquel; Villena, Fernando; Aguilar, Luis F.; Sotomayor, Carlos P.; Bolognin, Silvia; Zatta, Paolo

    2010-06-25

    Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl{sub 3} was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 {mu}M; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 {mu}M concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 {mu}m-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 {mu}M to 100 {mu}M.

  11. Aryl Hydrocarbon Receptor Downregulates MYCN Expression and Promotes Cell Differentiation of Neuroblastoma

    PubMed Central

    Wu, Pei-Yi; Liao, Yung-Feng; Juan, Hsueh-Fen; Huang, Hsuan-Cheng; Wang, Bo-Jeng; Lu, Yen-Lin; Yu, I-Shing; Shih, Yu-Yin; Jeng, Yung-Ming; Hsu, Wen-Ming; Lee, Hsinyu

    2014-01-01

    Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation. PMID:24586395

  12. An image-based model of calcium waves in differentiated neuroblastoma cells.

    PubMed Central

    Fink, C C; Slepchenko, B; Moraru, I I; Watras, J; Schaff, J C; Loew, L M

    2000-01-01

    Calcium waves produced by bradykinin-induced inositol-1,4, 5-trisphosphate (InsP(3))-mediated release from endoplasmic reticulum (ER) have been imaged in N1E-115 neuroblastoma cells. A model of this process was built using the "virtual cell," a general computational system for integrating experimental image, biochemical, and electrophysiological data. The model geometry was based on a cell for which the calcium wave had been experimentally recorded. The distributions of the relevant cellular components [InsP(3) receptor (InsP(3)R)], sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pumps, bradykinin receptors, and ER] were based on 3D confocal immunofluorescence images. Wherever possible, known biochemical and electrophysiological data were used to constrain the model. The simulation closely matched the spatial and temporal characteristics of the experimental calcium wave. Predictions on different patterns of calcium signals after InsP(3) uncaging or for different cell geometries were confirmed experimentally, thus helping to validate the model. Models in which the spatial distributions of key components are altered suggest that initiation of the wave in the center of the neurite derives from an interplay of soma-biased ER distribution and InsP(3) generation biased toward the neurite. Simulations demonstrate that mobile buffers (like the indicator fura-2) significantly delay initiation and lower the amplitude of the wave. Analysis of the role played by calcium diffusion indicated that the speed of the wave is only slightly dependent on the ability of calcium to diffuse to and activate neighboring InsP(3) receptor sites. PMID:10866945

  13. Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells.

    PubMed Central

    Bisogno, T; Sepe, N; Melck, D; Maurelli, S; De Petrocellis, L; Di Marzo, V

    1997-01-01

    The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2

  14. The inhibition of 45A ncRNA expression reduces tumor formation, affecting tumor nodules compactness and metastatic potential in neuroblastoma cells.

    PubMed

    Penna, Ilaria; Gigoni, Arianna; Costa, Delfina; Vella, Serena; Russo, Debora; Poggi, Alessandro; Villa, Federico; Brizzolara, Antonella; Canale, Claudio; Mescola, Andrea; Daga, Antonio; Russo, Claudio; Nizzari, Mario; Florio, Tullio; Menichini, Paola; Pagano, Aldo

    2017-01-31

    We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA.45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach.

  15. Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity.

    PubMed

    Rickard, Steve; Ono, Santa Jeremy

    2008-12-01

    A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

  16. 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.

  17. Differential role of RIP1 in Smac mimetic-mediated chemosensitization of neuroblastoma cells

    PubMed Central

    Czaplinski, Sebastian; Abhari, Behnaz Ahangarian; Torkov, Alica; SeggewiΔ, Dominik; Hugle, Manuela; Fulda, Simone

    2015-01-01

    We explored the potential of Smac mimetics, which antagonize Inhibitor of Apoptosis (IAP) proteins, for chemosensitization of neuroblastoma (NB). Here, we report that Smac mimetics, e.g. BV6, prime NB cells for chemotherapeutics including the topoisomerase II inhibitor doxorubicin (DOX) and vinca alkaloids such as Vincristine (VCR), Vinblastine (VBL) and Vinorelbine (VNR). Additionally, BV6 acts in concert with DOX or VCR to suppress long-term clonogenic growth. While BV6 causes rapid downregulation of cellular IAP (cIAP)1 protein and nuclear factor-kappaB (NF-κB) activation, DOX/BV6- or VCR/BV6-induced apoptosis occurs independently of NF-κB or TNFα signaling, since overexpression of dominant-negative IκBα superrepressor or the Tumor Necrosis Factor (TNF)α-blocking antibody Enbrel fail to block cell death. Mechanistic studies reveal that Receptor-interacting protein (RIP)1 is required for DOX/BV6-, but not for VCR/BV6-induced apoptosis, since transient or stable knockdown of RIP1 or the pharmacological RIP1 inhibitor necrostatin-1 significantly reduce apoptosis. By comparison, VCR/BV6-mediated apoptosis critically depends on the mitochondrial pathway. VCR/BV6 cotreatment causes phosphorylation of BCL-2 during mitotic arrest, enhanced activation of BAX and BAK and loss of mitochondrial membrane potential (MMP). Additionally, overexpression of BCL-2 profoundly suppresses VCR/BV6-induced apoptosis. Thus, BV6 sensitizes NB cells to chemotherapy-induced apoptosis via distinct initial signaling mechanisms depending on the chemotherapeutic drug. These findings provide novel mechanistic insights into Smac mimetic-mediated chemosensitization of NB. PMID:26575016

  18. The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells

    SciTech Connect

    Briggs, D.J.

    1989-01-01

    The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a three hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.

  19. Long noncoding RNAs and neuroblastoma

    PubMed Central

    Pandey, Gaurav Kumar; Kanduri, Chandrasekhar

    2015-01-01

    Neuroblastoma is a disease that affects infants and despite intense multimodal therapy, high-risk patients have low survival rates (<50%). In recent years long noncoding RNAs (lncRNAs) have become the cutting edge of cancer research with inroads made in understanding their roles in multiple cancer types, including prostate and breast cancers. The roles of lncRNAs in neuroblastoma have just begun to be elucidated. This review summarises where we are with regards to lncRNAs in neuroblastoma. The known mechanistic roles of lncRNAs during neuroblastoma pathogenesis are discussed, as well as the relationship between lncRNA expression and the differentiation capacity of neuroblastoma cells. We speculate about the use of some of these lncRNAs, such as those mapping to the 6p22 hotspot, as biomarkers for neuroblastoma prognosis and treatment. This novel way of thinking about both neuroblastoma and lncRNAs brings a new perspective to the prognosis and treatment of high-risk patients. PMID:26087192

  20. Long noncoding RNAs and neuroblastoma.

    PubMed

    Pandey, Gaurav Kumar; Kanduri, Chandrasekhar

    2015-07-30

    Neuroblastoma is a disease that affects infants and despite intense multimodal therapy, high-risk patients have low survival rates (<50%). In recent years long noncoding RNAs (lncRNAs) have become the cutting edge of cancer research with inroads made in understanding their roles in multiple cancer types, including prostate and breast cancers. The roles of lncRNAs in neuroblastoma have just begun to be elucidated. This review summarises where we are with regards to lncRNAs in neuroblastoma. The known mechanistic roles of lncRNAs during neuroblastoma pathogenesis are discussed, as well as the relationship between lncRNA expression and the differentiation capacity of neuroblastoma cells. We speculate about the use of some of these lncRNAs, such as those mapping to the 6p22 hotspot, as biomarkers for neuroblastoma prognosis and treatment. This novel way of thinking about both neuroblastoma and lncRNAs brings a new perspective to the prognosis and treatment of high-risk patients.

  1. 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.

  2. 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.

  3. 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

  4. Genetics Home Reference: neuroblastoma

    MedlinePlus

    ... Help Me Understand Genetics Home Health Conditions neuroblastoma neuroblastoma Enable Javascript to view the expand/collapse boxes. Download PDF Open All Close All Description Neuroblastoma is a type of cancer that most often ...

  5. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ.

  6. Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells

    PubMed Central

    Kim, Ki Jung; Jeun, Seung Hyun

    2017-01-01

    Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization. PMID:28280410

  7. Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells.

    PubMed

    Kim, Ki Jung; Jeun, Seung Hyun; Sung, Ki-Wug

    2017-03-01

    Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization.

  8. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  9. Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

    PubMed

    Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

    2016-12-20

    Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

  10. 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

  11. Targeting tachykinin receptors in neuroblastoma

    PubMed Central

    Szymansky, Annabell; Seiler, Marleen; Althoff, Kristina; Beckers, Anneleen; Speleman, Frank; Schäfers, Simon; De Preter, Katleen; Astrahanseff, Kathy; Struck, Joachim; Schramm, Alexander; Eggert, Angelika; Bergmann, Andreas; Schulte, Johannes H.

    2017-01-01

    Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma. PMID:27888795

  12. The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status.

    PubMed

    Stenger, Christophe; Naves, Thomas; Verdier, Mireille; Ratinaud, Marie-Helene

    2011-09-01

    Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (∆Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.

  13. Active uptake and extravesicular storage of m-iodobenzylguanidine in human neuroblastoma SK-N-SH cells

    SciTech Connect

    Smets, L.A.; Loesberg, C.; Janssen, M.; Metwally, E.A.; Huiskamp, R.

    1989-06-01

    Radioiodinated m-iodobenzylguanidine (MIBG), an analogue of the neurotransmitter norepinephrine (NE), is increasingly used in the diagnosis and treatment of neural crest tumors. Active uptake and subsequent retention of MIBG and NE was studied in human neuroblastoma SK-N-SH cells. Neuron-specific uptake of (125I)MIBG and (3H)NE saturated at extracellular concentration of 10(-6) M and exceeded by 20-30-fold that by passive diffusion alone. A minimum of 50% of accumulated MIBG remained permanently stored but the SK-N-SH cells were incapable of retaining recaptured (3H)NE. (125I)MIBG was displaced from intracellular binding sites by unlabeled MIBG with 10-fold higher potency than by unlabeled NE. MIBG stored in SK-N-SH cells was insensitive to depletion by the inhibitor of granular uptake reserpine (RSP) and was not precipitated in a granular fraction by differential centrifugation. Only few electron-dense granules were found in these cells by electron microscopy. In contrast, MIBG storage in PC-12 pheochromocytoma cells which contained many storage granules, was sensitive to RSP and part of accumulated drug was recovered in a granular fraction. Accordingly, storage of MIBG in the SK-N-SH neuroblastoma cells is predominantly extravesicular and thus essentially different from that of biogenic amines in normal adrenomedullary tissue or in pheochromocytoma tumors, while sharing with these tissues a common mechanism of active uptake.

  14. 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.

  15. Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells.

    PubMed

    Mailloux, Ryan J; Yumvihoze, Emmanuel; Chan, Hing Man

    2015-12-15

    The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O2(-)), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O2(-)mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O2(-) in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O2(-) production by mitochondria. Both rotenone and PQ, which increase O2(-) in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O2(-) into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O2(-) emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O2(-), specifically within the matrix of mitochondria when O2(-) is in adequate supply. Our results also show that O2(-) amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein.

  16. Environment-Mediated Drug Resistance in Neuroblastoma

    DTIC Science & Technology

    2013-10-01

    Neuroblastoma PRINCIPAL INVESTIGATOR: Yves A. DeClerck, MD CONTRACTING ORGANIZATION: Children’s Hospital Los Angeles Los Angeles, CA...3. DATES COVERED 30September2012 – 29September2013 4. TITLE AND SUBTITLE Environment-Mediated Drug Resistance in Neuroblastoma 5a. CONTRACT...demonstrating that interleukin-6 protects neuroblastoma cells from drug-induced apoptosis via activation of signal transduction and activator of

  17. Thymoquinone inhibits the migration of mouse neuroblastoma (Neuro-2a) cells by down-regulating MMP-2 and MMP-9.

    PubMed

    Arumugam, Paramasivam; Subramanian, Raghunandhakumar; Priyadharsini, Jayaseelan Vijayashree; Gopalswamy, Jayaraman

    2016-12-01

    Thymoquinone (TQ), an active component derived from the medial plant Nigella sativa, has been used for medical purposes for more than 2 000 years. Recent studies have reported that TQ blocked angiogenesis in animal model and reduced migration, adhesion, and invasion of glioblastoma cells. We have recently shown that TQ could exhibit a potent cytotoxic effect and induce apoptosis in mouse neuroblastoma (Neuro-2a) cells. In the present study, TQ treatment markedly decreased the adhesion and migration of Neuro-2a cells. TQ down-regulated MMP-2 and MMP-9 protein expression and mRNA levels and their activities. Furthermore, TQ significantly down-regulated the protein expression of transcription factor NF-κB (p65) but not significantly altered the expression of N-Myc. Taken together, our data indicated that TQ's inhibitory effect on the migration of Neuro-2a cells was mediated through the suppression of MMP-2 and MMP-9 expression, suggesting that TQ treatment can be a promising therapeutic strategy for human malignant neuroblastoma.

  18. In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells.

    PubMed

    Ferreira, Martiña; Blanco, Lucía; Garrido, Alejandro; Vieites, Juan M; Cabado, Ana G

    2013-05-01

    The toxic effects of the organotin compounds (OTCs) monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were evaluated in vitro in a neuroblastoma human cell line. Mechanisms of cell death, apoptosis versus necrosis, were studied by using several markers: inhibition of cell viability and proliferation, F-actin, and mitochondrial membrane potential changes as well as reactive oxygen species (ROS) production and DNA fragmentation. The most toxic effects were detected with DBT and TBT even at very low concentrations (0.1-1 μM). In contrast, MBT induced lighter cytotoxic changes at the higher doses tested. None of the studied compounds stimulated propidium iodide uptake, although the most toxic chemical, TBT, caused lactate dehydrogenase release at the higher concentrations tested. These findings suggest that in neuroblastoma, OTC-induced cytotoxicity involves different pathways depending on the compound, concentration, and incubation time. A screening method for DBT and TBT quantification based on cell viability loss was developed, allowing a fast detection alternative to complex methodology.

  19. The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    SciTech Connect

    Mei, Lin.

    1989-01-01

    The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({sup 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.

  20. 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.

  1. 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

  2. MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis

    PubMed Central

    Lao, Yuanzhi; Liao, Weijie; Liao, Meijian; Luo, Xuan; Wu, Jiangbin; Xie, Weidong; Zhang, Yaou; Xu, Naihan

    2016-01-01

    Damage to mitochondria often results in the activation of both mitophagy and mitochondrial apoptosis. The elimination of dysfunctional mitochondria is necessary for mitochondrial quality maintenance and efficient energy supply. Here we report that miR-181a is a novel inhibitor of mitophagy. miR-181a is downregulated by mitochondrial uncouplers in human neuroblastoma SH-SY5Y cells. Overexpression of miR-181a inhibits mitochondrial uncoupling agents-induced mitophagy by inhibiting the degradation of mitochondrial proteins without affecting global autophagy. Knock down of endogenous miR-181a accelerates the autophagic degradation of damaged mitochondria. miR-181a directly targets Parkin E3 ubiquitin ligase and partially blocks the colocalization of mitochondria and autophagosomes/lysosomes. Re-expression of exogenous Parkin restores the inhibitory effect of miR-181a on mitophagy. Furthermore, miR-181a increases the sensitivity of neuroblastoma cells to mitochondrial uncoupler-induced apoptosis, whereas miR-181a antagomir prevents cell death. Because mitophagy defects are associated with a variety of human disorders, these findings indicate an important link between microRNA and Parkin-mediated mitophagy and highlights a potential therapeutic strategy for human diseases. PMID:27281615

  3. CacyBP/SIP interacts with tubulin in neuroblastoma NB2a cells and induces formation of globular tubulin assemblies.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Kilanczyk, Ewa; Bieganowski, Paweł; Kuznicki, Jacek; Filipek, Anna

    2007-11-01

    CacyBP/SIP, originally identified as a S100A6 (calcyclin) target, was later shown to interact with some other members of the S100 family as well as with Siah-1 and Skp1 proteins. Recently, it has been shown that CacyBP/SIP is up-regulated during differentiation of cardiomyocytes. In this work we show that the level of CacyBP/SIP is higher in differentiated neuroblastoma NB2a cells than in undifferentiated ones and that in cells overexpressing CacyBP/SIP the level of GAP-43, a marker of differentiation, was increased. Since the process of differentiation is accompanied by an extensive rearrangement of microtubules, we examined whether CacyBP/SIP interacted with tubulin. By applying cross-linking experiments we found that these two proteins bind directly. The dissociation constant of the tubulin-CacyBP/SIP complex determined by the surface plasmon resonance technique is 1.57 x 10(-7 )M which suggests that the interaction is tight. The interaction and co-localization of CacyBP/SIP and tubulin was also demonstrated by co-immunoprecipitation, affinity chromatography and immunofluorescence methods. Light scattering measurements and electron microscopy studies revealed that CacyBP/SIP, but not its homologue, Sgt1, increased tubulin oligomerization. Altogether, our results suggest that CacyBP/SIP, via its interaction with tubulin, might contribute to the differentiation of neuroblastoma NB2a cells.

  4. D1 dopamine receptors of NS20Y neuroblastoma cells are functionally similar to rat striatal D1 receptors.

    PubMed

    Lovenberg, T W; Roth, R H; Nichols, D E; Mailman, R B

    1991-11-01

    Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.

  5. Presence in neuroblastoma cells of a mu 3 receptor with selectivity for opiate alkaloids but without affinity for opioid peptides.

    PubMed

    Cruciani, R A; Dvorkin, B; Klinger, H P; Makman, M H

    1994-12-26

    Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [3H]morphine and [3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including D-ala2-D-leu5-enkephalin (DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (DAGO) and dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [3H]morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly delta and also mu) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (delta and mu) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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

  7. 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.

  8. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    SciTech Connect

    Matsushima, H.; Bogenmann, E. )

    1990-09-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

  9. 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

  10. 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.

  11. Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells.

    PubMed

    Smith, Anna Grynfeld; Popov, Nikita; Imreh, Martha; Axelson, Håkan; Henriksson, Marie

    2004-08-15

    Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.

  12. ARID1A gene knockdown promotes neuroblastoma migration and invasion.

    PubMed

    Li, C; Xu, Z; Zhao, Z; An, Q; Wang, L; Yu, Y; Piao, D

    2017-03-03

    Neuroblastoma is the most common extracranial solid tumor in childhood which often acquires drug resistance and becomes aggressive phenotypes. The high-risk patients suffer from high mortality due to the limitation of the treatment strategies. ARID1A (AT-rich interactive domain-containing protein 1A), a subunit of SWI/SNF complexes, is considered as a tumor suppressor in many cancers. The aim of the present study was to investigate the effect of ARID1A on migration and invasion in neuroblastoma cells. The shRNA targeting ARID1A was designed and delivered into SK-N-SH cells to knock down ARID1A expression. Knockdown of ARID1A by shRNA significantly increased the viability and invasion ability, and caused G1 arrest inhibition and DNA synthesis increase in SK-N-SH cells. Moreover, Knockdown of ARID1A increased the activity and expression of matrix metalloproteinase (MMP)-2 and -9 in SK-N-SH cells. Furthermore, ARID1A knockdown caused diminished expression of E-cadherin, enhanced expression of N-cadherin and β-catenin nuclear translocation in SK-N-SH cells. These results suggest that loss of ARID1A may associate with the promotion of invasion and metastasis of neuroblastoma. Our findings indicate ARID1A is a tumor suppressor in neuroblastoma.

  13. Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    PubMed Central

    Sidhar, Himakshi; Giri, Ranjit K.

    2017-01-01

    Brain expressed X-linked (Bex) genes are newer group of pro-apoptotic genes. Role of any Bex gene in neuroblastoma and Bex4 and Bex6 in any cancer is completely unknown. Re-expression of all endogenous Bex genes by any nutraceutical is also unknown. Therefore, we investigated the induction of all endogenous Bex genes and associated mechanisms by curcumin using N2a, an aggressive neuroblastoma cell line. Curcumin induced all endogenous Bex genes prior to apoptosis in N2a cells in a dose- and time-dependent manner. Wortmannin (PI-3Kinases inhibitor), SP600125 (JNK inhibitor) and pifithrin-α (p53 inhibitor) abrogated curcumin-mediated induction of Bex genes. Inhibition of curcumin-mediated induction of Bex genes by pifithrin-α also inhibited N2a cells apoptosis suggesting, a direct role of Bex genes in N2a cells apoptosis and involvement of p53 in Bex genes induction. Curcumin treatment activated p53 through hyperphosphorylation at serine 15 before Bex genes induction indicating Bex genes are novel downstream targets of p53. Collectively, curcumin, a safe nutraceutical has the potential to induce all endogenous Bex genes to harness their anti-cancer properties in neuroblastoma cells. Re-expression of Bex genes by curcumin acts as tumor suppressors and may provide alternate strategy to treat neuroblastomas and other cancers with silenced Bex genes. PMID:28145533

  14. Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells.

    PubMed

    Sidhar, Himakshi; Giri, Ranjit K

    2017-02-01

    Brain expressed X-linked (Bex) genes are newer group of pro-apoptotic genes. Role of any Bex gene in neuroblastoma and Bex4 and Bex6 in any cancer is completely unknown. Re-expression of all endogenous Bex genes by any nutraceutical is also unknown. Therefore, we investigated the induction of all endogenous Bex genes and associated mechanisms by curcumin using N2a, an aggressive neuroblastoma cell line. Curcumin induced all endogenous Bex genes prior to apoptosis in N2a cells in a dose- and time-dependent manner. Wortmannin (PI-3Kinases inhibitor), SP600125 (JNK inhibitor) and pifithrin-α (p53 inhibitor) abrogated curcumin-mediated induction of Bex genes. Inhibition of curcumin-mediated induction of Bex genes by pifithrin-α also inhibited N2a cells apoptosis suggesting, a direct role of Bex genes in N2a cells apoptosis and involvement of p53 in Bex genes induction. Curcumin treatment activated p53 through hyperphosphorylation at serine 15 before Bex genes induction indicating Bex genes are novel downstream targets of p53. Collectively, curcumin, a safe nutraceutical has the potential to induce all endogenous Bex genes to harness their anti-cancer properties in neuroblastoma cells. Re-expression of Bex genes by curcumin acts as tumor suppressors and may provide alternate strategy to treat neuroblastomas and other cancers with silenced Bex genes.

  15. 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.

  16. Cell surface-mediated cellular interactions: effects of B104 neuroblastoma surface determinants on C6 glioma cellular properties.

    PubMed

    Ciment, G; de Vellis, J

    1982-01-01

    To study the influence of cell surface-associated molecules on intercellular communication, C6 glioma cells were cultured both on plastic and on substrata of paraformaldehyde-fixed B104 neuroblastoma cells. By then comparing the phenotypic expression of these "cocultured" C6 cells with cells cultured on tissue culture plastic, the influence of the cellular substratum was determined. The beta-adrenergic-responsive cyclic AMP-generating system of C6 cells was compared on these various substrata. We found that fixed beds of dibutyryl cyclic AMP (dbcAMP)-treated B104 cells uncoupled beta-receptors from adenylate cyclase, whereas fixed beds of similarly treated C6 cells did not. However, other cellular properties were not affected by growth atop fixed dbcAMP-treated B104 cell beds including the rate of C6 cellular proliferation and their rate of protein synthesis. The cell surface-associated determinant on B104 cells capable of uncoupling the beta-responsive cyclase system of C6 cells is probably a protein, as judged by its susceptibility to protease treatment. Other properties of C6 cells were also affected by the various substrata including basal and hydrocortisone-induced levels of glycerol phosphate dehydrogenase (GPDH; an oligodendroglial marker) and the rate of RNA synthesis in these cells.

  17. The metastatic microenvironment: lung-derived factors control the viability of neuroblastoma lung metastasis.

    PubMed

    Maman, Shelly; Edry-Botzer, Liat; Sagi-Assif, Orit; Meshel, Tsipi; Yuan, Weirong; Lu, Wuyuan; Witz, Isaac P

    2013-11-15

    Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.

  18. 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

  19. MYCN is retained in single copy at chromosome 2 band p23-24 during amplification in human neuroblastoma cells

    SciTech Connect

    Corvi, R.; Amler, L.C.; Savelyeva, L.; Gehring, M.; Schwab, M. )

    1994-06-07

    Amplification of the human N-myc protooncogene, MYCN, is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions of aggressively growing neuroblastomas. MYCN maps to chromosome 2 band p23-24, but homogeneously staining regions have never been observed at this band, suggesting transposition of MYCN during amplification. The authors have employed fluorescence in situ hybridization to determine the status of MYCN at 2p23-24 in five human neuroblastoma cell lines. All five lines carried, in addition to amplified MYCN in homogeneously staining regions or double minutes, single-copy MYCN at the normal position. In one line there was coamplification of MYCN together with DNA of the host chromosome 12, to which MYCN had been transposed. The results suggest a model of amplification where MYCN is retained at its original location. They further sustain the view that either the initial events of MYCN amplification or the further evolution of amplified MYCN copies follow mechanisms different from those leading to amplification of drug-resistance genes.

  20. Olfactory neuroblastoma

    SciTech Connect

    O'Connor, T.A.; McLean, P.; Juillard, G.J.; Parker, R.G.

    1989-06-15

    Fifteen patients with olfactory neuroblastoma were treated during the 17-year period of 1969 to 1986. Data was analyzed with respect to age at presentation, sex, presenting signs and symptoms, stage, and results of treatment. Age ranged from 4 to 67 years with the median age being 27 years. Median follow-up was 8 years. Local control was achieved in nine of nine patients or 100% with successful surgical resection, i.e., minimal residual disease, followed by postoperative radiation therapy (45 to 65 Gy) was employed. There were no distant failures when the primary site was controlled. Regional lymph node metastases were infrequent: only 13% (two of 15 patients) presented with positive nodes. Three of four patients treated initially with surgery alone had a local recurrence, two of which were successfully salvaged by combined therapy. There were four patients treated with radiation therapy alone: three had persistent disease after radiation therapy, and one patient was controlled with 65 Gy. Olfactory neuroblastoma has a propensity to recur locally when treated with surgery alone. The authors' experience suggests excellent local control can be achieved with surgery immediately followed by radiation therapy. Thus the authors recommend planned combined treatment for all resectable lesions.

  1. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

    PubMed

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, M Teresa

    2015-12-21

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

  2. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

    PubMed Central

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, Mª Teresa

    2015-01-01

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome. PMID:26687764

  3. Improved Mitochondrial and Methylglyoxal-Related Metabolisms Support Hyperproliferation Induced by 50 Hz Magnetic Field in Neuroblastoma Cells.

    PubMed

    Falone, Stefano; Santini, Silvano; di Loreto, Silvia; Cordone, Valeria; Grannonico, Marta; Cesare, Patrizia; Cacchio, Marisa; Amicarelli, Fernanda

    2016-09-01

    Extremely low frequency magnetic fields (ELF-MF) are common environmental agents that are suspected to promote later stages of tumorigenesis, especially in brain-derived malignancies. Even though ELF magnetic fields have been previously linked to increased proliferation in neuroblastoma cells, no previous work has studied whether ELF-MF exposure may change key biomolecular features, such as anti-glycative defence and energy re-programming, both of which are currently considered as crucial factors involved in the phenotype and progression of many malignancies. Our study investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field is supported by an improved defense towards methylglyoxal (MG), which is an endogenous cancer-static and glycating α-oxoaldehyde, and by rewiring of energy metabolism. Our findings show that not only the ELF magnetic field interfered with the biology of neuron-derived malignant cells, by de-differentiating further the cellular phenotype and by increasing the proliferative activity, but also triggered cytoprotective mechanisms through the enhancement of the defense against MG, along with a more efficient management of metabolic energy, presumably to support the rapid cell outgrowth. Intriguingly, we also revealed that the MF-induced bioeffects took place after an initial imbalance of the cellular homeostasis, which most likely created a transient unstable milieu. The biochemical pathways and molecular targets revealed in this research could be exploited for future approaches aimed at limiting or suppressing the deleterious effects of ELF magnetic fields. J. Cell. Physiol. 231: 2014-2025, 2016. © 2016 Wiley Periodicals, Inc.

  4. Induction of cereblon by NF-E2-related factor 2 in neuroblastoma cells exposed to hypoxia-reoxygenation.

    PubMed

    Lee, Kyung Jin; Lee, Kwang Min; Jo, Sooyeon; Kang, Keon Wook; Park, Chul-Seung

    2010-09-03

    Cereblon is a protein encoded by the CRBN gene, which has been associated with human autosomal recessive nonsyndromic mental retardation. However, little is known about the regulation of CRBN expression. Following exposure of mouse neuroblastoma N2A cells to hypoxia/reoxygenation (H/R), mRNA and protein expression of CRBN were increased. To better understand how CRBN expression is regulated, the promoter region of the mouse CRBN gene was characterized functionally. Deletion mutations and site-directed mutagenesis led to the identification of a functional NF-E2-related factor 2 (Nrf2)-binding site. Electrophoretic mobility shift analysis indicated that Nrf2 binds to a putative binding site in the CRBN promoter. Nrf2 overexpression and tert-butylhydroquinone treatment enhanced CRBN protein expression. These results imply that Nrf2 stimulates CRBN gene transcription under H/R conditions in neuronal cells.

  5. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

    PubMed Central

    Fang, M.; Jin, A.; Zhao, Y.; Liu, X.

    2016-01-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  6. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    PubMed

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  7. 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.

  8. Honokiol induces autophagy of neuroblastoma cells through activating the PI3K/Akt/mTOR and endoplasmic reticular stress/ERK1/2 signaling pathways and suppressing cell migration.

    PubMed

    Yeh, Poh-Shiow; Wang, Weu; Chang, Ya-An; Lin, Chien-Ju; Wang, Jhi-Joung; Chen, Ruei-Ming

    2016-01-01

    In children, neuroblastomas are the most common and deadly solid tumor. Our previous study showed that honokiol, a small-molecule polyphenol, can traverse the blood-brain barrier and kill neuroblastoma cells. In this study, we further investigated the mechanisms of honokiol-induced insults to neuroblastoma cells. Treatment of neuroblastoma neuro-2a cells with honokiol elevated the levels of microtubule-associated protein light chain 3 (LC3)-II and induced cell autophagy in time- and concentration-dependent manners. Interestingly, pretreatment with 3-methyladenine (3-MA), an inhibitor of autophagy, led to the simultaneous attenuation of honokiol-induced cell autophagy and apoptosis but did not influence cell necrosis. As to the mechanisms, exposure of neuro-2a cells to honokiol time-dependently decreased the amount of phosphatidylinositol 3-kinase (PI3K). Sequentially, honokiol downregulated phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in neuro-2a cells. Furthermore, honokiol elevated the levels of glucose-regulated protein (GpR)78, an endoplasmic reticular stress (ERS)-associated protein, and amounts of intracellular reactive oxygen species (ROS). In contrast, reducing production of intracellular ROS using N-acetylcysteine, a scavenger of ROS, concurrently suppressed honokiol-induced cellular autophagy. Consequently, honokiol stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2. However, pretreatment of neuro-2a cells with PD98059, an inhibitor of ERK1/2, lowered honokiol-induced autophagy. The effects of honokiol on inducing autophagy and apoptosis of neuroblastoma cells were further confirmed using mouse neuroblastoma NB41A3 cells as our experimental model. Fascinatingly, treatment of neuroblastoma neuro-2a and NB41A3 cells with honokiol for 12 h did not affect cell autophagy or apoptosis but caused significant suppression of cell migration. Taken together, this study showed that honokiol can induce

  9. 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

  10. Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

    PubMed

    Robbins, J; Trouslard, J; Marsh, S J; Brown, D A

    1992-01-01

    1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine

  11. In vitro propagation of the scrapie agent. I. Transformation of mouse glia and neuroblastoma cells after infection with the mouse-adapted scrapie strain c-506.

    PubMed

    Markovits, P; Dautheville, C; Dormont, D; Dianoux, L; Latarjet, R

    1983-01-01

    Seven cell lines including glia cells from mouse brains and mouse neuroblastoma cells were infected with the mouse-adapted scrapie strain c-506. During the early in vitro passages, a stimulation of growth was already observed but cellular morphology and differentiation did not alter. Later on, after 12-16 passages, six of the seven infected lines displayed cell proliferation and morphological alterations, suggesting an in vitro morphological transformation. At this stage, differentiation was no longer observed in the scrapie-infected neuroblastoma cells and all the scrapie-infected cells formed two to four times more colonies in liquid medium than the controls, and developed large tridimensional colonies in agar. The part played by the scrapie agent in these changes is discussed.

  12. Do We Know What Causes Neuroblastoma?

    MedlinePlus

    ... Factors, and Prevention Do We Know What Causes Neuroblastoma? The causes of most neuroblastomas are not known. ... Causes Neuroblastoma? Can Neuroblastoma Be Prevented? More In Neuroblastoma About Neuroblastoma Causes, Risk Factors, and Prevention Early ...

  13. What Are the Key Statistics about Neuroblastoma?

    MedlinePlus

    ... About Neuroblastoma What Are the Key Statistics About Neuroblastoma? Neuroblastoma is by far the most common cancer ... New in Neuroblastoma Research and Treatment? More In Neuroblastoma About Neuroblastoma Causes, Risk Factors, and Prevention Early ...

  14. 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.

  15. Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and {gamma}-irradiation on viability of neuroblastoma cells

    SciTech Connect

    Roessler, Jochen . E-mail: jochen.roessler@uniklinik-freiburg.de; Zambrzycka, Izabella; Lagodny, Jeanette; Kontny, Udo; Niemeyer, Charlotte Marie

    2006-04-21

    Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-{alpha} and -{beta}-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and {gamma}-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 {mu}M STI-571 for 72 h, two cell lines responding already to 10 {mu}M. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-{alpha} and -{beta}. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and {gamma}-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed {gamma}-irradiation therapy.

  16. 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

  17. Cytoprotection of pyruvic acid and reduced beta-nicotinamide adenine dinucleotide against hydrogen peroxide toxicity in neuroblastoma cells.

    PubMed

    Mazzio, Elizabeth A; Soliman, Karam F A

    2003-05-01

    Elevated production of hydrogen peroxide (H2O2) in the central nervous system has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, ischemic reperfusion, stroke, and Alzheimer's disease. Pyruvic acid has a critical role in energy metabolism and a capability to nonenzymatically decarboxylate H2O2 into H2O. This study examined the effects of glycolytic regulation of pyruvic acid on H2O2 toxicity in murine neuroblastoma cells. Glycolytic energy substrates including D-(+)-glucose, D-(-) fructose and the adenosine transport blocker dipyridamole, were not effective in providing protection against H2O2 toxicity, negating energy as a factor. On the other hand, pyruvic acid completely prevented H2O2 toxicity, restoring the loss of ATP and cell viability. H2O2 toxicity was also attenuated by D-fructose 1,6 diphosphate (FBP), phospho (enol) pyruvate (PEP), niacinamide, beta-nicotinamide adenine dinucleotide (beta-NAD+), and reduced form (beta-NADH). Both FBP and PEP exerted positive kinetic effects on pyruvate kinase (PK) activity. Interestingly, only pyruvic acid and beta-NADH exhibited powerful stoichiometric H2O2 antioxidant properties. Further, beta-NADH may exert positive effects on PK activity. Subsequent pyruvic acid accumulation can lead to the recycling of beta-NAD+ through lactate dehydrogenase and beta-NADH through glyceraldehyde-3-phosphate dehydrogenase. It was concluded from these studies that intracellular pyruvic acid and beta-NADH appear to act in concert through glycolysis, to enhance H2O2 intracellular antioxidant capacity in neuroblastoma cells. Future research will be required to examine whether similar effects are observed in primary neuronal culture or intact tissue.

  18. 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.

  19. Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells

    PubMed Central

    Aveic, Sanja; Pantile, Marcella; Seydel, Anke; Esposito, Maria Rosaria; Zanon, Carlo; Li, Gary; Tonini, Gian Paolo

    2016-01-01

    Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib. PMID:26735175

  20. MEG3, HCN3 and linc01105 influence the proliferation and apoptosis of neuroblastoma cells via the HIF-1α and p53 pathways

    PubMed Central

    Tang, Weitao; Dong, Kuiran; Li, Kai; Dong, Rui; Zheng, Shan

    2016-01-01

    The purpose of this study was to investigate the differential expression and functional roles of long non-coding RNAs (lncRNAs) in neuroblastoma tissue. LncRNA microarrays were used to identify differentially expressed lncRNAs between tumor and para-tumor tissues. In total, in tumor tissues, 3,098 and 1,704 lncRNAs were upregulated and downregulated, respectively. HCN3 and linc01105 exhibited the higher expression (P < 0.05; P < 0.01, respectively) in neuroblastoma tissue, whereas MEG3 displayed the lower expression (P < 0.01). HIF-1α expression was negatively correlated with cell proliferation in the linc01105 KD group. In addition, Noxa and Bid expression was positively correlated with cell apoptosis. Moreover, linc01105 knockdown promoted cell proliferation, whereas MEG3 overexpression inhibited proliferation. Finally, linc01105 knockdown, MEG3 overexpression and HCN3 knockdown all increased apoptosis. The correlation coefficients between those three lncRNAs and the International Neuroblastoma Staging System (INSS) stage were −0.48, −0.58 and −0.55, respectively. In conclusion, we have identified lncRNAs that are differentially expressed in neuroblastoma tissues. The lncRNAs HCN3, linc01105, and MEG3 may be important in biological behaviors of neuroblastoma through mechanisms involving p53 pathway members such as HIF-1α, Noxa, and Bid. The expressions of MEG3, HCN3 and linc01105 are all negatively correlated with the INSS stage. PMID:27824082

  1. Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells

    PubMed Central

    2010-01-01

    Background Neuroblastoma (NB) is the second most common solid malignancy of childhood that usually undergoes rapid progression with a poor prognosis upon metastasis. The Src-family tyrosine kinases (SFKs) are a group of proteins involved in cancer development and invasiveness that seem to play an important role in the NB carcinogenesis. Methods To determine cell proliferation, the growth rate was evaluated by both MTT test and cells counted. Analysis of DNA content was performed for the evaluation of the cell cycle and apoptosis. To characterize the mechanisms underlying the antiproliferative effects induced by SI 34, a novel pyrazolo-pyrimidine derivative provided with Src inhibitory activity, the involvement of some cellular pathways that are important for cell proliferation and survival was investigated by western blot assays. In particular, the contribution of cyclins, Src and ERK were examined. Finally, experiments of cell adhesion and invasiveness were performed. Results Treatment of SH-SY5Y human NB cells and CHP100 human neuroepithelioma (NE) cultures with three novel pyrazolo[3,4-d]pyrimidine derivatives, namely SI 34, SI 35 and SI 83, inhibits the cell proliferation in a time and concentration-dependent manner. The maximal effect was obtained after 72 hours incubation with SI 34 10 μM. Fluorescence microscopy experiments, flow cytometry analysis and determination of caspase-3 activity by fluorimetric assays showed that SI 34 induced SH-SY5Y apoptosis. Moreover, SI 34 determined cell cycle arrest at the G0/G1 phase, paralleled by a decreased expression of cyclin D1. Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness. Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells. Conclusions Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a

  2. A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

    PubMed Central

    Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D.; Sung, Derek C.; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D.; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

    2016-01-01

    MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation. PMID:27764804

  3. 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

  4. 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.

  5. 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

  6. Moderate superoxide production is an early promoter of mitochondrial biogenesis in differentiating N2a neuroblastoma cells.

    PubMed

    Valero, T; Moschopoulou, G; Mayor-Lopez, L; Kintzios, S

    2012-12-01

    Reactive oxygen species (ROS) have been widely considered as harmful for cell development and as promoters of cell aging by increasing oxidative stress. However, ROS have an important role in cell signaling and they have been demonstrated to be beneficial by triggering hormetic signals, which could protect the organism from later insults. In the present study, N2a murine neuroblastoma cells were used as a paradigm of cell-specific (neural) differentiation partly mediated by ROS. Differentiation was triggered by the established treatments of serum starvation, forskolin or dibutyryl cyclic AMP. A marked differentiation, expressed as the development of neurites, was detected by fixation and staining with coomassie brilliant blue after 48 h treatment. This was accompanied by an increase in mitochondrial mass detected by mitotracker green staining, an increased expression of the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1-alpha (PGC-1α) and succinate dehydrogenase activity as detected by MTT. In line with these results, an increase in free radicals, specifically superoxide anion, was detected in differentiating cells by flow cytometry. Superoxide scavenging by MnTBAP and MAPK inhibition by PD98059 partially reversed differentiation and mitochondrial biogenesis. In this way, we demonstrated that mitochondrial biogenesis and differentiation are mediated by superoxide and MAPK cues. Our data suggest that differentiation and mitochondrial biogenesis in N2a cells are part of a hormetic response which is triggered by a modest increase of superoxide anion concentration within the mitochondria.

  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. Implication of limonene and linalyl acetate in cytotoxicity induced by bergamot essential oil in human neuroblastoma cells.

    PubMed

    Russo, Rossella; Ciociaro, Antonella; Berliocchi, Laura; Cassiano, Maria Gilda Valentina; Rombolà, Laura; Ragusa, Salvatore; Bagetta, Giacinto; Blandini, Fabio; Corasaniti, Maria Tiziana

    2013-09-01

    Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a widely used plant extract showing anxiolytic, analgesic and neuroprotective effects in rodents; also, BEO activates multiple death pathways in cancer cells. Despite detailed knowledge of its chemical composition, the constituent/s responsible for these pharmacological activities remain largely unknown. Aim of the present study was to identify the components of BEO implicated in cell death. To this end, limonene, linalyl acetate, linalool, γ-terpinene, β-pinene and bergapten were individually tested in human SH-SY5Y neuroblastoma cultures at concentrations comparable with those found in cytotoxic dilutions of BEO. None of the tested compounds elicited cell death. However, significant cytotoxicity was observed when cells were cotreated with limonene and linalyl acetate whereas no other associations were effective. Only cotreatment, but not the single exposure to limonene and linalyl acetate, replicated distinctive morphological and biochemical changes induced by BEO, including caspase-3 activation, PARP cleavage, DNA fragmentation, cell shrinkage, cytoskeletal alterations, together with necrotic and apoptotic cell death. Collectively, our findings suggest a major role for a combined action of these monoterpenes in cancer cell death induced by BEO.

  9. Targeted inhibition of HDAC8 increases the doxorubicin sensitivity of neuroblastoma cells via up regulation of miR-137.

    PubMed

    Zhao, Gang; Wang, Guoliang; Bai, Hongmin; Li, Tiandong; Gong, Fanghe; Yang, Huan; Wen, Jinchong; Wang, Weimin

    2017-02-20

    Histone deacetylases (HDACs) have been suggested to be potential therapeutic targets for cancer treatment. Recent studies revealed that HDAC8 expression was associated with poor prognostic markers and poor overall survival rate of neuroblastoma (NB). Our present study revealed that among the four members of class I HDACs, HDAC8 is significantly over expressed in NB cells as compared with the normal fibroblast 3T3 cells or primary normal human astrocytes (NHA) cells. Targeted inhibition of HDAC8 by its specific siRNA (si-HDAC8) can inhibit the in vitro growth of NB cells. Furthermore, si-HDAC8 significantly increases the sensitivity of NB cells to doxorubicin (Dox). Silencing of HDAC8 can increase the expression of miR-137, which has been suggested to mediate the Dox sensitivity of NB cells. Knockdown of miR-137 can attenuate si-HDAC8 enhanced Dox sensitivity. Further, si-HDAC8 can also inhibit the expression of multi-drug resistance gene 1 (MDR1). While knockdown of miR-137 can attenuate si-HDAC8 induced down regulation of MDR1. Collectively, our data revealed that targeted inhibition of HDAC8 can suppress the growth of NB cells and increase Dox sensitivity via up regulation of miR-137 and suppression of MDR1. Therefor, combination of HDAC8 inhibitor will be helpful to elevate the treatment outcome of NB patients.

  10. Ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein regulates neuroblastoma cell proliferation through p21.

    PubMed

    Jung, Heekyung; Shin, Joo-Hyun; Park, Young-Seok; Chang, Mi-Sook

    2014-12-31

    Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G1 phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G1 phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.

  11. 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

  12. Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

    PubMed Central

    Webster, W A; Charlton, K M; Casey, G A

    1989-01-01

    Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus. PMID:2590871

  13. Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells.

    PubMed

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Acierno, Giovanni; Andolfo, Immacolata; Capasso, Mario; Iolascon, Achille

    2015-06-09

    Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

  14. Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

    PubMed Central

    2012-01-01

    Background Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. Results We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. Conclusions In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells. PMID:22853823

  15. Hydrogen sulfide generation from l-cysteine in the human glioblastoma-astrocytoma U-87 MG and neuroblastoma SHSY5Y cell lines.

    PubMed

    Bronowicka-Adamska, Patrycja; Bentke, Anna; Wróbel, Maria

    2017-01-01

    Hydrogen sulfide (H2S) is endogenously synthesized from l-cysteine in reactions catalyzed by cystathionine beta-synthase (CBS, EC 4.2.1.22) and gamma-cystathionase (CSE, EC 4.4.1.1). The role of 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) in H2S generation is also considered; it could be important for tissues with low CTH activity, e.g. cells of the nervous system. The expression and activity of CBS, CTH, and MPST were detected in the human glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cell lines. In both cell lines, the expression and activity of MPST were the highest among the investigated enzymes, suggesting its possible role in the generation of H2S. The RP-HPLC method was used to determine the concentration of cystathionine and alpha-ketobutyrate, products of the CBS- and CTH-catalyzed reactions. The difference in cystathionine levels between cell homogenates treated with totally CTH-inhibiting concentrations of dl-propargylglycine and without the inhibitor was used to evaluate the activity of CBS. The higher expression and activity of CBS, CTH and MPST in the neuroblastoma cells were associated with more intensive generation of H2S in the presence of 2 mM cysteine. A threefold higher level of sulfane sulfur, a potential source of hydrogen sulfide, was detected in the astrocytoma cells in comparison to the neuroblastoma cells.

  16. Unveiling MYCN regulatory networks in neuroblastoma via integrative analysis of heterogeneous genomics data

    PubMed Central

    Hsu, Chia-Lang; Chang, Hsin-Yi; Chang, Jen-Yun; Hsu, Wen-Ming; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2016-01-01

    MYCN, an oncogenic transcription factor of the Myc family, is a major driver of neuroblastoma tumorigenesis. Due to the difficulty in drugging MYCN directly, revealing the molecules in MYCN regulatory networks will help to identify effective therapeutic targets for neuroblastoma therapy. Here we perform ChIP-sequencing and small RNA-sequencing of neuroblastoma cells to determine the MYCN-binding sites and MYCN-associated microRNAs, and integrate various types of genomic data to construct MYCN regulatory networks. The overall analysis indicated that MYCN-regulated genes were involved in a wide range of biological processes and could be used as signatures to identify poor-prognosis MYCN-non-amplified patients. Analysis of the MYCN binding sites showed that MYCN principally served as an activator. Using a computational approach, we identified 32 MYCN co-regulators, and some of these findings are supported by previous studies. Moreover, we investigated the interplay between MYCN transcriptional and microRNA post-transcriptional regulations and identified several microRNAs, such as miR-124-3p and miR-93-5p, which may significantly contribute to neuroblastoma pathogenesis. We also found MYCN and its regulated microRNAs acted together to repress the tumor suppressor genes. This work provides a comprehensive view of MYCN regulations for exploring therapeutic targets in neuroblastoma, as well as insights into the mechanism of neuroblastoma tumorigenesis. PMID:27167114

  17. Gene therapy as a potential tool for treating neuroblastoma-a focused review.

    PubMed

    Kumar, M D; Dravid, A; Kumar, A; Sen, D

    2016-05-01

    Neuroblastoma, a solid tumor caused by rapid division of undifferentiated neuroblasts, is the most common childhood malignancy affecting children aged <5 years. Several approaches and strategies developed and tested to cure neuroblastoma have met with limited success due to different reasons. Many oncogenes are deregulated during the onset and development of neuroblastoma and thus offer an opportunity to circumvent this disease if the expression of these genes is restored to normalcy. Gene therapy is a powerful tool with the potential to inhibit the deleterious effects of oncogenes by inserting corrected/normal genes into the genome. Both viral and non-viral vector-based gene therapies have been developed and adopted to deliver the target genes into neuroblastoma cells. These attempts have given hope to bringing in a new regime of treatment against neuroblastoma. A few gene-therapy-based treatment strategies have been tested in limited clinical trials yielding some positive results. This mini review is an attempt to provide an overview of the available options of gene therapy to treat neuroblastoma.

  18. The mitochondrial apoptotic pathway is induced by Cu(II) antineoplastic compounds (Casiopeínas(®)) in SK-N-SH neuroblastoma cells after short exposure times.

    PubMed

    García-Ramos, Juan Carlos; Gutiérrez, Anllely Grizett; Vázquez-Aguirre, Adriana; Toledano-Magaña, Yanis; Alonso-Sáenz, Ana Luisa; Gómez-Vidales, Virginia; Flores-Alamo, Marcos; Mejía, Carmen; Ruiz-Azuara, Lena

    2017-02-01

    The family of Copper(II) coordination compounds Casiopeínas(®) (Cas) has shown antiproliferative activity in several tumour lines by oxidative cellular damage and mitochondrial dysfunction that lead to cell death through apoptotic pathways. The goal of this work is looking for the functional mechanism of CasIIgly, CasIIIia and CasIIIEa in neuroblastoma metastatic cell line SK-N-SH, a paediatric extra-cranial tumour which is refractory to several anti-carcinogenic agents. All Cas have shown higher antiproliferative activity than cisplatin (IC50 = 123 μM) with IC50 values of 18, 22 and 63 µM for CasIIgly, CasIIIEa and CasIIIia, respectively. At low concentrations and early times (4 h), these compounds cause a disruption of the mitochondrial transmembrane potential (Δψm). Concomitantly, an important depletion of intracellular glutathione and an increase of reactive oxygen species (ROS) hydrogen peroxide and radical superoxide were observed. On the other side, the lower cytotoxic effect of Casiopeínas on cultures of human peripheral blood lymphocytes (IC50CasIIgly  = 1720 µM, IC50 CasIIIEa  = 3860 µM and IC50 CasIIIia  = 4700 µM) show the selectivity of these compounds over the tumour cells compared with the non-transformed cells. Chemically, glutathione (GSH) interacts with Casiopeínas(®) through the coordination of sulphur atom to the metal centre, process which facilitates the electron transfer to get Cu(I), GSSG and the posterior production of ROS. Additionally, the molecular structure of CasIIIia as nitrate is reported. These results have shown that the anticarcinogenic activity of Casiopeínas(®) on neuroblastoma SK-N-SH is through mitochondrial apoptosis due to the enhanced pro-oxidant environment promoted by the presence of the coordination copper compounds.

  19. Nardostachys jatamansi Root Extract Modulates the Growth of IMR-32 and SK-N-MC Neuroblastoma Cell Lines Through MYCN Mediated Regulation of MDM2 and p53

    PubMed Central

    Suryavanshi, Snehal; Raina, Prerna; Deshpande, Rashmi; Kaul-Ghanekar, Ruchika

    2017-01-01

    Aim: The present study evaluated the effect of ethanolic extract of Nardostachys jatamansi roots (NJet) on MYCN mediated regulation of expression of MDM2 and p53 proteins in neuroblastoma cell lines, IMR-32 and SK-N-MC. Materials and Methods: The effect of NJet on cell viability was determined by MTT; and on growth kinetics was evaluated by trypan blue dye exclusion method and soft agar assay. The expression of p53, MDM2 and MYCN proteins in response to NJet treatment was evaluated by immunoblotting. Results: NJet decreased the viability of neuroblastoma cells without affecting the viability of non-cancerous, HEK-293 cells. It altered the growth kinetics of the cancer cells in a dose-dependent manner. NJet down regulated the expression of MYCN and MDM2 proteins with a simultaneous increase in the expression of tumor suppressor protein p53. Conclusions: The present data demonstrated that NJet regulated the growth of IMR-32 and SK-N-MC through reduction in MYCN expression that lead to down regulation of MDM2 protein and increase in p53 expression. These preliminary results warrant further in depth studies to explore the therapeutic potential of Nardostachys jatamansi in the management of neuroblastoma. SUMMARY NJet reduced the viability of human neuroblastoma cell lines without affecting the viability of non-cancerous, HEK-293 cells.NJet regulated the growth kinetics of the cancer cells.NJet decreased the expression of MYCN and MDM2 proteins and simultaneously increased the expression of tumor suppressor protein p53. Abbreviation used: NJet: Ethanolic extract of Nardostachys jatamansi MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide HPTLC: High performance thin layer chromatography PMID:28216878

  20. Shogaols from Zingiber officinale protect IMR32 human neuroblastoma and normal human umbilical vein endothelial cells from beta-amyloid(25-35) insult.

    PubMed

    Kim, Darrick S H L; Kim, Dong-Seon; Oppel, Marissa N

    2002-04-01

    From the rhizome of Zingiber officinale L. (Zingiberaceae), four shogaols that protect IMR32 human neuroblastoma and normal human umbilical vein endothelial cells from beta-amyloid(25 - 35) insult at EC50 = 4.5 - 81 microM were isolated. The efficacy of cell protection from beta-amyloid(25 - 35) insult by these shogaols was shown to improve as the length of the side chain increases.

  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. The Involvement of Microtubules and Actin during the Infection of Japanese Encephalitis Virus in Neuroblastoma Cell Line, IMR32

    PubMed Central

    Henry Sum, Magdline Sia

    2015-01-01

    The role of the cytoskeleton, actin, and microtubules were examined during the process of Japanese encephalitis (JEV) infection in a human neuroblastoma cell line, IMR32. Cytochalasin D and nocodazole were used to depolymerise the cellular actin and microtubules, respectively, in order to study the effect of JEV infection in the cell. This study shows that depolymerisation of the actin cytoskeleton at early process of infection inhibits JEV infection in the cell; however infection was not inhibited when depolymerisation occurred at the later stage of infection. The microtubules, on the other hand, are required at 2 points in infection. The antigen production in the cells was inhibited when the infected cells were treated at time up to 2 hours after inoculation and there was no significant effect at later times, while the viable virus released continued to be affected until 10 hours after inoculation. In conclusion, infection of JEV in IMR32 cells required actin to facilitate early process in infection and the microtubular network is utilised as the transport system to the virus replication site and the release of mature virus. PMID:25705678

  3. 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.

  4. Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells.

    PubMed

    Rosińska, Sara; Leśniak, Wiesława; Filipek, Anna

    2016-07-01

    CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation.

  5. Neuroblastoma and Its Zebrafish Model.

    PubMed

    Zhu, Shizhen; Thomas Look, A

    2016-01-01

    Neuroblastoma, an important developmental tumor arising in the peripheral sympathetic nervous system (PSNS), accounts for approximately 10 % of all cancer-related deaths in children. Recent genomic analyses have identified a spectrum of genetic alterations in this tumor. Amplification of the MYCN oncogene is found in 20 % of cases and is often accompanied by mutational activation of the ALK (anaplastic lymphoma kinase) gene, suggesting their cooperation in tumor initiation and spread. Understanding how complex genetic changes function together in oncogenesis has been a continuing and daunting task in cancer research. This challenge was addressed in neuroblastoma by generating a transgenic zebrafish model that overexpresses human MYCN and activated ALK in the PSNS, leading to tumors that closely resemble human neuroblastoma and new opportunities to probe the mechanisms that underlie the pathogenesis of this tumor. For example, coexpression of activated ALK with MYCN in this model triples the penetrance of neuroblastoma and markedly accelerates tumor onset, demonstrating the interaction of these modified genes in tumor development. Further, MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. In the context of MYCN overexpression, activated ALK provides prosurvival signals that block this apoptotic response, allowing continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. This application of the zebrafish model illustrates its value in rational assessment of the multigenic changes that define neuroblastoma pathogenesis and points the way to future studies to identify novel targets for therapeutic intervention.

  6. High frequency of p53/MDM2/p14ARF pathway abnormalities in relapsed neuroblastoma

    PubMed Central

    Carr-Wilkinson, Jane; O' Toole, Kieran; Wood, Katrina M.; Challen, Christine C.; Baker, Angela G.; Board, Julian R.; Evans, Laura; Cole, Michael; Cheung, Nai-Kong V.; Boos, Joachim; Köhler, Gabriele; Leuschner, Ivo; Pearson, Andrew D.J.; Lunec, John; Tweddle, Deborah A.

    2010-01-01

    Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14ARF pathway in 9/17 (53%) neuroblastoma cell lines established at relapse. Hypothesis: Inactivation of the p53/MDM2/p14ARF pathway develops during treatment and contributes to neuroblastoma relapse. Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14ARF methylation and deletion by methylation-specific PCR and duplex PCR respectively, and MDM2 amplification by fluorescent in-situ hybridisation. Results: Abnormalities in the p53 pathway were identified in 20/41(49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6/41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, post chemotherapy and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio 3.4, 95% confidence interval 1.2, 9.9; p = 0.02). Upstream defects were present in 35% cases: MDM2 amplification in 3 cases, all at diagnosis & relapse and p14ARF inactivation in 12/41 (29%) cases: 3 had p14ARF methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects where p53 independent therapies are indicated. PMID:20145180

  7. Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

    PubMed Central

    Walter, U; Costa, M R; Breakefield, X O; Greengard, P

    1979-01-01

    Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells. Images PMID:226964

  8. Neuroblastoma cell death is induced by inorganic arsenic trioxide (As(2)O(3)) and inhibited by a normal human bone marrow cell-derived factor.

    PubMed

    Gesundheit, Benjamin; Malach, Lea; Or, Reuven; Hahn, Talia

    2008-12-01

    Three phenotypically distinct cell types are present in human neuroblastomas (NB) and NB cell lines: I-type stem cells, N-type neuroblastic precursors, and S-type Schwannian/melanoblastic precursors. The stimulation of human N-type neuroblastoma cell proliferation by normal human bone marrow monocytic cell conditioned medium (BMCM) has been demonstrated in vitro, a finding consistent with the high frequency of bone marrow (BM) metastases in patients with advanced NB. Inorganic arsenic trioxide (As(2)O(3)), already clinically approved for the treatment of several hematological malignancies, is currently under investigation for NB. Recent studies show that As(2)O(3) induces apoptosis in NB cells. We examined the impact of BMCM on growth and survival of As(2)O(3)-treated NB cell lines, to evaluate the response of cultured NB cell variants to regulatory agents. We studied the effect of BMCM on survival and clonogenic growth of eleven As(2)O(3)-treated NB cell lines grown in sparsely seeded, non-adherent, semi-solid cultures. As(2)O(3) had a strong inhibitory effect on survival of all tested NB cell lines. BMCM augmented cell growth and survival and reversed the inhibitory action of As(2)O(3) in all tested cell lines, but most strongly in N-type cells(.) While As(2)O(3) effectively reduced survival of all tested NB cell lines, BMCM effectively impacted its inhibitory action. Better understanding of micro-environmental regulators affecting human NB tumor cell growth and survival may be seminal to the development of therapeutic strategies and clinically effective agents for this condition.

  9. 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.

  10. [COMPARISON OF CYTOPROTECTIVE EFFECTS OF HEMANTANE AND AMANTADINE UNDER CONDITIONS OF 6-HYDROXYDOPAMINE NEUROTOXIN ACTION ON CULTURED HUMAN NEUROBLASTOMA CELLS].

    PubMed

    Logvinov, I O; Antipova, T A; Nepoklonov, A V; Valdman, E A

    2016-01-01

    Potential neuroprotective activity of the novel antiparkinsonian drug hemantane (hydrochloride N-2-(adamantyl)-hexamethylenimine) in comparison to amantadine has been studied in various regimes of administration on human neuroblastoma SH-SY5Y cell line injury induced by 6-hydroxydopamine (6-OHDA), which is used as in vitro model of dopaminergic neurons for Parkinson's disease. Two regimes of hemantane and amantadine administration in a range of final concentrations 10⁻⁶-10⁻⁸ M were used either prior to or immediately after 6-OHDA introduction. MTT colorimetric assay was used to assess the viability of test cells. Significant decrease in viability of SH-SY5Y cells treated with 6-OHDA was observed. The addition of hemantane to cell medium produced cytoprotective effects in both regimes of administration--before and after 6-OHDA--at concentrations 10⁻⁷ M and 10⁻⁶-10⁻⁸ M, respectively. Amantadine in con- centrations 10⁻⁷-10⁻⁸ M was effective to increase cell survival only when administered after 6-OHDA. These results show that hemantane has a greater neu-roprotective potential in comparison to amantadine.

  11. Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells.

    PubMed

    Földi, István; Tóth, Anikó M; Szabó, Zoltán; Mózes, Emese; Berkecz, Róbert; Datki, Zsolt L; Penke, Botond; Janáky, Tamás

    2013-01-01

    Disturbances in intraluminal endoplasmic reticulum (ER) Ca(2+) concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca(2+) homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.

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

    PubMed Central

    2013-01-01

    Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

  13. Morphine and endomorphins differentially regulate micro-opioid receptor mRNA in SHSY-5Y human neuroblastoma cells.

    PubMed

    Yu, Xin; Mao, Xin; Blake, Allan D; Li, Wen Xin; Chang, Sulie L

    2003-08-01

    A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure micro-opioid receptor (MOR) mRNA expression in SHSY-5Y neuroblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 microM) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antagonist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists' regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA expression paralleled changes in MOR receptor function, intracellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomorphin-1 or -2 than in untreated control cells. Together, our studies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells.

  14. Cloning of a brain-type isoform of human Rab GDI and its expression in human neuroblastoma cell lines and tumor specimens.

    PubMed

    Nishimura, N; Goji, J; Nakamura, H; Orita, S; Takai, Y; Sano, K

    1995-11-15

    Rab proteins, a family of Ras-related small GTP-binding proteins, play a key role in regulating intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins and thereby prevents the exchange of GDP for GTP. Recently, two isoforms of Rab GDI cDNA were isolated from rats and mice. In this study, we have isolated a brain-type isoform of human Rab GDI cDNA and examined its expression in neuroblastoma. We tentatively designate it as human Rab GDI alpha (hu GDI alpha) and another human Rab GDI, as human Rab GDI beta (hu GDI beta). Hu GDI alpha cDNA encodes a protein of 447 amino acids with a deduced molecular weight of 50,200. Northern blot analysis revealed that hu GDI alpha gene is expressed abundantly in the brain but much less in other tissues, while hu GDI beta gene is ubiquitously expressed. All human neuroblastoma cell lines and tumor specimens examined express hu GDI alpha gene to various extents, while a human T cell leukemia cell line, MOLT3, does not. The levels of both hu GDI alpha and beta mRNA were constant in a human neuroblastoma cell line, NB1, during its neuronal differentiation, while Rab3A and neurofilament-L gene expression and the number of neurosecretory granules were elevated at this condition. These results suggest that hu GDI alpha gene expression is not related to the differentiation state of neuronal cells.

  15. Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells.

    PubMed

    Perez-Alvarez, Sergio; Iglesias-Guimarais, Victoria; Solesio, María E; Melero-Fernandez de Mera, Raquel María; Yuste, Víctor J; Galindo, María F; Jordán, Joaquín

    2011-04-01

    Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors.

  16. Bone Marrow-Infiltrating Human Neuroblastoma Cells Express High Levels of Calprotectin and HLA-G Proteins

    PubMed Central

    Gallo, Fabio; Stigliani, Sara; Moretti, Stefano; Bonassi, Stefano; Gambini, Claudio; Mazzocco, Katia; Fardin, Paolo; Haupt, Riccardo; Arcamone, Giampaolo; Pistoia, Vito; Tonini, Gian Paolo; Corrias, Maria Valeria

    2012-01-01

    Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients. PMID:22253825

  17. Outgrowth of Neurites from NIE-115 Neuroblastoma Cells Is Prevented on Repulsive Substrates through the Action of PAK

    PubMed Central

    Marler, Katharine J. M.; Kozma, Robert; Ahmed, Sohail; Dong, Jing-Ming; Hall, Christine; Lim, Louis

    2005-01-01

    In the central nervous system (CNS), damaged axons are inhibited from regeneration by glial scars, where secreted chondroitin sulfate proteoglycan (CSPG) and tenascin repulse outgrowth of neurites, the forerunners of axons and dendrites. During differentiation, these molecules are thought to form boundaries for guiding neurons to their correct targets. In neuroblastoma NIE-115 cells, outgrowth of neurites on laminin could be induced by serum starvation or inhibition of RhoA by Clostridium botulinum C3 toxin. The outgrowing neurites avoided crossing onto the repulsive substrate CSPG or tenascin. This avoidance response was partially overcome on expression of membrane-targeted and kinase-inactive forms of PAK. In these cells, the endogenous PAK isoforms colocalized with actin in distinctive sites, αPAK in the cell center as small clusters and along the neurite shaft and βPAK and γPAK in areas with membrane ruffles and filopodia, respectively. When isoform-specific N-terminal PAK sequences were introduced to interfere with PAK function, substantially more neurites crossed onto CSPG when cells contained a γPAK-derived peptide but not the corresponding αPAK- or βPAK-derived peptide. Thus, while neurite outgrowth can be promoted by RhoA inhibition, overcoming the accompanying repulsive guidance response will require modulation of PAK activity. These results have therapeutic implications for CNS repair processes. PMID:15923637

  18. [Pt(O,O'-acac)(γ-acac)(DMS)] versus cisplatin: apoptotic effects in B50 neuroblastoma cells.

    PubMed

    Grimaldi, Maddalena; Santin, Giada; Insolia, Violetta; Dal Bo, Veronica; Piccolini, Valeria Maria; Veneroni, Paola; Barni, Sergio; Verri, Manuela; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Bernocchi, Graziella; Bottone, Maria Grazia

    2016-05-01

    Cisplatin is one of the most active chemotherapeutic agents used in the treatment of childhood and adult malignancies. Cisplatin induces cell death through different pathways. Despite its effectiveness, the continued clinical use of cisplatin is limited by onset of severe side effects (nephrotoxicity, ototoxicity and neurotoxicity) and drug resistance. Therefore, one of the main experimental oncology purpose is related to the search for new platinum-based drugs to create different types of adducts or more specific and effective subcellular targets. Thus, [Pt(O,O'-acac)(γ-acac)(DMS)], which reacts preferentially with protein thiols or thioether, was synthesized. In our research, different approaches were used to compare cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)] effects in B50 rat neuroblastoma cells. Our results, using immunocytochemical, cytometric and morphological techniques, showed that these compounds exert a cytostatic action and activate apoptosis with different pathways. Long-term effects demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] exerts cytotoxic effects in neuronal B50 cell line not inducing drug resistance. Analysis was performed both to compare the ability of these platinum compounds to induce cell death and to investigate the intracellular mechanisms at the basis of their cytotoxicity.

  19. 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.

  20. Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells.

    PubMed

    Sulpizio, Marilisa; Falone, Stefano; Amicarelli, Fernanda; Marchisio, Marco; Di Giuseppe, Fabrizio; Eleuterio, Enrica; Di Ilio, Carmine; Angelucci, Stefania

    2011-12-01

    Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.

  1. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells.

    PubMed

    Xu, Qingqiang; Zhu, Naiwei; Chen, Shenglin; Zhao, Ping; Ren, Hao; Zhu, Shiying; Tang, Hailin; Zhu, Yongzhe; Qi, Zhongtian

    2017-03-28

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection.

  2. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells

    PubMed Central

    Xu, Qingqiang; Zhu, Naiwei; Chen, Shenglin; Zhao, Ping; Ren, Hao; Zhu, Shiying; Tang, Hailin; Zhu, Yongzhe; Qi, Zhongtian

    2017-01-01

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection. PMID:28349961

  3. 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.

  4. Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma

    PubMed Central

    Corrias, M V; Parodi, S; Haupt, R; Lacitignola, L; Negri, F; Sementa, A R; Dau, D; Scuderi, F; Carlini, B; Bianchi, M; Casale, F; Faulkner, L; Garaventa, A

    2008-01-01

    The impact of bone marrow (BM) GD2-positive cells on survival has been evaluated in 145 Italian children with localised neuroblastoma (NB) evaluated at diagnosis by anti-GD2 immunocytochemistry. Nineteen of these (13.1%) were found to be BM GD2-positive, with the number of positive cells ranging between 1 and 155 out of 1 × 106 total cells analysed. Seven/19 (38.8%) GD2-positive vs 12/126 (9.5%) GD2-negative patients relapsed. The 5-year event-free survival (EFS) and overall survival of the GD2-positive patients was significantly worse than that of the GD2-negative ones (62.2 vs 89.9%, P<0.001; and 74.9 vs 95.9%, P=0.005, respectively). GD2 positivity was not associated to other known risk factors, and in particular to Myc-N amplification and 1p deletion. Among Myc-N-negative patients, the EFS of those negative for both GD2 and 1p deletion was significantly better than in children positive for either one of these two markers (EFS=96.9 vs 66.0%, P<0.001). In conclusion, GD2 positivity may represent a prognostic marker for patients with non-metastatic NB without Myc-N amplification, and its combination with genetic alterations might help identifying patients that require a more careful follow-up. PMID:18182983

  5. Fifty hertz extremely low-frequency electromagnetic field causes changes in redox and differentiative status in neuroblastoma cells.

    PubMed

    Falone, Stefano; Grossi, Maria R; Cinque, Benedetta; D'Angelo, Barbara; Tettamanti, Enzo; Cimini, Annamaria; Di Ilio, Carmine; Amicarelli, Fernanda

    2007-01-01

    The current study was designed to establish whether extremely low-frequency electromagnetic fields might affect neuronal homeostasis through redox-sensitive mechanisms. To this end, intracellular reactive oxygen species production, antioxidant and glutathione-based detoxifying capability and genomic integrity after extremely low-frequency electromagnetic fields exposure were investigated. Moreover, we also studied potential extremely low-frequency electromagnetic fields-dependent changes in the proliferative and differentiative cellular status. Results seem to support redox-mediated extremely low-frequency electromagnetic fields effects on biological models as, although no major oxidative damage was detected, after exposure we observed a positive modulation of antioxidant enzymatic expression, as well as a significant increase in reduced glutathione level, indicating a shift of cellular environment towards a more reduced state. In addition, extremely low-frequency electromagnetic fields treatment induced a more differentiated phenotype as well as an increased expression in peroxisome proliferators-activated receptor isotype beta, a class of transcription factors related to neuronal differentiation and cellular stress response. As second point, to deepen how extremely low-frequency electromagnetic fields treatment could affect neuroblastoma cell antioxidant capacity, we examined the extremely low-frequency electromagnetic fields-dependent modifications of cell susceptibility to pro-oxidants. Results clearly showed that 50 Hz extremely low-frequency electromagnetic fields exposure reduces cell tolerance towards oxidative attacks.

  6. 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

  7. 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.

  8. Influence of carrier cells on the clinical outcome of children with neuroblastoma treated with high dose of oncolytic adenovirus delivered in mesenchymal stem cells.

    PubMed

    Melen, Gustavo J; Franco-Luzón, Lidia; Ruano, David; González-Murillo, África; Alfranca, Arantzazu; Casco, Fernando; Lassaletta, Álvaro; Alonso, Mercedes; Madero, Luís; Alemany, Ramón; García-Castro, Javier; Ramírez, Manuel

    2016-02-28

    We report here our clinical experience of a program of compassionate use of Celyvir--autologous marrow-derived mesenchymal stem cells (MSCs) carrying an oncolytic adenovirus--for treating children with advanced metastatic neuroblastoma. Children received weekly doses of Celyvir with no concomitant treatments. The tolerance was excellent, with very mild and self-limited viral-related symptoms. Patients could be distinguished based on their response to therapy: those who had a clinical response (either complete, partial or stabilization) and those who did not respond. We found differences between patients who responded versus those who did not when analyzing their respective MSCs, at the expression levels of adhesion molecules (CCR1, CXCR1 and CXCR4) and in migration capacities in transwell assays, and in immune-related molecules (IFNγ, HLA-DR). These results suggest interpatient differences in the homing and immune modulation capacities of the therapy administered. In addition, the pretherapy immune T cell status and the T effector response were markedly different between responders and non-responders. We conclude that multidoses of Celyvir have an excellent safety profile in children with metastatic neuroblastoma. Intrinsic patients' and MSCs' factors appear to be related to clinical outcome.

  9. Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways.

    PubMed

    Durbas, Małgorzata; Horwacik, Irena; Boratyn, Elżbieta; Rokita, Hanna

    2016-08-01

    We have recently shown that mRNA and protein of PHLDA1 (pleckstrin-homology-like domain family A, member  1) were by far the most upregulated molecules upon treatment of IMR-32 cells with the anti-GD2 ganglioside monoclonal antibody 14G2a. Hence, we decided to study functions of PHLDA1 using human neuroblastoma IMR-32 cells as a model. Here, we show that constitutive expression of mRNA and protein of the PHLDA1 gene in IMR-32 cells was inversely correlated with transcript of the AURKA gene and Aurora A oncoprotein. Next, we silenced PHLDA1 expression in IMR-32 cells using an shRNA interference method. We report that IMR-32 cells with stable downregulation of PHLDA1 showed enhanced cellular ATP levels and an increase in mitochondrial membrane potential, as compared to control and non-transduced cells. We demonstrated that downregulation of PHLDA1 leads to a significant increase in expression of Aurora A and TRKB that are markers of poor prognosis in neuroblastoma. Also, we measured an increase in Aurora A and Akt kinases phosphorylation in the cells. Most importantly, PHLDA1-silenced cells were less susceptible to apoptosis than control cells, as shown by the lower expression of cleaved caspase-3 and PARP as well as a decreased activity of caspase-3 and -7. Our study negatively correlates expression of PHLDA1 and Aurora A in IMR-32 cells and sheds new light on functions of PHLDA1 in the neuroblastoma tumor cells, suggesting its role as a pro-apoptotic protein. Additionally, our results show possible links of the protein to regulation of features of mitochondria and formation of autophagosomes.

  10. 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

  11. 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.

  12. Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

    PubMed

    Salani, Monica; Anelli, Tonino; Tocco, Gabriella Augusti; Lucarini, Elena; Mozzetta, Chiara; Poiana, Giancarlo; Tata, Ada Maria; Biagioni, Stefano

    2009-02-01

    Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

  13. Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy.

    PubMed

    Veith, Nathalie M; Plattner, Helmut; Stuermer, Claudia A O; Schulz-Schaeffer, Walter J; Bürkle, Alexander

    2009-01-01

    The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrPC/PrPSc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.

  14. Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

    PubMed Central

    Angrisano, T.; Sacchetti, S.; Natale, F.; Cerrato, A.; Pero, R.; Keller, S.; Peluso, S.; Perillo, B.; Avvedimento, V. E.; Fusco, A.; Bruni, C. B.; Lembo, F.; Santoro, M.; Chiariotti, L.

    2011-01-01

    Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci. PMID:20952403

  15. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

    PubMed Central

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations. PMID:25692239

  16. 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.

  17. 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

  18. 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.

  19. 17β-estradiol downregulated the expression of TASK-1 channels in mouse neuroblastoma N2A cells.

    PubMed

    Hao, Xuran; Li, Xiaoqing; Li, Xiantao

    2014-03-01

    TASK channels, an acid-sensitive subgroup of two pore domain K⁺ (K2P) channels family, were widely expressed in a variety of neural tissues, and exhibited potent functions such as the regulation of membrane potential. The steroid hormone estrogen was able to interact with K⁺ channels, including voltage-gated K⁺ (Kv) and large conductance Ca²⁺-activated (BK) K⁺ channels, in different types of cells like cardiac myocytes and neurons. However, it is unclear about the effects of estrogen on TASK channels. In the present study, the expressions of two members of acid-sensitive TASK channels, TASK-1 and TASK-2, were detected in mouse neuroblastoma N2A cells by RT-PCR. Extracellular acidification (pH 6.4) weakly but statistically significantly inhibited the outward background current by 22.9 % at a holding potential of 0 mV, which inactive voltage-gated K⁺ currents, suggesting that there existed the functional TASK channels in the membrane of N2A cells. Although these currents were not altered by the acute application of 100 nM 17β-estradiol, incubation with 10 nM 17β-estradiol for 48 h reduced the mRNA level of TASK-1 channels by 40.4 % without any effect on TASK-2 channels. The proliferation rates of N2A cells were also increased by treatment with 10 nM 17β-estradiol for 48 h. These data implied that N2A cells expressed functional TASK channels and chronic exposure to 17β-estradiol downregulated the expression of TASK-1 channels and improved cell proliferation. The effect of 17β-estradiol on TASK-1 channels might be an alternative mechanism for the neuroprotective action of 17β-estradiol.

  20. Effects of inorganic lead on voltage-sensitive calcium channels in N1E-115 neuroblastoma cells.

    PubMed

    Audesirk, G; Audesirk, T

    1991-01-01

    N1E-115 mouse neuroblastoma cells have been reported to possess two types of voltage-sensitive calcium channels: Low voltage activated, rapidly inactivating T-type (type I) and high voltage activated, slowly inactivating L-type (type II). We studied the effects of acute in vitro exposure to inorganic lead on these calcium channels, using the whole-cell variant of patch clamping. Using salines with a high lead-buffering capacity, we found that both T-type and L-type channels are reversibly inhibited in a dose-dependent manner at free Pb2+ concentrations ranging from 20 nM to 14 microM. L-type channels are somewhat more sensitive to Pb2+ than T-type channels are (L-type: IC50 approx. 0.7 microM; T-type: IC50 approx. 1.3 microM). Both channels show small but significant inhibition (approx. 10%) at 20 nM free Pb2+. Pb2+ affects neither activation nor inactivation of T-type channels, but enhances inactivation of L-type channels at holding potentials around -60 to -40 mV. A peculiar phenomenon was observed in cells exposed to 2.3 microM free Pb2+. T-type channels were inhibited in all 20 cells studied. In 15 cells, L-type channels were also inhibited, but in the remaining 5 cells, current flow through L-type channels was enhanced by Pb2+ exposure.

  1. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA).

    PubMed

    Gotoh, Mari; Sano-Maeda, Katsura; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2012-01-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2)) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl(2) induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2)-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2). Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1), LPA(2), and LPA(6), which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1) and LPA(2) have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1) and LPA(2) on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1) and LPA(3) antagonist, was adopted to know the LPA(1) function and siRNA was used to knockdown the expression of LPA(2). On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2)-induced hypoxia damage is mediated via LPA(2).

  2. Neuroblastoma: A Tough Nut to Crack.

    PubMed

    Speleman, Frank; Park, Julie R; Henderson, Tara O

    2016-01-01

    Neuroblastoma, an embryonal tumor arising from neural crest-derived progenitor cells, is the most common solid tumor in childhood, with more than 700 cases diagnosed per year in the United States. In the past several decades, significant advances have been made in the treatment of neuroblastoma. Treatment advances reflect improved understanding of the biology of neuroblastoma. Although amplification of MYCN was discovered in the early 1980s, our understanding of neuroblastoma oncogenesis has advanced in the last decade as a result of high-throughput genomic analysis, exome and whole-genome sequencing, genome-wide association studies, and synthetic lethal drug screens. Our refined understanding of neuroblastoma biology and genetics is reflected in improved prognostic stratification and appropriate tailoring of therapy in recent clinical trials. Moreover, for high-risk neuroblastoma, a disease that was uniformly fatal 3 decades ago, recent clinical trials incorporating autologous hematopoietic transplant and immunotherapy utilizing anti-GD2 antibody plus cytokines have shown improved event-free and overall survival. These advances have resulted in a growing population of long-term survivors of neuroblastoma. Examination of the late effects and second malignant neoplasms (SMNs) in both older generations of survivors and more recently treated survivors will inform both design of future trials and surveillance guidelines for long-term follow-up. As a consequence of advances in understanding of the biology of neuroblastoma, successful clinical trials, and refined understanding of the late effects and SMNs of survivors, the promise of precision medicine is becoming a reality for patients with neuroblastoma.

  3. Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics.

    PubMed

    Bao, Lei; Chen, Shu-Jen; Conrad, Kathleen; Keefer, Kerry; Abraham, Thomas; Lee, John P; Wang, JuFang; Zhang, Xue-Qian; Hirschler-Laszkiewicz, Iwona; Wang, Hong-Gang; Dovat, Sinisa; Gans, Brian; Madesh, Muniswamy; Cheung, Joseph Y; Miller, Barbara A

    2016-11-18

    Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.

  4. Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

    PubMed

    Di Scala, Coralie; Mazzarino, Morgane; Yahi, Nouara; Varini, Karine; Garmy, Nicolas; Fantini, Jacques; Chahinian, Henri

    2017-04-04

    Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

  5. Calcium-independent effects of TMB-8. Modification of phospholipid metabolism in neuroblastoma cells by inhibition of choline uptake.

    PubMed Central

    Palmer, F B; Byers, D M; Spence, M W; Cook, H W

    1992-01-01

    TMB-8 [8-(NN-diethylamino)-octyl-3,4,5-trimethoxybenzoate] blocks agonist-stimulated release of Ca2+ from intracellular sites in many cell lines and is often used to distinguish between dependence on extracellular and intracellular Ca2+. In N1E-115 neuroblastoma cells, TMB-8 did not alter the resting cytosolic Ca2+ concentration in unstimulated cells, yet phospholipid metabolism was greatly affected. At concentrations of TMB-8 (25-150 microM) that inhibit Ca2+ release, phosphatidylcholine formation was inhibited, whereas synthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine was stimulated. Unlike other cationic amphipathic compounds, TMB-8 did not inhibit phosphatidate phosphatase or enzymes in the pathway from choline to phosphatidylcholine. Choline transport was the major site of action. TMB-8 was a competitive inhibitor (Ki = 10 microM) of low-affinity (Kt = 20 microM) choline transport. When added at the same time as labelled precursor, TMB-8 also decreased cellular uptake of phosphate and inositol, but not that of ethanolamine or serine. In prelabelled cells, continued uptake and incorporation of phosphate and inositol were not affected. Under these conditions phosphatidylinositol synthesis was increased 2-fold and, like the effect on phosphatidylcholine, reached a plateau at 100 microM-TMB-8. Phosphatidylglycerol synthesis increased linearly with TMB-8 concentration to 40-fold stimulation at 150 microM, suggesting a selective effect on synthesis of phosphatidylglycerol from CDP-diacylglycerol. Phosphatidylserine synthesis was also increased up to 3-fold. These Ca(2+)-independent effects limit the use of TMB-8 in studies of cell signalling that involve stimulated phosphatidylinositol and phosphatidylcholine metabolism. PMID:1530583

  6. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

    PubMed

    Luessen, Deborah J; Hinshaw, Tyler P; Sun, Haiguo; Howlett, Allyn C; Marrs, Glen; McCool, Brian A; Chen, Rong

    2016-11-01

    Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

  7. 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.

  8. Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence

    PubMed Central

    Taschner-Mandl, Sabine; Schwarz, Magdalena; Blaha, Johanna; Kauer, Maximilian; Kromp, Florian; Frank, Nelli; Rifatbegovic, Fikret; Weiss, Tamara; Ladenstein, Ruth; Hohenegger, Martin; Ambros, Inge M.; Ambros, Peter F.

    2016-01-01

    Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified. We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21WAF/CIP1 up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP. This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles. PMID:26657295

  9. Low dose of arsenic trioxide inhibits multidrug resistant-related P-glycoprotein expression in human neuroblastoma cell line.

    PubMed

    Liu, Ling; Li, Yang; Xiong, Xilin; Qi, Kai; Zhang, Chi; Fang, Jianpei; Guo, Haixia

    2016-12-01

    This study investigated arsenic trioxide (As2O3), cisplatin (DDP) and etoposide (Vp16) on the anticancer effects and P-glycoprotein (P-gp) expression in neuroblastoma (NB) SK-N-SH cells. The potential influence of As2O3, DDP and Vp16 currently included in NB routine treatment protocols on cytotoxicity in SK-N-SH cells was measured by flow cytometry and drug half-maximal inhibitory concentration (IC50) was established. Moreover, chemotherapeutic agent-mediated changes of cellular expression levels of resistant-related P-gp, was monitored using western blotting. The data showed that As2O3, DDP and Vp16 significantly inhibited the growth and survival of the SK-N-SH cells at different concentration. Notably, the levels of apoptosis were upregulated in SK-N-SH cells with an acceleration of the exposure time and the concentration of As2O3, DDP and Vp16. As2O3, DDP and Vp16 were observed with their IC50 values on SK-N-SH cells being 3 µM, 8 and 100 µg/ml, respectively. Flow cytometry analysis showed that As2O3 at low concentrations in SK-N-SH cells led to enhanced accumulation of cell populations in G2/M phase with increasing the exposure time, and increased levels of apoptosis. In contrast, we observed that SK-N-SH cell populations arrested in S phase by DDP and Vp16. In vitro examination revealed that following pretreatment of SK-N-SH cells with As2O3, the expression of P-gp was not increased. The expression of P-gp downregulation were noted following the group treated by As2O3 at 2 and 3 µM. Exposed to As2O3 at 3 µM for 72 h, SK-N-SH cells exhibited lower expression of P-gp than 2 µM As2O3 for 72 h. In contrast, the expression of P-gp was upregulated by DDP and VP16. In summary, SK-N-SH cells were responsive to chemotherapeutic agent-induced apoptosis in a dose-dependent and time-dependent manner. In particular, ours findings showed that low dose of As2O3 markedly reduced the P-gp expression and increased apoptotic cell death in human NB cell line.

  10. Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a.

    PubMed

    Horwacik, Irena; Rokita, Hanna

    2017-05-01

    Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1β1 heterodimer), BIO 1211 (inhibiting active α4β1 heterodimer), cilengitide and SB273005 (inhibitors of αVβ3, αVβ5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.

  11. 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

  12. Short-term regulation of muscarinic acetylcholine receptors: An assessment utilizing mouse brain and mouse neuroblastoma cells

    SciTech Connect

    Cioffi, C.L.

    1988-01-01

    The effects of muscarinic agonists and diisopropylfluorophosphate (DFP) on muscarinic receptor density and muscarinic receptor-mediated responses was assessed in mouse brain and mouse neuroblastoma cells (clone N1E-115). Utilizing the antagonist ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB), there was no difference in the maximal binding capacity (B{sub max}) or equilibrium dissociation constant (K{sub d}) between untreated and 24 hour DFP-treated mice. However, one administration of DFP produced a 24% and 33% decrease in B{sub max} measured by ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) after 18 and 24 hours which was rapidly reversible within 36 hours after DFP treatment. The loss of ({sup 3}H)NMS binding sites following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment.

  13. Osthol is a use-dependent blocker of voltage-gated Na+ channels in mouse neuroblastoma N2A cells.

    PubMed

    Leung, Yuk-Man; Kuo, Yueh-Hsiung; Chao, Chia-Chia; Tsou, Yi-Huan; Chou, Chun-Hsiao; Lin, Chia-Huei; Wong, Kar-Lok

    2010-01-01

    Osthol, a Chinese herbal compound, has been shown to possess vasorelaxant and neuroprotective properties. Not much is known about the effects of osthol on ionic channels, activities of which are implicated in vasorelaxation and neuroprotection. In this work we report that osthol could inhibit voltage-gated Na (+) currents with state-dependence in mouse neuroblastoma N2A cells (IC (50) = 12.3 microM and 31.5 microM at holding potentials of - 70 mV and - 100 mV, respectively). Current blockade was equally effective in both extracellular and intracellular application of osthol. Osthol (18 microM) did not significantly affect the kinetics and voltage-dependence of Na (+) channel activation, but left-shifted the steady-state inactivation curve (V (1/2) = - 60.5 mV and - 78.7 mV in the absence and presence of osthol, respectively). Osthol also mildly but significantly retarded channel recovery from inactivation (recovery time constant = 19.9 ms and 35.6 ms in the absence and presence of osthol, respectively). In addition, osthol blocked Na (+) currents in a frequency-dependent fashion: blockades of 17 %, 34 % and 49 % when currents were triggered at 0.33 Hz, 1 Hz and 3.33 Hz, respectively. Taken together, our results therefore suggest that osthol blocked voltage-gated Na (+) channels intracellularly with state- and frequency-dependence.

  14. The Tat antagonist neomycin B hexa-arginine conjugate inhibits gp-120-induced death of human neuroblastoma cells.

    PubMed

    Catani, Maria Valeria; Corasaniti, Maria Tiziana; Ranalli, Marco; Amantea, Diana; Litovchick, Alexander; Lapidot, Aviva; Melino, Gerry

    2003-03-01

    Several patients with acquired immunodeficiency syndrome (AIDS) develop neurological complications, which are referred to as human immunodeficiency virus (HIV)-associated dementia (HAD). The HIV-1 coat glycoprotein gp-120 has been proposed as the major etiologic agent for neuronal loss reported postmortem in the brain of AIDS patients. Chemokine receptors may play a role in gp-120-triggered neurotoxicity, both in vitro and in vivo, thus being an intriguing target for developing therapeutic strategies aimed to prevent or reduce neuronal damage occurring during HIV infection. We have previously shown that human CHP100 neuroblastoma cells express CXCR4 and CCR5 chemokine receptors and that interaction between gp-120 and these receptors contributes to cytotoxicity elicited by the protein. Here, we examined the neuroprotective potential of neomycin B hexa-arginine conjugate (NeoR), a recently synthesized compound with anti-HIV activity. We found that gp-120-triggered death is significantly reduced by NeoR, and this protective effect seems related to the ability of NeoR to interact with CXCR4 receptors. The ability of NeoR to cross the blood-brain barrier, as demonstrated in mice by systemic administration of the fluorescein conjugate drug, makes this compound a powerful and attractive therapeutic agent.

  15. 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.

  16. Stress Conditions Increase Vimentin Cleavage by Omi/HtrA2 Protease in Human Primary Neurons and Differentiated Neuroblastoma Cells.

    PubMed

    Lucotte, Bérangère; Tajhizi, Mehdi; Alkhatib, Dareen; Samuelsson, Eva-Britt; Wiehager, Birgitta; Schedin-Weiss, Sophia; Sundström, Erik; Winblad, Bengt; Tjernberg, Lars O; Behbahani, Homira

    2015-12-01

    Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

  17. 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.

  18. 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

  19. Uptake of silica nanoparticles: neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells.

    PubMed

    Yang, Xifei; He, Chun'e; Li, Jie; Chen, Hongbin; Ma, Quan; Sui, Xiaojing; Tian, Shengli; Ying, Ming; Zhang, Qian; Luo, Yougen; Zhuang, Zhixiong; Liu, Jianjun

    2014-08-17

    Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 μg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular β-amyloid 1-42 (Aβ(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-β-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3β at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3β. This is the first

  20. Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation.

    PubMed

    Gómez-Villafuertes, Rosa; Pintor, Jesús; Miras-Portugal, María Teresa; Gualix, Javier

    2014-11-01

    Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn(2+) ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells

  1. 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.

  2. The long non-coding RNA GAS5 differentially regulates cell cycle arrest and apoptosis through activation of BRCA1 and p53 in human neuroblastoma

    PubMed Central

    Mazar, Joseph; Rosado, Amy; Shelley, John; Marchica, John; Westmoreland, Tamarah J

    2017-01-01

    The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death. PMID:28035057

  3. HMJ-53A accelerates slow inactivation gating of voltage-gated K+ channels in mouse neuroblastoma N2A cells.

    PubMed

    Chao, Chia-Chia; Shieh, Jeffrey; Kuo, Sheng-Chu; Wu, Bor-Tsang; Hour, Mann-Jen; Leung, Yuk-Man

    2008-06-01

    Voltage-gated K(+) (Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely understood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding the selectivity filter. Pharmacological tools probing this slow inactivation process are scarce. In this work we reported that bath application of HMJ-53A (30 microM), a novel compound, could drastically speed up the slow decay (decay tau=1677+/-120 ms and 85.6+/-7.7 ms, respectively, in the absence and presence of HMJ-53A) of Kv currents in neuroblastoma N2A cells. HMJ-53A also significantly left-shifted the steady-state inactivation curve by 12 mV. HMJ-53A, however, did not affect voltage-dependence of activation and the kinetics of channel activation. Intracellular application of this drug through patch pipette dialysis was ineffective at all in accelerating the slow current decay, suggesting that HMJ-53A acted extracellularly. Blockade of currents by HMJ-53A did not require an open state of channels. In addition, the inactivation time constants and percentage block of Kv currents in the presence of HMJ-53A were independent of the (i) degree of depolarization and (ii) intracellular K(+) concentration. Therefore, this drug did not appear to directly occlude the outer channel pore during stimulation (depolarization). Taken together, our results suggest that HMJ-53A selectively affected (accelerated) the slow inactivation gating process of Kv channels, and could thus be a selective and novel probe for the inactivation gate.

  4. Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells.

    PubMed

    Provansal, Monique; Roche, Stéphane; Pastore, Manuela; Casanova, Danielle; Belondrade, Maxime; Alais, Sandrine; Leblanc, Pascal; Windl, Otto; Lehmann, Sylvain

    2010-01-01

    Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.

  5. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    PubMed

    Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; De Poli, Angela; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio

    2014-01-01

    Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

  6. TDP-43 Inclusion Bodies Formed in Bacteria Are Structurally Amorphous, Non-Amyloid and Inherently Toxic to Neuroblastoma Cells

    PubMed Central

    Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; De Poli, Angela; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio

    2014-01-01

    Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein. PMID:24497973

  7. Radiolabeled Metaiodobenzylguanidine for the Treatment of Neuroblastoma

    PubMed Central

    DuBois, Steven G.; Matthay, Katherine K.

    2008-01-01

    Introduction Neuroblastoma is the most common pediatric extracranial solid cancer. This tumor is characterized by metaiodobenzylguanidine (MIBG) avidity in 90% of cases, prompting the use of radiolabeled MIBG for targeted radiotherapy in these tumors. Methods The available English language literature was reviewed for original research investigating in vitro, in vivo, and clinical applications of radiolabeled MIBG for neuroblastoma. Results MIBG is actively transported into neuroblastoma cells by the norepinephrine transporter. Preclinical studies demonstrate substantial activity of radiolabeled MIBG in neuroblastoma models, with 131I-MIBG showing enhanced activity in larger tumors compared to 125I-MIBG. Clinical studies of 131I-MIBG in patients with relapsed or refractory neuroblastoma have identified myelosuppression as the main dose-limiting toxicity, necessitating stem cell reinfusion at higher doses. Most studies report a response rate of 30–40% with 131I-MIBG in this population. More recent studies have focused on the use of 131I-MIBG in combination with chemotherapy or myeloablative regimens. Conclusions 131I-MIBG is an active agent for the treatment of patients with neuroblastoma. Future studies will need to define the optimal role of this targeted radiopharmaceutical in the therapy of this disease. PMID:18707633

  8. Didymin: an orally active citrus flavonoid for targeting neuroblastoma.

    PubMed

    Singhal, Sharad S; Singhal, Sulabh; Singhal, Preeti; Singhal, Jyotsana; Horne, David; Awasthi, Sanjay

    2017-02-08

    Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma.

  9. A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ2

    PubMed Central

    Dunham, Ann; Chen, Paula X.; Chen, Michelle; Huynh, Milan; Rheingold, Evan; Prosper, Olivia

    2016-01-01

    Researchers have observed that response of tumor cells to treatment varies depending on whether the cells are grown in monolayer, as in vitro spheroids or in vivo. This study uses data from the literature on monolayer treatment of SK-N-SH neuroblastoma cells with 15-deoxy-PGJ2 and couples it with data on growth rates for untreated SK-N-SH neuroblastoma cells grown as multicellular spheroids. A linear model is constructed for untreated and treated monolayer data sets, which is tuned to growth, death, and cell cycle data for the monolayer case for both control and treatment with 15-deoxy-PGJ2. The monolayer model is extended to a five-dimensional nonlinear model of in vitro tumor spheroid growth and treatment that includes compartments of the cell cycle (G1, S, G2/M) as well as quiescent (Q) and necrotic (N) cells. Monolayer treatment data for 15-deoxy-PGJ2 is used to derive a prediction of spheroid response under similar treatments. For short periods of treatment, spheroid response is less pronounced than monolayer response. The simulations suggest that the difference in response to treatment of monolayer versus spheroid cultures observed in laboratory studies is a natural consequence of tumor spheroid physiology rather than any special resistance to treatment. PMID:28044089

  10. PARP inhibitors enhance replication stress and cause mitotic catastrophe in MYCN-dependent neuroblastoma.

    PubMed

    Colicchia, V; Petroni, M; Guarguaglini, G; Sardina, F; Sahún-Roncero, M; Carbonari, M; Ricci, B; Heil, C; Capalbo, C; Belardinilli, F; Coppa, A; Peruzzi, G; Screpanti, I; Lavia, P; Gulino, A; Giannini, G

    2017-04-10

    High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.Oncogene advance online publication, 10 April 2017; doi:10.1038/onc.2017.40.

  11. KIR and HLA Genotypes are Associated with Disease Progression and Survival following Autologous Hematopoietic Stem Cell Transplantation for High-Risk Neuroblastoma

    PubMed Central

    Venstrom, Jeffrey M.; Zheng, Junting; Noor, Nabila; Danis, Karen E.; Yeh, Alice W.; Cheung, Irene Y.; Dupont, Bo; O’Reilly, Richard J.; Cheung, Nai-Kong V.; Hsu, Katharine C.

    2009-01-01

    Purpose Natural killer (NK) cells exhibit cytotoxicity against neuroblastoma. Gene polymorphisms governing NK cell function, therefore, may influence prognosis. Two highly polymorphic genetic loci instrumental in determining NK cell responses encode the NK cell killer immunoglobulin-like receptors (KIR) and their class I human leukocyte antigen (HLA) ligands. We hypothesized that patients with a “missing ligand” KIR-HLA compound genotype may uniquely benefit from autologous hematopoietic stem cell transplantation (HSCT). Experimental Design 169 patients treated with autologous HSCT for stage 4 neuroblastoma underwent KIR and HLA genotyping. Patients were segregated according to presence or absence of HLA ligands for autologous inhibitory KIR. Univariate and multivariate analyses were performed for overall and progression-free survival. Results 64% of patients lacked one or more HLA ligands for inhibitory KIR. Patients lacking an HLA ligand had a 46% lower risk of death (HR 0.54; 95% CI, 0.35–0.85, P=.007) and a 34% lower risk of progression (HR 0.66; 95% CI, 0.44–1.0; P=.047) at 3 years compared with patients who possessed all ligands for his/her inhibitory KIR. Among all KIR-HLA combinations, 16 patients lacking the HLA-C1 ligand for KIR2DL2/2DL3 experienced the highest 3-year survival rate of 81% (95% CI: 64–100). Survival was more strongly associated with “missing ligand” than with tumor MYCN gene amplification. Conclusion KIR-HLA immunogenetics represents a novel prognostic marker for patients undergoing autologous HSCT for high-risk neuroblastoma. PMID:19934297

  12. Mu and Delta opioid receptors activate the same G protein