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Sample records for neuroblastoma nb-1 cells

  1. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells

    DEFF Research Database (Denmark)

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-01-01

    kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways....... The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1...... by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation....

  2. Cell Proliferation in Neuroblastoma

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    Stafman, Laura L.; Beierle, Elizabeth A.

    2016-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, continues to carry a dismal prognosis for children diagnosed with advanced stage or relapsed disease. This review focuses upon factors responsible for cell proliferation in neuroblastoma including transcription factors, kinases, and regulators of the cell cycle. Novel therapeutic strategies directed toward these targets in neuroblastoma are discussed. PMID:26771642

  3. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

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    Mori Isamu

    2006-12-01

    Full Text Available Abstract Background Recently it has been reported that, toll-like receptors (TLRs are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. Methods Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR and flow cytometric analysis, respectively. Activation of nuclear factor (NF-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP kinase and interferon regulatory factor (IRF-3 was detected by immunoblot analysis. Results Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. Conclusion Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.

  4. Cell Survival Signaling in Neuroblastoma

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    Megison, Michael L.; Gillory, Lauren A.; Beierle, Elizabeth A.

    2013-01-01

    Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Neuroblastoma tumorigenesis and malignant transformation is driven by overexpression and dominance of cell survival pathways and a lack of normal cellular senescence or apoptosis. Therefore, manipulation of cell survival pathways may decrease the malignant potential of these tumors and provide avenues for the development of novel therapeutics. This review focuses on several facets of cell survival pathways including protein kinases (PI3K, AKT, ALK, and FAK), transcription factors (NF-κB, MYCN and p53), and growth factors (IGF, EGF, PDGF, and VEGF). Modulation of each of these factors decreases the growth or otherwise hinders the malignant potential of neuroblastoma, and many therapeutics targeting these pathways are already in the clinical trial phase of development. Continued research and discovery of effective modulators of these pathways will revolutionize the treatment of neuroblastoma. PMID:22934706

  5. Calmodulin interacts with PAC1 and VPAC2 receptors and regulates PACAP-induced FOS expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Falktoft, B.; Georg, B.; Fahrenkrug, J.

    2009-01-01

    is a well-known marker of neuronal activation, so we used a human neuroblastoma cell line NB-1 to explore the role of calmodulin in PACAP-induced FOS gene expression. We observed both short-term and prolonged altered PACAP-mediated activation of the FOS gene in the presence of the calmodulin-antagonist W-7...

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

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    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.

  7. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

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    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

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

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    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.

    2015-01-01

    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. PMID:26452036

  9. Rare MYC-amplified Neuroblastoma With Large Cell Histology.

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    Matsuno, Ryosuke; Gifford, Andrew J; Fang, Junming; Warren, Mikako; Lukeis, Robyn E; Trahair, Toby; Sugimoto, Tohru; Marachelian, Araz; Asgharzadeh, Shahab; Maris, John M; Ikegaki, Naohiko; Shimada, Hiroyuki

    2018-01-01

    Background Although MYCN (aka N-myc) amplification is reported in ∼20% of neuroblastomas, MYC (aka C-myc) amplification appears to be a rare event in this disease. As of today, only 2 MYC-amplified neuroblastomas have been briefly mentioned in the literature. Methods We studied here the clinicopathological features of 3 MYC-amplified neuroblastomas. Results All 3 patients (2 females and 1 male) had stage 4 disease. One female is currently alive and well 52 months after the diagnosis, while the other female and male patients died of disease 24 and 20 months after the diagnosis, respectively. Further analysis on 2 tumors revealed unfavorable histology with MYC protein overexpression but with neither MYCN amplification nor MYCN protein overexpression. Both of these tumors exhibited "large cell neuroblastoma" histology with enlarged, uniquely open nuclei and nucleolar hypertrophy, along with "aberrant" desmin expression. Conclusions MYC-amplified neuroblastomas are extremely rare and seem to present with distinct clinicopathological features.

  10. Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

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    Boeva, Valentina; Louis-Brennetot, Caroline; Peltier, Agathe; Durand, Simon; Pierre-Eugène, Cécile; Raynal, Virginie; Etchevers, Heather C; Thomas, Sophie; Lermine, Alban; Daudigeos-Dubus, Estelle; Geoerger, Birgit; Orth, Martin F; Grünewald, Thomas G P; Diaz, Elise; Ducos, Bertrand; Surdez, Didier; Carcaboso, Angel M; Medvedeva, Irina; Deller, Thomas; Combaret, Valérie; Lapouble, Eve; Pierron, Gaelle; Grossetête-Lalami, Sandrine; Baulande, Sylvain; Schleiermacher, Gudrun; Barillot, Emmanuel; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2017-09-01

    Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

  11. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

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    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.

  12. Transcriptional Profiling Reveals a Common Metabolic Program in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells

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    Mengling Liu

    2016-10-01

    Full Text Available High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here, we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of the TH-MYCN mouse, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma.

  13. Neuroblastoma

    International Nuclear Information System (INIS)

    Hall-Craggs, M.A.; Finn, J.P.; Dicks-Mireaux, C.; Kiely, E.M.; Pritchard, J.

    1989-01-01

    Twenty-one children with neuroblastoma (mean age, 36.7 months) were examined with high-field strength (1.5 T) MR imaging to define how accurately disease could be documented and to establish optimum sequences. Twenty-eight studies were obtained with T1- and T2-weighted spin-echo and short inversion-recovery (STIR) sequences. Thirteen children underwent surgery, 16 CT. MR imaging exactly predicted tumor extent and involvement of adjacent organs, vessels, and the spine in all patients undergoing surgery. STIR images defined tumor margins and node involvement most clearly. Following chemotherapy, MR imaging could not differentiate active tumor from maturing ganglioneuroma or residual hyperplasia. MR imaging was superior to CT in assessing intraabdominal, marrow, and spinal disease

  14. Neuroblastoma

    International Nuclear Information System (INIS)

    Thomas, P.R.M.

    1987-01-01

    Neuroblastoma is a complex disease with prognosis varying not only with stage, but also with age, site, and histology. Dissemination is the main problem. For stage I tumors, complete surgical excision alone is likely to be curative. For stage II tumors, postoperative irradiation is not clearly established, but it should be considered for patients over 1 year of age with incompletely resected tumors or positive lymph nodes. For stage III tumors, radiation therapy is indicated, with possible second-look surgery after treatment. Adjuvant chemotherapy is being tested. The prognosis is uncertain and may depend partly on the lymph node status. For stage IV tumors, chemotherapy is preferred. Remissions are usually obtained, although cure is very unlikely, except in infants. Radiation has a definite palliative role. For stage IV-S tumors, a conservative approach is preferred, with active treatment being reserved for complications of tumor bulk or obvious progression. New approaches include the use of total-body irradiation with chemotherapy, with or without transplantation, in stage IV patients in an attempt to maintain remissions. Tumor market such as N-myc help to define the indications for aggressive therapy. Once it is possible to maintain complete remissions, a dramatic improvement in the overall prognosis is likely

  15. Probenecid Sensitizes Neuroblastoma Cancer Stem Cells to Cisplatin.

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    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.

  16. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2012-07-01

    or tumor necrosis in 4/8 subjects with refractory/relapsed NB1. Although encouraging, this study also revealed that the signal from the transgenic... neonatal thymectomized mice results in the onset of systemic autoimmune diseases, such as colitis, gastritis, insulin-dependent autoimmune diabetes, and...Tregs prevents their migration to the intestine in an inflammatory bowel disease disease model, with consequent significant increase in intestinal in

  17. Neuroblastoma Treatment

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    ... adrenal glands. Signs and symptoms of neuroblastoma include bone pain and a lump in the abdomen, neck, ... options for neuroblastoma are also affected by tumor biology, which includes: The patterns of the tumor cells. ...

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

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    Emanuela Mari

    2016-11-01

    Full Text Available 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.

  19. Cell cycle control by the thyroid hormone in neuroblastoma cells

    International Nuclear Information System (INIS)

    Garcia-Silva, Susana; Perez-Juste, German; Aranda, Ana

    2002-01-01

    The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-β cells that overexpress the β1 isoform of the T3 receptor. An element in the region responsible for premature termination of transcription mediates a rapid repression of c-myc gene expression by T3. The hormone also causes a decrease of cyclin D1 gene transcription, and is able to antagonize the activation of the cyclin D1 promoter by Ras. In addition, a strong and sustained increase of the levels of the cyclin kinase inhibitor (CKI) p27 Kip1 are found in T3-treated cells. The increased levels of p27 Kip1 lead to a marked inhibition of the kinase activity of the cyclin-CDK2 complexes. As a consequence of these changes, retinoblastoma proteins are hypophosphorylated in T3-treated N2a-β cells, and progression through the restriction point in the cell cycle is blocked

  20. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

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    2015-10-01

    System (Promega). Firefly lucifer - ase activity was normalized to Renilla luciferase activity to evalu- ate the effect of the miRNAs. Biotinylated-miR...objective of Aim 1. We further validated the effect of miR-449a on the expression of molecular differentiation markers, on cell cycle distribution, and on...inducing effect in neuroblastoma cell lines regardless of the genetic backgrounds of the cell lines. We have completed the screen in the MYCN-amplified

  1. Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

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    Barbara Hämmerle

    Full Text Available Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2 neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

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

    NARCIS (Netherlands)

    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

    2015-01-01

    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

  3. Sodium valproate does not augment Prpsc in murine neuroblastoma cells.

    Science.gov (United States)

    Legendre, C; Casagrande, F; Andrieu, T; Dormont, D; Clayette, P

    2007-10-01

    Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 microM VPA. Higher VPA concentrations (240 and 600 microM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.

  4. Binimetinib inhibits MEK and is effective against neuroblastoma tumor cells with low NF1 expression.

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    Woodfield, Sarah E; Zhang, Linna; Scorsone, Kathleen A; Liu, Yin; Zage, Peter E

    2016-03-01

    Novel therapies are needed for children with high-risk and relapsed neuroblastoma. We hypothesized that MAPK/ERK kinase (MEK) inhibition with the novel MEK1/2 inhibitor binimetinib would be effective in neuroblastoma preclinical models. Levels of total and phosphorylated MEK and extracellular signal-regulated kinase (ERK) were examined in primary neuroblastoma tumor samples and in neuroblastoma cell lines by Western blot. A panel of established neuroblastoma tumor cell lines was treated with increasing concentrations of binimetinib, and their viability was determined using MTT assays. Western blot analyses were performed to examine changes in total and phosphorylated MEK and ERK and to measure apoptosis in neuroblastoma tumor cells after binimetinib treatment. NF1 protein levels in neuroblastoma cell lines were determined using Western blot assays. Gene expression of NF1 and MEK1 was examined in relationship to neuroblastoma patient outcomes. Both primary neuroblastoma tumor samples and cell lines showed detectable levels of total and phosphorylated MEK and ERK. IC50 values for cells sensitive to binimetinib ranged from 8 nM to 1.16 μM, while resistant cells did not demonstrate any significant reduction in cell viability with doses exceeding 15 μM. Sensitive cells showed higher endogenous expression of phosphorylated MEK and ERK. Gene expression of NF1, but not MEK1, correlated with patient outcomes in neuroblastoma, and NF1 protein expression also correlated with responses to binimetinib. Neuroblastoma tumor cells show a range of sensitivities to the novel MEK inhibitor binimetinib. In response to binimetinib, sensitive cells demonstrated complete loss of phosphorylated ERK, while resistant cells demonstrated either incomplete loss of ERK phosphorylation or minimal effects on MEK phosphorylation, suggesting alternative mechanisms of resistance. NF1 protein expression correlated with responses to binimetinib, supporting the use of NF1 as a biomarker to identify

  5. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

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    Yonatan Y Mahller

    Full Text Available Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

  6. Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells

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    Erica L Carpenter

    2014-07-01

    Full Text Available Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

  7. Identification of nuclear τ isoforms in human neuroblastoma cells

    International Nuclear Information System (INIS)

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

    1990-01-01

    The τ proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, τ 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 τ molecule in the isolated nuclei of neuroblastoma cells. Nuclear τ proteins, like the τ 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 τ may function in processes not directly associated with microtubules and that highly insoluble complexes of τ may also play a role in normal cellular physiology

  8. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2014-07-01

    curative treatment is available currently. Hai and colleagues found that the Hedgehog signaling pathway in salivary glands was activated during... salivary gland hypofunction by preserving salivary stem/progenitor cells and the parasympathetic innervation in mice. Similar effects on expression of genes...cytotoxic T lymphocytes without enhancement of regulatory T-cell inhibition. Clin Cancer Res. Clin Cancer Res. 2014 Jan 1;20(1):131-9. Highlited article

  9. Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma.

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    Agnes Rasmuson

    Full Text Available BACKGROUND: Prostaglandin E(2 (PGE(2 is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2 has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE(2 production, the expression pattern and localization of PGE(2 receptors and intracellular signal transduction pathways activated by PGE(2. PRINCIPAL FINDINGS: A high expression of the PGE(2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE(2 and stimulation of serum-starved neuroblastoma cells with PGE(2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE(2 (dmPGE(2 increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE(2. Similarly, PGE(2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner. CONCLUSIONS: These findings demonstrate that PGE(2 acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE(2 signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.

  10. N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma

    NARCIS (Netherlands)

    Akeson, R.; Bernards, R.A.

    1990-01-01

    In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the

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

    NARCIS (Netherlands)

    Boes, Marianne; Meyer-Wentrup, Friederike

    2015-01-01

    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

  12. In vitro assessment of curcumin against murine neuroblastoma cells.

    Science.gov (United States)

    Vanisree, Arambakkam Janardhanam; Ramanan, Ramya

    2007-04-01

    Neuroblastoma (NB) is a well-known malignant disease in infants, which comprises 10% of childhood malignancies. Despite recent advances in understanding the neuro-oncology, NB still accounts for more death in childhood than any other cancer. Research in childhood tumors should not only be focused on the malignant signatures of cancer cells but also novel drug prototypes using phytochemicals. The present study was aimed to determine the role of curcumin against murine neuroblastoma cell line (N2a). The in vitro assessment of curcumin against was made in N2a cell line in a dose-dependent manner (group I (control) and group II - IX (10 microM-80 microM). The efficacy of the drug was evaluated by estimating the levels of protein bound carbohydrates, glycoprotein, genomic DNA, total RNA levels, and inhibition of MMP-9 were studied. The gap junctional communication in the cells was also assessed. The levels of protein bound carbohydrates, DNA, RNA levels, glycoprotein were found to be altered on drug supplementation in NB cells. Inhibition of MMP-9 in curcumin-supplemented N2a cells was revealed by zymographic analysis. Assessment of Lucifer yellow dye uptake in curcumin-supplemented N2a cells showed the up-regulation of GJIC. These observations suggest that the curcumin, the active principle of curcuma longa, could be developed into an effective chemo preventive and chemotherapeutic agent. This selected concentration range needs further studies at molecular level, for conforming its role and its action against uncontrolled proliferation of NB.

  13. Time-lapse imaging of neuroblastoma cells to determine cell fate upon gene knockdown.

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    Richa Batra

    Full Text Available Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at https://ichip.bioquant.uni-heidelberg.de.

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

    Science.gov (United States)

    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.

  15. Molecular mechanism of action of opioids in human neuroblastoma cells

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    Yu, V.C.K.

    1987-01-01

    A series of human neuroblastoma cell lines was screened for the presence of opioid receptor sites. Of these cell lines, SK-N-SH was found to express approximately 50,000 ..mu.. and 10,000 delta opioid receptor sites/cell. In vitro characterization revealed that the binding properties of these receptor sites closely resembled those of human and rodent brain. Phosphatidylinositol turnover as a potential second messenger system for the ..mu.. receptor was examined in SK-N-SH cells. Neurotransmitter receptor systems were determined in the three sub-clones of SK-N-SH cells. Cells of the SH-SY5Y line, a phenotypically stable subclone of SK-N-SH cells, were induced to differentiate by treatment with various inducing agents, and changes of several neurotransmitter receptor systems were determined. Nerve growth factor (NGF) and retinoic acid (RA) up-regulated, while dBcAMP down-regulated opioid receptor sites. (/sup 3/H)Dopamine uptake was slightly enhanced only in RA-treated cells. Strikingly, the efficacy of PGE/sub 1/-stimulated accumulation of cAMP was enhanced by 15- to 30-fold upon RA treatment.

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

    Science.gov (United States)

    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.

  17. Genome-wide analysis of HOXC9-induced neuronal differentiation of neuroblastoma cells

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    Xiangwei Wang

    2014-12-01

    Full Text Available Induction of differentiation is a therapeutic strategy in neuroblastoma, a common pediatric cancer of the sympathetic nervous system. The homeobox protein HOXC9 is a key regulator of neuroblastoma differentiation. To gain a molecular understanding of the function of HOXC9 in promoting differentiation of neuroblastoma cells, we conducted a genome-wide analysis of the HOXC9-induced differentiation program by microarray gene expression profiling and chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-seq. Here we describe in detail the experimental system, methods, and quality control for the generation of the microarray and ChIP-seq data associated with our recent publication [1].

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

    Science.gov (United States)

    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.

  19. Acute application of cisplatin affects methylation status in neuroblastoma cells.

    Science.gov (United States)

    Tabata, Keiichi; Sakai, Hayato; Nakajima, Ryosuke; Saya-Nishimura, Reiko; Motani, Kou; Okano, Soichiro; Shibata, Yasuko; Abiko, Yoshimitsu; Suzuki, Takashi

    2011-06-01

    The pharmacological mechanism of the anti-cancer effect of cisplatin is well known to be DNA intercalation, but the direct or indirect effects of cisplatin on protein expression in cancer cells remain to be explained. In this study, we used a proteomic approach to clarify the early impact of cisplatin on protein expression. In a 2-dimensional gel electrophoresis proteomic experiment, the application of cisplatin for 24 h increased the expression of four proteins and decreased the levels of one protein in neuroblastoma IMR-32 cells. Levels of S-adenosyl-L-homocysteine hydrolase, a key enzyme in methylation metabolism, were increased the most. Therefore, we examined the methylation status of histone proteins. Histone H3K9 methylation was reduced by the application of cisplatin for 24 h. These results suggest that acute cisplatin treatment alters methylation status. Thus, these data can help clarify the unknown pharmacological mechanisms of cisplatin, including the anticancer effect, adverse effects and/or the mechanism of drug resistance.

  20. Classing it up to get noticed : MHC class 1 antigen display in dendritic cells and neuroblastoma

    NARCIS (Netherlands)

    Spel, Lotte

    2018-01-01

    In this thesis I have explored the process of MHC-1-mediated antigen presentation in two distinctive cell types: dendritic cells and neuroblastoma tumor cells. Dendritic cells (DCs) are pivotal players that bridge innate and adaptive immunity. DCs are able to engulf tumor-derived material and

  1. LMNA knock-down affects differentiation and progression of human neuroblastoma cells.

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    Giovanna Maresca

    Full Text Available BACKGROUND: Neuroblastoma (NB is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma. METHODOLOGY/PRINCIPAL FINDINGS: Knock-down of Lamin A/C (LMNA-KD in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.

  2. Functional dissection of HOXD cluster genes in regulation of neuroblastoma cell proliferation and differentiation.

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    Yunhong Zha

    Full Text Available Retinoic acid (RA can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13 that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1 or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13 on BE(2-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

  3. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    International Nuclear Information System (INIS)

    Yang, Qiwei; Tian, Yufeng; Ostler, Kelly R; Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Godley, Lucy A; Cohn, Susan L

    2010-01-01

    Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

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

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    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    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.

  6. Multi-omic profiling of MYCN-amplified neuroblastoma cell-lines

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    Erik Dassi

    2015-12-01

    Full Text Available Neuroblastoma is the most common pediatric cancer, arising from the neural crest cells of the sympathetic nervous system. Its most aggressive subtype, characterized by the amplification of the MYCN oncogene, has a dismal prognosis and no effective treatment is available. Understanding the alterations induced by the tumor on the various layers of gene expression is therefore important for a complete characterization of this neuroblastoma subtype and for the discovery of new therapeutic opportunities. Here we describe the profiling of 13 MYCN-amplified neuroblastoma cell lines at the genome (copy number, transcriptome, translatome and miRome levels (GEO series GSE56654, GSE56552 and GSE56655. We provide detailed experimental and data analysis procedures by means of which we derived the results described in [1].

  7. Inhibition of H3K9 Methyltransferase G9a Repressed Cell Proliferation and Induced Autophagy in Neuroblastoma Cells

    Science.gov (United States)

    Ke, Xiao-Xue; Zhang, Dunke; Zhu, Shunqin; Xia, Qingyou; Xiang, Zhonghuai; Cui, Hongjuan

    2014-01-01

    Histone methylation plays an important role in gene transcription and chromatin organization and is linked to the silencing of a number of critical tumor suppressor genes in tumorigenesis. G9a is a histone methyltransferase (HMTase) for histone H3 lysine 9. In this study, we investigated the role of G9a in neuroblastoma tumor growth together with the G9a inhibitor BIX01294. The exposure of neuroblastoma cells to BIX01294 resulted in the inhibition of cell growth and proliferation, and BIX01294 treatment resulted in the inhibition of the tumorigenicity of neuroblastoma cells in NOD/SCID mice. Therefore, G9a may be a potential therapeutic target in neuroblastoma. Moreover, we found several specific characteristics of autophagy after BIX01294 treatment, including the appearance of membranous vacuoles and microtubule-associated protein light chain 3 (LC3B). Similar results were observed in G9a-knockdown cells. In conclusion, our results demonstrated that G9a is a prognostic marker in neuroblastoma, and revealed a potential role of G9a in regulating the autophagy signaling pathway in neuroblastoma. PMID:25198515

  8. SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT.

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    Praveen Bhoopathi

    Full Text Available Secreted protein acidic and rich in cysteine (SPARC is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.

  9. 1;17 translocations and other chromosome 17 rearrangements in human primary neuroblastoma tumors and cell lines

    NARCIS (Netherlands)

    van Roy, N.; Laureys, G.; Cheng, N. C.; Willem, P.; Opdenakker, G.; Versteeg, R.; Speleman, F.

    1994-01-01

    We report on the finding of a t(1;17) in two primary neuroblastomas. Subsequent fluorescence in situ hybridization (FISH) analysis revealed the presence of 1;17 translocations in four out of nine neuroblastoma cell lines. The chromosome 1 short arm breakpoints were determined using region-specific

  10. Chemoresistance, Cancer Stem Cells, and miRNA Influences: The Case for Neuroblastoma

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    Alfred Buhagiar

    2015-01-01

    Full Text Available Neuroblastoma is a type of cancer that develops most often in infants and children under the age of five years. Neuroblastoma originates within the peripheral sympathetic ganglia, with 30% of the cases developing within the adrenal medulla, although it can also occur within other regions of the body such as nerve tissue in the spinal cord, neck, chest, abdomen, and pelvis. MicroRNAs (miRNAs regulate cellular pathways, differentiation, apoptosis, and stem cell maintenance. Such miRNAs regulate genes involved in cellular processes. Consequently, they are implicated in the regulation of a spectrum of signaling pathways within the cell. In essence, the role of miRNAs in the development of cancer is of utmost importance for the understanding of dysfunctional cellular pathways that lead to the conversion of normal cells into cancer cells. This review focuses on highlighting the recent, important implications of miRNAs within the context of neuroblastoma basic research efforts, particularly concerning miRNA influences on cancer stem cell pathology and chemoresistance pathology for this condition, together with development of translational medicine approaches for novel diagnostic tools and therapies for this neuroblastoma.

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

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    M Emmy M Dolman

    Full Text Available 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.

  12. Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells

    NARCIS (Netherlands)

    Ruigt, G.S.F.; Neyt, H.C.; Zalm, J.M. van der; Bercken, J. van der

    1987-01-01

    The effects of 4 different pyrethroid insecticides on sodium channel gating in internally perfused, cultured mouse neuroblastoma cells (N1E-115) were studied using the suction pipette, voltage clamp technique. Pyrethroids increased the amplitude of the sodium current, sometimes by more than 200%.

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

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    Shoji, Wataru [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Suenaga, Yusuke, E-mail: ysuenaga@chiba-cc.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Yokoi, Sana [Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Nio, Masaki [Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Nakagawara, Akira, E-mail: nakagawara-a@koseikan.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan)

    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.

  14. Identification of two distinct chromosome 12-derived amplification units in neuroblastoma cell line NGP

    NARCIS (Netherlands)

    van Roy, N.; Forus, A.; Myklebost, O.; Cheng, N. C.; Versteeg, R.; Speleman, F.

    1995-01-01

    The neuroblastoma cell line NGP contains two homogeneously staining regions (hsr). One of these hsrs contains MYCN sequences. Reverse painting experiments demonstrated that the second HSR consisted of two chromosome 12-derived amplification units, located at 12q14-15 and 12q24. Southern blot and

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  16. Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-01-01

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

  17. Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-06-27

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

  18. Neuroblastoma: A neurochemical approach

    International Nuclear Information System (INIS)

    Schor, N.F.

    1991-01-01

    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

  19. Combined cisplatin and aurora inhibitor treatment increase neuroblastoma cell death but surviving cells overproduce BDNF

    Directory of Open Access Journals (Sweden)

    Alessio Polacchini

    2016-07-01

    Full Text Available Drug-resistance to chemotherapics in aggressive neuroblastoma (NB is characterized by enhanced cell survival mediated by TrkB and its ligand, brain-derived neurotrophic factor (BDNF; thus reduction in BDNF levels represent a promising strategy to overcome drug-resistance, but how chemotherapics regulate BDNF is unknown. Here, cisplatin treatment in SK-N-BE neuroblastoma upregulated multiple BDNF transcripts, except exons 5 and 8 variants. Cisplatin increased BDNF mRNA and protein, and enhanced translation of a firefly reporter gene flanked by BDNF 5′UTR exons 1, 2c, 4 or 6 and 3′UTR-long. To block BDNF translation we focused on aurora kinases inhibitors which are proposed as new chemotherapeutics. NB cell survival after 24 h treatment was 43% with cisplatin, and 22% by cisplatin+aurora kinase inhibitor PHA-680632, while the aurora kinases inhibitor alone was less effective; however the combined treatment induced a paradoxical increase of BDNF in surviving cells with strong translational activation of exon6-3′UTR-long transcript, while translation of BDNF transcripts 1, 2C and 4 was suppressed. In conclusion, combined cisplatin and aurora kinase inhibitor treatment increases cell death, but induces BDNF overproduction in surviving cells through an aurora kinase-independent mechanism.

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

    Science.gov (United States)

    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.

  1. Olfactory Neuroblastoma: Diagnostic Difficulty

    Directory of Open Access Journals (Sweden)

    Vidya MN,

    2011-01-01

    Full Text Available Olfactory neuroblastoma is an uncommon malignant tumor of sinonasal tract arising from the olfactory neuro epithelium. The olfactory neuroblastomas presenting with divergent histomorphologies like, epithelial appearance of cells, lacking a neuro fibrillary background and absence of rosettes are difficult to diagnose. Such cases require immunohistochemistry to establish the diagnosis. We describe the clinical features, pathological and immunohistochemical findings of grade IV Olfactory neuroblastoma in a 57 year old man

  2. Prognostic value of partial genetic instability in Neuroblastoma with ≤ 50% neuroblastic cell content.

    OpenAIRE

    Piqueras , Marta; Navarro , Samuel; Cañete , Adela; Castel , Victoria; Noguera , Rosa

    2011-01-01

    Abstract Aims. Better understanding of neuroblastoma genetics will improve with genome-wide techniques. However it is not adequated to perform these analyses in samples with less than 60% neuroblastic cell content. We evaluated the utility of FISH on tissue microarrays (TMA) in detecting partial genetic instability (PGI), focussing on samples with ? 50% neuroblastic cells. Methods and results. Alterations of 11q and 17q were detected by FISH on 369 neuroblastic samples included...

  3. N-myc oncogene amplification is correlated to trace metal concentrations in neuroblastoma cultured cells

    International Nuclear Information System (INIS)

    Gouget, B.; Sergeant, C.; Benard, J.; Llabador, Y.; Simonoff, M.

    2000-01-01

    N-myc oncogene amplification is a powerful predictor of aggressive behavior of neuroblastoma (NB), the most common solid tumor of the early childhood. Since N-myc overexpression - subsequent to amplification - determines a phenotype of invasiveness and metastatic spreading, it is assumed that N-myc amplified neuroblasts synthesize zinc metalloenzymes leading to tumor invasion and formation of metastases. In order to test a possible relation between N-myc oncogene amplification and trace metal contents in human NB cells, Fe, Cu and Zn concentrations have been measured by nuclear microprobe analysis in three human neuroblastoma cell lines with various degrees of N-myc amplification. Elemental determinations show uniform distribution of trace metals within the cells, but variations of intracellular trace metal concentrations with respect to the degree of N-myc amplification are highly dependent on the nature of the element. Zinc concentration is higher in both N-myc amplified cell lines (IMR-32 and IGR-N-91) than in the non-amplified cells (SK-N-SH). In contrast, intracellular iron content is particularly low in N-myc amplified cell lines. Moreover, copper concentrations showed an increase with the degree of N-myc amplification. These results indicate that a relationship exists between intracellular trace metals and N-myc oncogene amplification. They further suggest that trace metals very probably play a determinant role in mechanisms of the neuroblastoma invasiveness

  4. N- myc oncogene amplification is correlated to trace metal concentrations in neuroblastoma cultured cells

    Science.gov (United States)

    Gouget, B.; Sergeant, C.; Benard, J.; Llabador, Y.; Simonoff, M.

    2000-10-01

    N- myc oncogene amplification is a powerful predictor of aggressive behavior of neuroblastoma (NB), the most common solid tumor of the early childhood. Since N- myc overexpression - subsequent to amplification - determines a phenotype of invasiveness and metastatic spreading, it is assumed that N- myc amplified neuroblasts synthesize zinc metalloenzymes leading to tumor invasion and formation of metastases. In order to test a possible relation between N- myc oncogene amplification and trace metal contents in human NB cells, Fe, Cu and Zn concentrations have been measured by nuclear microprobe analysis in three human neuroblastoma cell lines with various degrees of N- myc amplification. Elemental determinations show uniform distribution of trace metals within the cells, but variations of intracellular trace metal concentrations with respect to the degree of N- myc amplification are highly dependent on the nature of the element. Zinc concentration is higher in both N- myc amplified cell lines (IMR-32 and IGR-N-91) than in the non-amplified cells (SK-N-SH). In contrast, intracellular iron content is particularly low in N- myc amplified cell lines. Moreover, copper concentrations showed an increase with the degree of N- myc amplification. These results indicate that a relationship exists between intracellular trace metals and N- myc oncogene amplification. They further suggest that trace metals very probably play a determinant role in mechanisms of the neuroblastoma invasiveness.

  5. HIF2A and IGF2 Expression Correlates in Human Neuroblastoma Cells and Normal Immature Sympathetic Neuroblasts

    Directory of Open Access Journals (Sweden)

    Sofie Mohlin

    2013-03-01

    Full Text Available During normal sympathetic nervous system (SNS development, cells of the ganglionic lineage can malignantly transform and develop into the childhood tumor neuroblastoma. Hypoxia-inducible transcription factors (HIFs mediate cellular responses during normal development and are central in the adaptation to oxygen shortage. HIFs are also implicated in the progression of several cancer forms, and high HIF-2α expression correlates with disseminated disease and poor outcome in neuroblastoma. During normal SNS development, HIF2A is transiently expressed in neuroblasts and chromaffin cells. SNS cells can, during development, be distinguished by distinct gene expression patterns, and insulin-like growth factor 2 (IGF2 is a marker of sympathetic chromaffin cells, whereas sympathetic neuroblasts lack IGF2 expression. Despite the neuronal derivation of neuroblastomas, we show that neuroblastoma cell lines and specimens express IGF2 and that expression of HIF2A and IGF2 correlates, with the strongest correlation in high-stage tumors. In neuroblastoma, both IGF2 and HIF2A are hypoxia-driven and knocking down IGF2 at hypoxia resulted in downregulated HIF2A levels. HIF-2α and IGF2 were strongly expressed in subsets of immature neuroblastoma cells, suggesting that these two genes could be co-expressed also at early stages of SNS development. We show that IGF2 is indeed expressed in sympathetic chain ganglia at embryonic week 6.5, a developmental stage when HIF-2α is present. These findings provide a rationale for the unexpected IGF2 expression in neuroblastomas and might suggest that IGF2 and HIF2A positive neuroblastoma cells are arrested at an embryonic differentiation stage corresponding to the stage when sympathetic chain ganglia begins to coalesce.

  6. Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression.

    Science.gov (United States)

    Messi, Elio; Florian, Maria C; Caccia, Claudio; Zanisi, Mariarosa; Maggi, Roberto

    2008-01-29

    Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment. We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively. Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness

  7. Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

    Directory of Open Access Journals (Sweden)

    Zanisi Mariarosa

    2008-01-01

    Full Text Available Abstract Background Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem. Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment. Methods We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively. Results Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. Conclusion a Doublecortin is expressed in human

  8. Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

    International Nuclear Information System (INIS)

    Messi, Elio; Florian, Maria C; Caccia, Claudio; Zanisi, Mariarosa; Maggi, Roberto

    2008-01-01

    Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem. Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment. We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively. Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness; b

  9. Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

    Science.gov (United States)

    Muñoz, Jorge; Lázcoz, Paula; Inda, María Mar; Nistal, Manuel; Pestaña, Angel; Encío, Ignacio J; Castresana, Javier S

    2004-05-01

    Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing. Copyright 2004 Wiley-Liss, Inc.

  10. Reversible adaptive plasticity: A mechanism for neuroblastoma cell heterogeneity and chemo-resistance

    Directory of Open Access Journals (Sweden)

    Lina eChakrabarti

    2012-08-01

    Full Text Available We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered, anchorage dependent (AD or sphere forming, anchorage independent (AI growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin, self-renewal capacity and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2, β-catenin and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice, tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity, respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic, dynamic and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

  11. Hypoxia promotes etoposide (VP-16) resistance in neuroblastoma CHP126 cells.

    Science.gov (United States)

    Wang, Duoduo; Zhu, Qionghua; Zhang, Xiayan; Zhang, Lei; He, Qiaojun; Yang, Bo

    2010-01-01

    Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature, which is recognized to play a role in the resistance of cancer cells to chemotherapy. Etoposide (VP-16), a drug commonly used in chemotherapy, leads to enhanced accumulation of cell populations in G2/M phase and increases levels of apoptosis as a topoisomerase II inhibitor. We evaluated the effects of hypoxia on the response of the neuroblastoma cell line CHP126 to VP-16, in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance of this clinically conventional anti-cancer agent, with an insight to determining potential indications in neuroblastoma therapy. In this study, physiological hypoxia was shown to attenuate G2/M arrest and apoptosis induced in CHP126 cells by VP-16. It suppressed drug-related Cdk1 activity with a less elevation of regulator proteins such as cyclin B1, Cdk7 and reduced caspase activation and PARP cleavage compared to the efficiency observed in normoxic condition, which were significantly relative with hypoxia-driven inhibition of p53 and p-ERK1/2 activation. These results clearly demonstrated that hypoxia had a protective effect against VP-16-induced cytotoxicity, which is likely to provide a further therapeutic knowledge in neuroblastomas.

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

    Science.gov (United States)

    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.

  13. Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

    Science.gov (United States)

    Marciano-Cabral, F M; Fulford, D E

    1986-05-01

    The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.

  14. Identification of compounds that selectively target highly chemotherapy refractory neuroblastoma cancer stem cells.

    Science.gov (United States)

    Díaz-Carballo, David; Acikelli, Ali Haydar; Bardenheuer, Walter; Gustmann, Sebastian; Malak, Sascha; Stoll, Raphael; Kedziorski, Thorsten; Nazif, Mhd Ali; Jastrow, Holger; Wennemuth, Gunter; Dammann, Philip; Feigel, Martin; Strumberg, Dirk

    2014-09-01

    Relapse of cancer months or years after an apparently successful therapy is probably caused by cancer stem cells (CSCs) due to their intrinsic features like dormant periods, radiorefraction, and acquired multidrug resistance (MDR) phenotypes, among other mechanisms of cellular drug evasiveness. Thus, the lack of currently efficacious interventions remains a major problem in the treatment of malignancies, together with the inability of existing drugs to destroy specifically CSCs. Neuroblastomas per se are highly chemotherapy-refractory extracranial tumors in infants with very low survival rates. So far, no effective cytostatics against this kind of tumors are clinically available. Therefore, we have put much effort into the development of agents to efficiently combat this malignancy. For this purpose, we tested several compounds isolated from Cuban propolis on induced CSCs (iCSC) derived from LAN-1 neuroblastoma cells which expressed several characteristics of tumor-initiating cells both in in-vitro and in-vivo models. Some small molecules such as flavonoids and polycyclic polyprenylated acylphloroglucinols (PPAP) were isolated using successive RT-HPLC cycles and identified employing mass spectrometry and NMR spectroscopic techniques. Their cytotoxicity was first screened in sensitive cell systems by MTT proliferation assays and afterwards studied in less sensitive neuroblastoma iCSC models. We found several compounds with considerable anti-iCSC activity, most of them belonging to the PPAP class. The majority of the compounds act in a pleiotropic manner on the molecular biology of tumors although their specific targets remain unclear. Nevertheless, two substances, one of them a flavonoid, induced a strong disruption of tubulin polymerization. In addition, an unknown compound strongly inhibited replicative enzymes like toposimerases I/II and DNA polymerase. Here, we report for the first time cytotoxic activities of small molecules isolated from Caribbean propolis

  15. Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

    Directory of Open Access Journals (Sweden)

    L.A. Casulari

    2006-09-01

    Full Text Available Glucocorticoids (Gc influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1 the presence and the binding characteristics of Gc receptors in these cells, 2 the effect of dexamethasone (Dex on the migration of SK-N-SH cells, and 3 the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1 receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell are present in cytosolic preparations of SK-N-SH cells, and 2 treatment with Dex (in the range of 10 nM to 1 µM has an inhibitory effect (from 100% to 74 and 43%, respectively on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM, and 3 as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s of action of Gc on cells originating in the neural crest.

  16. The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Sennvik, Kristina; Nilsberth, Camilla; Stenh, Charlotte

    2002-01-01

    The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT...... their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease....

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Young-Sang; Son, Hyeong-Guk; Kim, Dae-Hoon; Oh, Hong-Gi; Lee, Da-Som; Kim, Min-Hye; Lim, Ki-Moo; Song, Kwang-Soup, E-mail: kssong10@kumoh.ac.kr

    2016-01-15

    Graphical abstract: - Highlights: • The nanocrystalline diamond (NCD) surface is functionalized with F or O. • The cell adhesion and growth are evaluated on the functionalized NCD surface. • The cell adhesion and growth depend on the wettability of the surface. • Cell patterning was achieved by using of hydrophilic and hydrophobic surfaces. • Neuroblastoma cells were arrayed on the micro-patterned NCD surface. - Abstract: Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O{sub 2} or C{sub 3}F{sub 8} 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.

  18. Etoposide-induced apoptosis in murine neuroblastoma (N2A cells infected with Paramyxoviruses Apoptose induzida por etoposídeo em células de neuroblastoma murino (N2A infectadas por paramixovírus

    Directory of Open Access Journals (Sweden)

    L. Moro

    2003-02-01

    Full Text Available The present study aimed to determine whether measles virus can induce apoptosis in murine neuroblastoma cells and the behavior of these cells under acute infection with measles virus or persistent infection with canine distemper virus upon treatment with etoposide. Measles virus induced necrosis in murine neuroblastoma cells. Canine distemper virus-persistent infection did not alter murine neuroblastoma cells behavior when treated with etoposide.O presente trabalho foi realizado tendo como objetivo determinar se o vírus de sarampo induz apoptose em células de neuroblastoma murino e avaliar o comportamento de células de neuroblastoma murino agudamente infectadas com vírus do sarampo ou persistentemente infectadas com o vírus da cinomose canina quando tratadas com etoposídeo. A infecção pelo vírus de sarampo induziu principalmente necrose em células de neuroblastoma murino. A infecção persistente pelo vírus de cinomose canina não alterou o comportamento de células de neuroblastoma murino tratadas com etoposídeo.

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

    Science.gov (United States)

    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.

  20. Regulation of MYCN expression in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Jacobs, Joannes FM; Bokhoven, Hans van; Leeuwen, Frank N van; Hulsbergen-van de Kaa, Christina A; Vries, I Jolanda M de; Adema, Gosse J; Hoogerbrugge, Peter M; Brouwer, Arjan PM de

    2009-01-01

    Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (ΔMYCN) that was recently identified in fetal tissues. Both MYCNOS and ΔMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level. Expression of MYCN, MYCNOS and ΔMYCN was measured in human NB tissues of different stages. Transcript levels were quantified using a real-time reverse transcriptase polymerase chain reaction assay (QPCR). In addition, relative expression of these three transcripts was compared to the number of MYCN copies, which was determined by genomic real-time PCR (gQPCR). Both ΔMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN-amplification, these transcripts are significantly higher expressed. The ratio of MYCN:ΔMYCN expression was identical in all tested NBs. This indicates that ΔMYCN and MYCN are co-regulated, which suggests that ΔMYCN is not a regulator of MYCN in NB. However, the ratio of MYCNOS:MYCN expression is directly correlated with NB disease stage (p = 0.007). In the more advanced NB stages and NBs with MYCN-amplification, relatively more MYCNOS is present as compared to MYCN. Expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level. The MYCNOS:MYCN-ratio in NBs is significantly correlated with both MYCN-amplification and NB-stage. Our data indicate that in NB, MYCN expression levels might be influenced by MYCNOS but not by ΔMYCN

  1. Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

    OpenAIRE

    Marciano-Cabral, F M; Fulford, D E

    1986-01-01

    The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures o...

  2. Enhancing the GLP-1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells

    OpenAIRE

    Li, Yazhou; Tweedie, David; Mattson, Mark P.; Holloway, Harold W.; Greig, Nigel H.

    2010-01-01

    Increasing evidence suggests that glucagon-like peptide-1 (GLP-1), an incretin hormone of current interest in type 2 diabetes, is neuroprotective in both cell culture and animal models. To characterize the neuroprotective properties of GLP-1 and associated underlying mechanisms, we over-expressed the GLP-1 receptor (R) on human neuroblastoma SH-SY5Y cells to generate a neuronal culture system featuring enhanced GLP-1R signaling. In GLP-1R over-expressing SH-SY5Y (SH-hGLP-1R#9) cells, GLP-1 an...

  3. MIBG causes oxidative stress and up-regulation of anti-oxidant enzymes in the human neuroblastoma cell line SK-N-BE(2c)

    NARCIS (Netherlands)

    Cornelissen, J.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

    1997-01-01

    We report the effects of meta-iodobenzylguanidine (MIBG), a neuroblastoma-seeking agent, on cell proliferation and several oxidative stress-related parameters in the human neuroblastoma cell line SK-N-BE(2c). MIBG inhibited the proliferation of this cell line in micromolar concentrations.

  4. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Xie Li

    2009-02-01

    Full Text Available Abstract Background Astrocyte elevated gene-1 (AEG-1 was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

  5. Persistent positive metaiodobenzylguanidine scans after autologous peripheral blood stem cell transplantation may indicate maturation of stage 4 neuroblastoma.

    Science.gov (United States)

    Okamoto, Yasuhiro; Kodama, Yuichi; Nishikawa, Takuro; Rindiarti, Almitra; Tanabe, Takayuki; Nakagawa, Shunsuke; Yoshioka, Takako; Takumi, Koji; Kaji, Tatsuru; Kawano, Yoshifumi

    2017-04-01

    Metaiodobenzylguanidine (MIBG) scans are sensitive testing tools for neuroblastoma. Persistent positive MIBG scans in patients with stage 3 neuroblastoma have previously been found to indicate maturation rather than regression. We assessed the significance of this finding in stage 4 neuroblastoma in the present study. Fifteen consecutive pediatric patients with stage 4 neuroblastoma treated between 2004 and 2014 at the Kagoshima University Hospital were retrospectively examined. Treatment involved a combination of multiagent chemotherapy, resection, autologous peripheral blood stem cell transplantation (PBSCT), radiotherapy, and maintenance therapy with retinoic acid. The MIBG uptake in each patient during treatment was assessed using a Curie score. The 5-year event-free and overall survival rates in 15 patients were 38.9% and 58.7%, respectively. Four patients with persistent positive MIBG scans who underwent autologous PBSCT but experienced decreased 123 I-MIBG uptake during the clinical course survived without progression, and their event-free survival (EFS) was significantly superior to that of patients who showed negative MIBG scans after PBSCT (5-year EFS rate: 18.2%, p = 0.0176). Therefore, persistent positive MIBG scans with gradually decreased uptake after PBSCT do not always indicate neuroblastoma progression, and may instead indicate tumor maturation in some selected cases, if not all cases, of stage 4 neuroblastoma.

  6. Role of trace metals in cell proliferation in the human neuroblastoma: relations with the oncogene N-myc

    International Nuclear Information System (INIS)

    Moretto, Ph.; Michelet, C.; Gouget, B.; Ortega, R.; Sergiant, C.; Llabador, Y.; Simonoff, M.; Benard, J.

    1997-01-01

    Neuroblastoma is one of the most common tumors in young children. Iron is known to be necessary for cellular proliferation. Several studies have suggested that neuroblastoma cells appear to be relatively sensitive to growth inhibition by specific Fe chelators, in vitro. In addition, it appeared that an increased serum ferritin level at diagnosis was associated with a poorer outcome than a normal level. On the other hand it was reported that untreated primary neuroblastoma had multiple copies of the N-myc oncogene. A significant association between genomic amplification and rapid tumor progression after diagnosis has been demonstrated. In order to study the relationship between iron N-myc amplification, we propose to determine the trace metal content of neuroblastoma cells. Preliminary results obtained with two distinct cell lines: SK-N-SH, a neuroblastoma cell line with a single copy of N-myc and IGR-N-91, a metastatic cell line exhibiting 60 copies of N-myc are presented. (authors)

  7. Suppression of Cpn10 increases mitochondrial fission and dysfunction in neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    So Jung Park

    Full Text Available To date, several regulatory proteins involved in mitochondrial dynamics have been identified. However, the precise mechanism coordinating these complex processes remains unclear. Mitochondrial chaperones regulate mitochondrial function and structure. Chaperonin 10 (Cpn10 interacts with heat shock protein 60 (HSP60 and functions as a co-chaperone. In this study, we found that down-regulation of Cpn10 highly promoted mitochondrial fragmentation in SK-N-MC and SH-SY5Y neuroblastoma cells. Both genetic and chemical inhibition of Drp1 suppressed the mitochondrial fragmentation induced by Cpn10 reduction. Reactive oxygen species (ROS generation in 3-NP-treated cells was markedly enhanced by Cpn10 knock down. Depletion of Cpn10 synergistically increased cell death in response to 3-NP treatment. Furthermore, inhibition of Drp1 recovered Cpn10-mediated mitochondrial dysfunction in 3-NP-treated cells. Moreover, an ROS scavenger suppressed cell death mediated by Cpn10 knockdown in 3-NP-treated cells. Taken together, these results showed that down-regulation of Cpn10 increased mitochondrial fragmentation and potentiated 3-NP-mediated mitochondrial dysfunction in neuroblastoma cells.

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

    Science.gov (United States)

    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.

  9. Β-carotene inhibits neuroblastoma tumorigenesis by regulating cell differentiation and cancer cell stemness.

    Science.gov (United States)

    Lim, Ji Ye; Kim, Yoo-Sun; Kim, Kyung-Mi; Min, Soo Jin; Kim, Yuri

    2014-08-08

    Neuroblastoma (NB) is the most common extracranial solid cancer in young children and malignant NB cells have been shown to possess cancer stem cell (CSC) characteristics. Thus, the successful elimination of CSCs represents a strategy for developing an effective preventive and chemotherapeutic agent. CSCs are characterized by differentiation and tumorigenicity. β-Carotene (BC) has been associated with many anticancer mechanisms, although the efficacy of BC on CSCs remains unclear. In the present study, the effects of BC on tumor cell differentiation and tumorigenicity was investigated using a xenograft model. Mice were pretreated with BC for 21 days, then received a subcutaneous injection of SK-N-BE(2)C cells. Both tumor incidence and tumor growth were significantly inhibited for mice that received BC supplementation compared to the control group. Treatment with BC has also been shown to induce tumor cell differentiation by up-regulating differentiation markers, such as vimentin, peripherin, and neurofilament. Conversely, BC treatment has been shown to significantly suppress tumor stemness by down-regulating CSC markers such as Oct 3/4 and DLK1. BC treatment also significantly down-regulated HIF1-α expression and its downstream target, vascular endothelial growth factor (VEGF). Taken together, these results suggest that BC is a potential chemotherapeutic reagent for the treatment of NB, and mediates this effect by regulating the differentiation and stemness of CSCs, respectively. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y

    DEFF Research Database (Denmark)

    Plant, Leigh D; Boyle, John P; Thomas, Natasha M

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...

  11. Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Babich, J.W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States); Graham, W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States); Fischman, A.J. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States)

    1997-05-01

    The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [{sup 125}I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were with varying concentrations of {alpha}-agonist (clonidine, methoxamine, and xylazine), {alpha}-antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), {beta}-antagonist (propranolol, atenolol), {beta}-agonist (isoprenaline and salbutamol), mixed {alpha}/{beta} antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [{sup 125}I]MIBG (0.1 {mu}M). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [{sup 125}I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [{sup 125}I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. In conclusion, the results of this study establish that selected adrenergic ligands can significantly influence the pattern of uptake and storage of MIBG in cultured neuroblastoma cells, most likely through inhibition of uptake or through noncompetitive inhibition. The potential inplications of these findings justify further study. (orig./VHE). With 4 figs., 1 tab.

  12. Polyamine Metabolism Is Sensitive to Glycolysis Inhibition in Human Neuroblastoma Cells*

    Science.gov (United States)

    Ruiz-Pérez, M. Victoria; Medina, Miguel Ángel; Urdiales, José Luis; Keinänen, Tuomo A.; Sánchez-Jiménez, Francisca

    2015-01-01

    Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors. PMID:25593318

  13. Polyamine metabolism is sensitive to glycolysis inhibition in human neuroblastoma cells.

    Science.gov (United States)

    Ruiz-Pérez, M Victoria; Medina, Miguel Ángel; Urdiales, José Luis; Keinänen, Tuomo A; Sánchez-Jiménez, Francisca

    2015-03-06

    Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Involvement of triacylglycerol in the metabolism of fatty acids by cultured neuroblastoma and glioma cells

    International Nuclear Information System (INIS)

    Cook, H.W.; Clarke, J.T.; Spence, M.W.

    1982-01-01

    The metabolism (chain elongation, desaturation, and incorporation into complex lipids) of thirteen different radiolabeled fatty acids and acetate was examined in N1E-115 neuroblastoma and C-6 glioma cell lines in culture. During 6-hr incubations, all fatty acids were extensively (14-80%) esterified to complex lipids, mainly choline phosphoglycerides and triacylglycerol. With trienoic and tetraenoic substrates, inositol and ethanolamine phosphoglycerides also contained up to 30% of the labeled fatty acids; plasmalogen contained up to half of the label in the ethanolamine phosphoglyceride fraction of neuroblastoma cells. Chain elongation and delta 9, delta 6, and delta 5 desaturation occurred in both cell lines; delta 4 desaturation was not observed. Seemingly anomalous utilization of arachidic acid and some selectivity based on the geometric configuration of double bonds was observed. These studies indicate that these cell lines are capable of modulating cellular membrane composition by a combination of selective exclusion and removal of inappropriate acyl chains and of modification of other acyl chains by desaturation and chain elongation. The time courses and patterns of modification and incorporation of exogenous substrates into phospholipids and triacylglycerol suggest that exogenous unsaturated fatty acid may be incorporated into triacylglycerol and later released for further metabolism and incorporation into phospholipids. This supports a role for triacylglycerol in the synthesis of membrane complex lipids in cell lines derived from neural tissue

  15. A novel synthetic drug, LB-18, closely related to lembehyne-A derived from a marine sponge, induces caspase-independent cell death to human neuroblastoma cells.

    Science.gov (United States)

    Izumi, Moriatsu; Yogosawa, Shingo; Aoki, Shunji; Watanabe, Hirotsuna; Kamiyama, Jun; Takahara, Yoshinori; Sowa, Yoshihiro; Kobayashi, Motomasa; Hosoi, Hajime; Sugimoto, Tohru; Sakai, Toshiyuki

    2006-07-01

    Neuroblastoma is a common solid tumor of children that arises from the sympathetic nervous system. Much work has consequently focused on the possibility of inducing marked cell death in neuroblastoma, and the new effective drugs are required. We have newly synthesized LB-18, closely related to lembehyne A (LB-A), a polyacetylene derived from a kind of marine sponge. LB-A has been shown to induce p21/WAF1 and causes G1 phase arrest in mouse neuroblastoma Neuro2A cells; however, we show here that LB-18 causes cell death in human neuroblastoma KP-N-TK cells in a dose-dependent manner. TUNEL assay and flow cytometric analysis showed that the cell death caused by LB-18 was associated with the DNA damage but the pan-caspase inhibitor, zVAD-fmk, could not prevent the cell death. Western blot analysis and cleavage of the caspase-3 or -7 substrate assay showed that LB-18 could not activate caspases 3, 7, 8 and 9. These results suggest that LB-18 causes caspase-independent cell death in human neuroblastoma cells. In the future, LB-18 may be useful for cancer therapeutics, especially for neuroblastoma.

  16. Neuroblastoma cells undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression.

    Science.gov (United States)

    Rifatbegovic, Fikret; Frech, Christian; Abbasi, M Reza; Taschner-Mandl, Sabine; Weiss, Tamara; Schmidt, Wolfgang M; Schmidt, Iris; Ladenstein, Ruth; Ambros, Inge M; Ambros, Peter F

    2018-01-15

    Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive. The present RNA-Seq study of stage 4/M primary tumors, enriched BM-derived diagnostic and relapse DTCs, as well as the corresponding BM-derived mononuclear cells (MNCs) from 53 patients revealed 322 differentially expressed genes in DTCs as compared to the tumors (q 2). Particularly, the levels of transcripts encoded by mitochondrial DNA were elevated in DTCs, whereas, for example, genes involved in angiogenesis were downregulated. Furthermore, 224 genes were highly expressed in DTCs and only slightly, if at all, in MNCs (q  6). Interestingly, we found the transcriptome of relapse DTCs largely resembling those of diagnostic DTCs with only 113 differentially expressed genes under relaxed cut-offs (q 0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4 and GLTSCR2. This first RNA-Seq analysis of neuroblastoma DTCs revealed their unique expression profile in comparison to the tumors and MNCs, and less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected downregulation of genes encoded by chromosome 19. As these alterations might be associated with treatment failure and disease relapse, further functional studies on DTCs should be considered. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  17. Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells.

    Science.gov (United States)

    Lehti, Timo A; Pajunen, Maria I; Skog, Maria S; Finne, Jukka

    2017-12-04

    Eukaryotic organisms are continuously exposed to bacteriophages, which are efficient gene transfer agents in bacteria. However, bacteriophages are considered not to pass the eukaryotic cell membrane and enter nonphagocytic cells. Here we report the binding and penetration of Escherichia coli PK1A2 bacteriophage into live eukaryotic neuroblastoma cells in vitro. The phage interacts with cell surface polysialic acid, which shares structural similarity with the bacterial phage receptor. Using fluorescence and electron microscopy, we show that phages are internalized via the endolysosomal route and persist inside the human cells up to one day without affecting cell viability. Phage capsid integrity is lost in lysosomes, and the phage DNA is eventually degraded. We did not detect the entry of phage DNA into the nucleus; however, we speculate that this might occur as a rare event, and propose that this potential mechanism could explain prokaryote-eukaryote gene flow.

  18. Renal cell carcinoma in long-term survivors of advanced stage neuroblastoma in early childhood

    International Nuclear Information System (INIS)

    Fleitz, Julie M.; Wootton-Gorges, Sandra L.; Kurzrock, Eric A.; Wyatt-Ashmead, Josephine; McGavran, Loris; Koyle, Martin; Odom, Lorrie F.; West, Daniel C.; Martin, Kenneth W.

    2003-01-01

    Renal cell carcinoma (RCC) is rare in children and comprises only 1-3% of all pediatric primary renal tumors. Recently, several case reports have described RCC developing in patients previously treated for advanced stage neuroblastoma (NB). Our experience with four patients treated for advanced stage NB during early childhood who developed RCC later in life are added to 14 others in the literature. These patients and our review of the literature suggest an association between RCC and NB that warrants further study. (orig.)

  19. Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, L.; Liang, H. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China); Cao, W. [Department of Obstetrics, Qingdao Central Hospital, Qingdao (China); Xu, R.; Ju, X.L. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China)

    2014-05-23

    Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

  20. Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastoma tumor cells

    NARCIS (Netherlands)

    Molenaar, J. J.; Ebus, M. E.; Koster, J.; Santo, E.; Geerts, D.; Versteeg, R.; Caron, H. N.

    2010-01-01

    Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1

  1. Panobinostat synergistically enhances the cytotoxic effects of cisplatin, doxorubicin or etoposide on high-risk neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Guan Wang

    Full Text Available High-risk neuroblastoma remains a therapeutic challenge with a long-term survival rate of less than 40%. Therefore, new agents are urgently needed to overcome chemotherapy resistance so as to improve the treatment outcome of this deadly disease. Histone deacetylase (HDAC inhibitors (HDACIs represent a novel class of anticancer drugs. Recent studies demonstrated that HDACIs can down-regulate the CHK1 pathway by which cancer cells can develop resistance to conventional chemotherapy drugs. This prompted our hypothesis that combining HDACIs with DNA damaging chemotherapeutic drugs for treating neuroblastoma would result in enhanced anti-tumor activities of these drugs. Treatment of high-risk neuroblastoma cell lines with a novel pan-HDACI, panobinostat (LBH589, resulted in dose-dependent growth arrest and apoptosis in 4 high-risk neuroblastoma cell lines. Further, the combination of panobinostat with cisplatin, doxorubicin, or etoposide resulted in highly synergistic antitumor interactions in the high-risk neuroblastoma cell lines, independent of the sequence of drug administration. This was accompanied by cooperative induction of apoptosis. Furthermore, panobinostat treatment resulted in substantial down-regulation of CHK1 and its downstream pathway and abrogation of the G2 cell cycle checkpoint. Synergistic antitumor interactions were also observed when the DNA damaging agents were combined with a CHK1-specific inhibitor, LY2603618. Contrary to panobinostat treatment, LY2603618 treatments neither resulted in abrogation of the G2 cell cycle checkpoint nor enhanced cisplatin, doxorubicin, or etoposide-induced apoptosis in the high-risk neuroblastoma cells. Surprisingly, LY2603618 treatments caused substantial down-regulation of total CDK1. Despite this discrepancy between panobinostat and LY2603618, our results indicate that suppression of the CHK1 pathway by panobinostat is at least partially responsible for the synergistic antitumor interactions

  2. Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

    Science.gov (United States)

    2012-01-01

    Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

  3. Herpes simplex virus type 2 infection induces AD-like neurodegeneration markers in human neuroblastoma cells.

    Science.gov (United States)

    Kristen, Henrike; Santana, Soraya; Sastre, Isabel; Recuero, Maria; Bullido, Maria J; Aldudo, Jesus

    2015-10-01

    Herpes simplex virus (HSV) types 1 and 2 are neurotropic viruses that establish lifelong latent infections in neurons. Mounting evidence suggests that HSV-1 infection is involved in the pathogenesis of Alzheimer's disease (AD). The relationships between other herpesvirus infections and events associated with neurodegeneration have not, however, been extensively studied. The present work reports that HSV-2 infection leads to the strong accumulation of hyperphosphorylated tau and the amyloid-β peptides Aβ40 and Aβ42 (all major pathological hallmarks of AD) in human SK-N-MC neuroblastoma cells. Infection is also associated with a marked reduction in the amount of Aβ40 secreted and in the proteolytic fragments of the amyloid-β precursor protein (APP) (secreted APPα and the α-C-terminal fragment). These results indicate that HSV-2 infection inhibits the nonamyloidogenic pathway of APP processing and impairs Aβ secretion in these cells. In addition, HSV-2 induces the accumulation of intracellular autophagic compartments containing Aβ due to a failure in the late stages of autophagy. To our knowledge, this is the first report to show that HSV-2 infection strongly alters the tau phosphorylation state, APP processing, and autophagic process in human neuroblastoma cells, leading to the appearance of AD-like neurodegeneration markers. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Neuroblastoma and MYCN

    Science.gov (United States)

    Huang, Miller; Weiss, William A.

    2013-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells. Amplification of the MYC family member, MYCN, is found in ∼25% of cases and correlates with high-risk disease and poor prognosis. Currently, amplification of MYCN remains the best-characterized genetic marker of risk in neuroblastoma. This article reviews roles for MYCN in neuroblastoma and highlights recent identification of other driver mutations. Strategies to target MYCN at the level of protein stability and transcription are also reviewed. PMID:24086065

  5. The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

    Science.gov (United States)

    Lu, Jiaxiong; Guan, Shan; Zhao, Yanling; Yu, Yang; Woodfield, Sarah E; Zhang, Huiyuan; Yang, Kristine L; Bieerkehazhi, Shayahati; Qi, Lin; Li, Xiaonan; Gu, Jerry; Xu, Xin; Jin, Jingling; Muscal, Jodi A; Yang, Tianshu; Xu, Guo-Tong; Yang, Jianhua

    2017-08-01

    Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira

    2007-01-01

    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 (p53ΔC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53ΔC 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

  7. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in humanALKmutant neuroblastoma cell lines and xenograft models.

    Science.gov (United States)

    Wang, Hui Qin; Halilovic, Ensar; Li, Xiaoyan; Liang, Jinsheng; Cao, Yichen; Rakiec, Daniel P; Ruddy, David A; Jeay, Sebastien; Wuerthner, Jens U; Timple, Noelito; Kasibhatla, Shailaja; Li, Nanxin; Williams, Juliet A; Sellers, William R; Huang, Alan; Li, Fang

    2017-04-20

    The efficacy of ALK inhibitors in patients with ALK -mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Molecular galactose-galectin association in neuroblastoma cells: An unconventional tool for qualitative/quantitative screening.

    Science.gov (United States)

    Pastorino, Fabio; Ponzoni, Mirco; Simone, Giuseppina

    2017-05-01

    Galectin decorates the cell membrane and forms an extracellular molecular association with galactoside units. Here, galactoside probes have been used to study galectin expression in neuroblastoma cells. The hypothesis behind this investigation has been that the molecular mechanisms by which glycans modulate neural metastatic cells involve a protein-carbohydrate association, galectin-galactose. Preliminary screening to validate the hypothesis has been performed with galactose moieties anchored to beads. The molecular association has been studied by FACS. In vitro experiments reveal the molecular binding preferences of the metastatic neuroblastoma cells. Ex vivo, the galactose probes discriminate healthy tissues. The unconventional assay in microfluidics used in this study displayed results analogous to the above (GI-LI-N cell capture efficiency overcomes IMR-32). At the point of equilibrium of shear and binding forces, the capture yield inside the chamber was measured to 60 ± 4.4% in GI-LI-N versus 40 ± 2.1% in IMR-32. Staining of the fished cells and subsequent conjugation with red beads bearing the galactose also have evidenced that microfluidics can be used to study and quantify the molecular association of galectin-galactose. Most importantly, a crucial insight for obtaining single-cell qualitative/quantitative glycome analysis has been achieved. Finally, the specificity of the assay performed in microfluidics is demonstrated by comparing GI-LI-N fishing efficiency in galactose and fucose environments. The residual adhesion to fucose confirmed the existence of receptors for this glycan and that its eventual unspecific binding (i.e. due to electrostatic interactions) is insignificant compared with the molecular binding. Identification and understanding of this mechanism of discrimination can be relevant for diagnostic monitoring and for producing probes tailored to interfere with galectin activities associated with the malignant phenotype. Besides, the given

  10. Dichloroacetate stimulates changes in the mitochondrial network morphology via partial mitophagy in human SH-SY5Y neuroblastoma cells

    Czech Academy of Sciences Publication Activity Database

    Pajuelo-Reguera, David; Alán, Lukáš; Olejár, Tomáš; Ježek, Petr

    2015-01-01

    Roč. 46, č. 6 (2015), s. 2409-2418 ISSN 1019-6439 R&D Projects: GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : dichloroacetate * mitochondria * mitophagy * neuroblastoma SH-SY5Y cells * mitochondrial network Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.018, year: 2015

  11. Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers

    Czech Academy of Sciences Publication Activity Database

    Procházka, Pavel; Hraběta, J.; Vícha, A.; Cipro, S.; Stejskalová, E.; Musil, Z.; Vodička, Pavel; Eckschlager, T.

    2013-01-01

    Roč. 29, č. 6 (2013), s. 2415-2421 ISSN 1021-335X Grant - others:GA ČR(CZ) GAP301/10/0356 Institutional support: RVO:68378041 Keywords : high-risk neuroblastoma cell line * multiplex ligation-dependent probe amplification * fluorescent in situ hybridization Subject RIV: CE - Biochemistry Impact factor: 2.191, year: 2013

  12. Effect of ascorbic acid and hydrogen peroxide on mouse neuroblastoma cells

    Science.gov (United States)

    HARDAWAY, CHRISITNA M.; BADISA, RAMESH B.; SOLIMAN, KARAM F.A.

    2012-01-01

    Ascorbic acid is one of the antioxidant compounds widely used against free radical stress. The present study was undertaken to examine whether ascorbic acid and hydrogen peroxide (H2O2), alone or in combination, could influence cell viability. The murine neuroblastoma cell line, N2a, was used to perform a dose response curve for ascorbic acid. It was observed that ascorbic acid alone at physiological concentrations (0.1–0.4 mM) did not cause any cell death. However, at pharmacological concentrations (1–6 mM), ascorbic acid caused dose-dependent cell death. The lethal concentration at which 50% cells were killed (LC50) was determined to be approximately 3.141 mM ascorbic acid at 24 h. H2O2 up to 300 μM alone did not cause significant cell death. In the combined treatment, when the cells were treated with ascorbic acid at physiological concentrations (0.4 mM) and H2O2 at 400 μM, higher rates of cell death were observed compared to the cell death rates caused by either compound alone. Subsequent experiments revealed that cell death was partly mediated through the loss of total glutathione levels in the cells. These data suggest that the combination of ascorbic acid and H2O2 is disadvantageous for cancer cell survival. Further studies are required to ascertain the physiological significance of these observations. PMID:22469841

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

    Science.gov (United States)

    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.

  14. Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation

    DEFF Research Database (Denmark)

    Emdal, Kristina B; Pedersen, Anna-Kathrine; Bekker-Jensen, Dorte B

    2015-01-01

    SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells...... then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines...

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

    Science.gov (United States)

    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. Copyright © 2013 Wiley Periodicals, Inc.

  16. CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation.

    Science.gov (United States)

    Li, Xuan; Wu, Xiao-Qi; Deng, Rong; Li, Dan-Dan; Tang, Jun; Chen, Wen-Dan; Chen, Jing-Hong; Ji, Jiao; Jiao, Lin; Jiang, Shan; Yang, Fen; Feng, Gong-Kan; Senthilkumar, Ravichandran; Yue, Fei; Zhang, Hai-Liang; Wu, Rui-Yan; Yu, Yan; Xu, Xue-Lian; Mai, Jia; Li, Zhi-Ling; Peng, Xiao-Dan; Huang, Yun; Huang, Xiang; Ma, Ning-Fang; Tao, Qian; Zeng, Yi-Xin; Zhu, Xiao-Feng

    2017-10-27

    Autophagy is a degradative pathway that delivers cellular components to the lysosome for degradation. The role of autophagy in cell differentiation is poorly understood. Here we show that CaMKII can directly phosphorylate Beclin 1 at Ser90 to promote K63-linked ubiquitination of Beclin 1 and activation of autophagy. Meanwhile, CaMKII can also promote K63-linked ubiquitination of inhibitor of differentiation 1/2 (Id-1/2) by catalyzing phosphorylation of Id proteins and recruiting TRAF-6. Ubiquitinated Id-1/Id-2 can then bind to p62 and be transported to autolysosomes for degradation. Id degradation promotes the differentiation of neuroblastoma cells and reduces the proportion of stem-like cells. Our study proposes a mechanism by which autophagic degradation of Id proteins can regulate cell differentiation. This suggests that targeting of CaMKII and the regulation of autophagic degradation of Id may be an effective therapeutic strategy to induce cell differentiation in neuroblastoma.

  17. Anti-cancer effect of oncolytic adenovirus-armed shRNA targeting MYCN gene on doxorubicin-resistant neuroblastoma cells.

    Science.gov (United States)

    Li, Yuan; Zhuo, Baobiao; Yin, Yiyu; Han, Tao; Li, Shixian; Li, Zhengwei; Wang, Jian

    2017-09-09

    Chemotherapy is one of the few effective choices for patients with neuroblastoma. However, the development of muti-drug resistance (MDR) to chemotherapy is a major obstacle to the effective treatment of advanced or recurrent neuroblastoma. The muti-drug resistance-associated protein (MRP), which encodes a transmembrane glycoprotein, is a key regulator of MDR. The expression of MRP is a close correlation with MYCN oncogene in neuroblastoma. We have recently shown ZD55-shMYCN (oncolytic virus armed with shRNA against MYCN) can down-regulate MYCN to inhibit tumor cells proliferation and induce apoptosis in neuroblastoma. Here we further report ZD55-shMYCN re-sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and inhibited cell migration), and reduced the in vivo growth rate of neuroblastoma xenografts by down-regulation of MRP expression. Sequential therapy with doxorubicin did not affect the replication of ZD55-shMYCN in doxorubicin-resistant neuroblastoma cells, but decreased the expression of Bcl-2, Bcl-X L , MMP-1. Thus, this synergistic effect of ZD55-shMYCN in combination with doxorubicin provides a novel therapy strategy for doxorubicin-resistant neuroblastoma, and is a promising approach for further clinical development. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Nardou Katya

    2008-06-01

    Full Text Available Abstract Background Histone deacetylase inhibitors (HDACi are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. Results We show here that the HDACi Sodium Butyrate (NaB, suberoylanilide hydroxamic acid (SAHA and Trichostatin A (TSA strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. Conclusion HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

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

    International Nuclear Information System (INIS)

    Jung, Tae Woo; Lee, Ji Young; Shim, Wan Sub; Kang, Eun Seok; Kim, Soo Kyung; Ahn, Chul Woo; Lee, Hyun Chul; Cha, Bong Soo

    2006-01-01

    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-γ 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

  20. Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells.

    Science.gov (United States)

    Huang, L Y; Moran, N; Ehrenstein, G

    1982-01-01

    We have studied the effects of batrachotoxin (BTX) on sodium channels in hybrid mouse neuroblastoma cells NG108-15 by using the suction pipet voltage clamp method. BTX-modified sodium channels activate with first-order kinetics and, over most of the potential range, activate more slowly than normal sodium channels. The peak conductance-voltage curve and the time constant of activation-versus-voltage curve for BTX-modified sodium channels are shifted about 50 mV in the hyperpolarizing direction compared to the corresponding curves for normal sodium channels. There is no change in the slope of the conductance-voltage curve. These results suggest that BTX slows down one of the steps leading to channel opening, which consequently becomes rate-limiting. In addition, BTX eliminates both fast and slow inactivation. PMID:6281796

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

    Science.gov (United States)

    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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells

    Science.gov (United States)

    2013-01-01

    Background In order to gain insight into neuroprotective effects of ECa 233, a standardized extract of Centella asiatica, previously demonstrated in animal models of memory impairment induced by transient global ischemia or intracerebroventricular injection of β-amyloid, the effect of ECa 233 on neurite outgrowth of human IMR-32 neuroblastoma cell line was investigated. Methods Cells were seeded and incubated with various concentrations of ECa 233. Morphometric analysis was carried out by a measurement of the longest neurite growth of cells at 24 and 48 h. Contributing signaling pathways possibly involved were subsequently elucidated by western blot analysis. Results While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1–100 μg/ml. Western blot analysis revealed that ECa 233 significantly upregulated the level of activated ERK1/2 and Akt of the treated cells suggesting their involvement in the neuritogenic effect observed, which was subsequently verified by the finding that an addition of their respective inhibitors could reverse the effect of ECa 233 on these cells. Conclusions The present study clearly demonstrated neurite outgrowth promoting activity of ECa 233. ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed. In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases. PMID:23915016

  3. TLR4 receptor expression and function in F11 dorsal root ganglion × neuroblastoma hybrid cells.

    Science.gov (United States)

    Hashemian, Sanaz; Alhouayek, Mireille; Fowler, Christopher J

    2017-11-01

    TLR4 respond to bacterial LPS to produce inflammatory cytokines. TLR4 are expressed in dorsal root ganglia and play a role in pain. F11 dorsal root ganglia × mouse neuroblastoma cells possess many of the properties seen in nociceptive dorsal root ganglia neuronal cells. Here, we investigated the effect of 2 h and 6 h treatment with LPS upon the expression of inflammatory proteins in undifferentiated and differentiated F11 cells. The cells expressed mRNA for TRL4 (mouse, not rat) and proteins involved in TLR4 signaling. TLR4 expression was confirmed using immunohistochemistry. LPS produced modest increases in mouse and rat IL-6 and in mouse cyclooxygenase-2 levels in undifferentiated cells, but did not significantly affect mouse TNF-α expression. This contrasts with the robust effects of LPS upon cyclooxygenase-2 expression in cultured dorsal root ganglia neurons. F11 cells expressed the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and N-acylethanolamine acid amidase (both murine), which were functionally active. These data suggest that F11 cells are not a useful model for the study of LPS-mediated effects but may be useful for the study of endocannabinoid catabolism.

  4. Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

    Science.gov (United States)

    Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T; Bloom, Marshall E

    2017-01-15

    The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. Published by Elsevier Inc.

  5. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production......, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  6. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    Science.gov (United States)

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

  7. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

    2010-01-01

    signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels...... staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary...

  8. MEIS homeobox genes in neuroblastoma

    NARCIS (Netherlands)

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

    2005-01-01

    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

  9. Neuroblastoma | Office of Cancer Genomics

    Science.gov (United States)

    The TARGET Neuroblastoma projects elucidate comprehensive molecular characterization to determine the genetic changes that drive the initiation and progression of high-risk or hard-to-treat childhood cancers. Neuroblastoma (NBL) is a cancer that arises in immature nerve cells of the sympathetic nervous system, primarily affecting infants and children.

  10. Small molecule inhibitor regorafenib inhibits RET signaling in neuroblastoma cells and effectively suppresses tumor growthin vivo.

    Science.gov (United States)

    Chen, Zhenghu; Zhao, Yanling; Yu, Yang; Pang, Jonathan C; Woodfield, Sarah E; Tao, Ling; Guan, Shan; Zhang, Huiyuan; Bieerkehazhi, Shayahati; Shi, Yan; Patel, Roma; Vasudevan, Sanjeev A; Yi, Joanna S; Muscal, Jodi A; Xu, Guo-Tong; Yang, Jianhua

    2017-11-28

    Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo . We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Cunjin; Shi, Aiming; Cao, Guowen [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Tao, Tao [Department of Urology, Zhongda Hospital, Medical School of Southeast University, Nanjing, 210009 (China); Chen, Ruidong [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Zhanhong; Shen, Zhu; Tao, Hong; Cao, Bin [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Duanmin, E-mail: hudmsdfey@sina.com [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Bao, Junjie, E-mail: baojjsdfey@sina.com [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

    KAUST Repository

    Marinaro, Giovanni

    2015-01-01

    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. This journal is

  14. Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins

    NARCIS (Netherlands)

    Ouwehand, Krista; de Ruijter, Annemieke J. M.; van Bree, Chris; Caron, Huib N.; van Kuilenburg, André B. P.

    2005-01-01

    Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521

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

    Science.gov (United States)

    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.

  16. Investigation of in vitro cytotoxicity of the redox state of ionic iron in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Ajay Vikram Singh

    2012-01-01

    Full Text Available Background: there is an intimate relation between transition metals and cell homeostasis due to the physiological necessity of metals in vivo. Particularly, iron (ferrous and ferric state is utilized in many physiological processes of the cell but in excess has been linked with negative role contributing in many neurodegenerative processes. Objective: the aim of this study was to investigate which oxidation state of ionic iron (Ferrous (II versus Ferric (III is more toxic to neuronal cells (SHSY5Y. Materials and Methods: The neuroblastoma (SHSY5Y cells were exposed to varying concentration of ferric and ferrous iron. Morphological studies using immunofluorescence staining and microscopic analysis as confirmed by intracellular glutathione (GSH test demonstrated oxidative stress to cells in iron microenvironment. In addition, MTT assay was performed to evaluate the viability and metabolic state of the cells. Results: the results showed that ferrous form has significantly higher toxicity compared to the ferric ionic state of higher concentration. In addition, microscopic analysis shows cell fenestration at higher concentrations and swelling at intermediate ferric dosages as demonstrated by atomic force microscopy (AFM. Interestingly, the addition of a differentiation inducing factor, trans-retinoic rcid (RA retains significant viability and morphological features of the cells irrespective of the ionic state of the iron. AFM images revealed clustered aggregates arising from iron chelation with RA. Conclusions: the results indicate that Fe (II has more toxic effects on cells. In addition, it could be an interesting finding with respect to the antioxidant properties of RA as a chelating agent for the neurodegenerative therapeutics.

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

    Directory of Open Access Journals (Sweden)

    Lina Chakrabarti

    Full Text Available 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.

  18. Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y

    DEFF Research Database (Denmark)

    Plant, Leigh D; Boyle, John P; Thomas, Natasha M

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...... membrane distribution when the deltaE9 over-expressing cells were compared to control cells. Intracellular retention of Kv3.1 is consistent with the notion that PS1 can modulate the activity and trafficking of ion channels in central neurones and implicates a compromise in electrical signalling...

  19. Proteomic Alterations in Response to Hypoxia Inducible Factor 2α in Normoxic Neuroblastoma Cells.

    Science.gov (United States)

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Persano, Luca; Vitale, Monica; Sassi, Mauro; Bresolin, Silvia; Serafin, Valentina; Zambrano, Nicola; Scaloni, Andrea; Basso, Giuseppe; Iolascon, Achille; Capasso, Mario

    2016-10-07

    Hypoxia inducible factor (HIF)-2α protein expression in solid tumors promotes stem-like phenotype in cancer stem cells and increases tumorigenic potential in nonstem cancer cells. Recently, we have shown that HIF-1/2α gene expression is correlated to neuroblastoma (NB) poor survival and to undifferentiated tumor state; HIF-2α protein was demonstrated to enhance aggressive features of the disease. In this study, we used proteomic experiments on NB cells to investigate HIF-2α downstream-regulated proteins or pathways with the aim of providing novel therapeutic targets or bad prognosis markers. We verified that pathways mostly altered by HIF-2α perturbation are involved in tumor progression. In particular, HIF-2α induces alteration of central metabolism and splicing control pathways. Simultaneously, WNT, RAS/MAPK, and PI3K/AKT activity or expression are affected and may impact the sensitivity and the intensity of HIF-2α-regulated pathways. Furthermore, genes coding the identified HIF-2α-related markers built a signature able to stratify NB patients with unfavorable outcome. Taken together, our findings underline the relevance of dissecting the downstream effects of a poor survival marker in developing targeted therapy and improving patient stratification. Future prospective studies are needed to translate the use of these data into the clinical practice.

  20. Dioxin induces expression of hsa-miR-146b-5p in human neuroblastoma cells.

    Science.gov (United States)

    Xu, Tuan; Xie, Heidi Q; Li, Yunping; Xia, Yingjie; Sha, Rui; Wang, Lingyun; Chen, Yangsheng; Xu, Li; Zhao, Bin

    2018-01-01

    Dioxin can cause a series of neural toxicological effects. MicroRNAs (miRs) play important roles in regulating nervous system function and mediating cellular responses to environmental pollutants, such as dioxin. Hsa-miR-146b-5p appears to be involved in neurodegenerative diseases and brain tumors. However, little is known about effects of dioxin on the expression of hsa-miR-146b-5p. We found that the hsa-miR-146b-5p expression and its promoter activity were significantly increased in dioxin treated SK-N-SH cells, a human-derived neuroblastoma cell line. Potential roles of hsa-miR-146b-5p in mediating neural toxicological effects of dioxin may be due to the regulation of certain target genes. We further confirmed that hsa-miR-146b-5p significantly suppressed acetylcholinesterase (AChE) activity and targeted the 3'-untranslated region of the AChE T subunit, which has been down-regulated in dioxin treated SK-N-SH cells. Functional bioinformatic analysis showed that the known and predicted target genes of hsa-miR-146b-5p were involved in some brain functions or cyto-toxicities related to known dioxin effects, including synapse transmission, in which AChE may serve as a responsive gene for mediating the effect. Copyright © 2017. Published by Elsevier B.V.

  1. Sparstolonin B, a novel plant derived compound, arrests cell cycle and induces apoptosis in N-myc amplified and N-myc nonamplified neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Ambrish Kumar

    Full Text Available Neuroblastoma is one of the most common solid tumors and accounts for ∼ 15% of all the cancer related deaths in the children. Despite the standard therapy for advanced disease including chemotherapy, surgery, and radiation, the mortality rate remains high for these patients. Hence, novel therapeutic agents are desperately needed. Here we examined the anticancer activity of a novel plant-derived compound, sparstolonin B (SsnB; 8,5'-dihydroxy-4-phenyl-5,2'-oxidoisocoumarin using neuroblastoma cell lines of different genetics. SsnB was recently isolated from an aquatic Chinese herb, Sparganium stoloniferum, and tubers of this herb have been used in traditional Chinese medicine for the treatment of several inflammatory diseases and cancers. Our cell viability and morphological analysis indicated that SsnB at 10 µM concentration significantly inhibited the growth of both N-myc amplified (SK-N-BE(2, NGP, and IMR-32 cells and N-myc nonamplified (SH-SY5Y and SKNF-1 cells neuroblastoma cells. The flow cytometric analyses suggested that SsnB arrests the cell cycle progression at G2-M phase in all neuroblastoma cell lines tested. Exposure of SsnB inhibited the compact spheroid formation and reduced the tumorigenicity of SH-SY5Y cells and SK-N-BE(2 cells in in vitro 3-D cell culture assays (anchorage-independent colony formation assay and hanging drop assay. SsnB lowers the cellular level of glutathione (GSH, increases generation of reactive oxygen species and activates the cleavage of caspase-3 whereas co-incubation of a GSH precursor, N-acetylcysteine, along with SsnB attenuates the inhibitory effects of SsnB and increases the neuroblastoma cell viability. Our results for the first time demonstrate that SsnB possesses anticancer activity indicating that SsnB-induced reactive oxygen species generation promotes apoptotic cell death in neuroblastoma cells of different genetic background. Thus these data suggest that SsnB can be a promising drug candidate

  2. The protective effect of geniposide on human neuroblastoma cells in the presence of formaldehyde

    Science.gov (United States)

    2013-01-01

    Background Formaldehyde can induce misfolding and aggregation of Tau protein and β amyloid protein, which are characteristic pathological features of Alzheimer’s disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline. Methods In this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored. Results Cell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment. Conclusions Our results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione

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

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Md. Motarab [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States); Banik, Naren L. [Department of Neurosciences, Medical University of South Carolina, Charleston, SC (United States); Ray, Swapan K., E-mail: swapan.ray@uscmed.sc.edu [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States)

    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

  4. PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage.

    Directory of Open Access Journals (Sweden)

    Barbara Marengo

    Full Text Available Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH, which is crucial in counteracting the endogenous production of reactive oxygen species (ROS. We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO, a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.

  5. Reversible LSD1 inhibition with HCI-2509 induces the p53 gene expression signature and disrupts the MYCN signature in high-risk neuroblastoma cells.

    Science.gov (United States)

    Gupta, Sumati; Doyle, Kelly; Mosbruger, Timothy L; Butterfield, Andrew; Weston, Alexis; Ast, Allison; Kaadige, Mohan; Verma, Anupam; Sharma, Sunil

    2018-02-09

    Lysine-Specific Demethylase 1 (LSD1) over-expression correlates with poorly differentiated neuroblastoma and predicts poor outcome despite multimodal therapy. We have studied the efficacy of reversible and specific LSD1 inhibition with HCI-2509 in neuroblastoma cell lines and particularly the effect of HCI-2509 on the transcriptomic profile in MYCN amplified NGP cells. Cell survival assays show that HCI-2509 is cytotoxic to poorly differentiated neuroblastoma cell lines in low micromole or lower doses. Transcriptional profiling of NGP cells treated with HCI-2509 shows a significant effect on p53, cell cycle, MYCN and hypoxia pathway gene sets. HCI-2509 results in increased histone methyl marks and p53 levels along with cell cycle arrest in the G2/M phase and inhibition of colony formation of NGP cells. Our findings indicate that LSD1 inhibition with HCI-2509 has a multi-target effect in neuroblastoma cell lines, mediated in part via p53. MYCN-amplified neuroblastoma cells have a targeted benefit as HCI-2509 downregulates the MYCN upregulated gene set.

  6. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

    Directory of Open Access Journals (Sweden)

    Murphy Derek M

    2010-04-01

    Full Text Available Abstract Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  7. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2.

    Science.gov (United States)

    Foley, Niamh H; Bray, Isabella M; Tivnan, Amanda; Bryan, Kenneth; Murphy, Derek M; Buckley, Patrick G; Ryan, Jacqueline; O'Meara, Anne; O'Sullivan, Maureen; Stallings, Raymond L

    2010-04-21

    Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  8. Effect of heavy metals on silencing of engineered long interspersed element-1 retrotransposon in nondividing neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Motamedi, Mahdieh; Akrami, Seyed Mohammad

    2013-01-01

    L1 retrotransposons are the most active mobile DNA elements in human genome. Unregulated L1 retrotransposition may have deleterious effect by disrupting vital genes and inducing genomic instabilities. Therefore, human cells control L1 elements by silencing their activities through epigenetic mechanisms. It has been shown that cell division and heavy metals stimulate the frequency of L1 activities. Removal of silencing by L1 motivators may restart L1 element functions. Here, we have proposed that weather neurotoxic environmental heavy metals (as L1 stimulating factors) have a role in removing L1 silencing and restating its activities in nondividing neuronal cells. L1-RP green fluorescent protein (GFP)-tagged knock-in human neuroblastoma clones were prepared. Single-cell clone was treated with mitomycin-c combined with nontoxic and toxic concentrations of iron (Fe), copper (Cu), and mercury (Hg). Silencing status of engineered L1 elements in dividing and nondividing cells was determined through measuring the amount of GFP expressing cells with flow cytometry. The cytotoxic effect of mitomycin-c combined with metals was measured by MTT assay. Hg in nondividing cells and Fe, Cu, and Hg in dividing neuroblastoma cells could significantly remove L1 silencing. Also, mitomycin-c treatment did not have any effect on metal toxicity status in neuroblastoma cells. Totally, our findings have shown that cell division has a role in removing L1 silencing as well as L1 retrotransposition induced by environmental heavy metals. It has been also indicated that Hg at all concentrations could remove silencing of engineered L1 element regardless of cell cycle state.

  9. Effect of gamma radiation on sodium channels in different conformations in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Freschi, J.E.; Moran

    1986-01-01

    The dose-response relationship between gamma radiation and batrachotoxin-stimulated sodium influx in neuroblastoma cells in tissue culture was studied. Also tested was the hypothesis that changes in sodium-channel conformation may alter the radiosensitivity of the channel. It was found that gamma radiation inhibited toxin-stimulated /sup 22/Na uptake at doses beyond a threshold of 200-300 Gy. No effects were seen following doses below 100 Gy. This inhibition of sodium permeability was seen when the cells were irradiated with sodium channels in the closed or inactivated, nonconducting states. However, when the channels were in the toxin-opened, conducting state, gamma radiation had no effect at doses up to 2000 Gy. Results support earlier electrophysiological studies that showed that high doses of ionizing radiation are required to produce a measureable decrease in sodium permeability. In addition, the data suggest that by changing the sodium-channel conformation, batrachotoxin appears to alter radiosensitive chemical bonds in the gating or ion-conducting portion of the channel.

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

    Directory of Open Access Journals (Sweden)

    Stefania Cocco

    Full Text Available 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.

  11. 1070 myeloablative megatherapy procedures followed by stem cell rescue for neuroblastoma: 17 years of European experience and conclusions

    Energy Technology Data Exchange (ETDEWEB)

    Chauvin, F. [Centre Leon Berard, Unit of Biostatistics, 28, rue Laennec, 69373 Lyon (France); Rosti, G. [Ospedale Civile, Oncologia Medica, Via Le Randi 5, 48100 Ravenna (Italy); Bernard, J.L. [Hopital d' Enfants de la Timone, Department of Paediatrics, Boulevard Jean Moulin, 13385 Marseille (France); Kremens, B. [Universitats Klinikum Essen, Department of Paediatrics, Hufelandstrasse, 55, 45122 Essen (Germany); Garaventa, A. [Istituto Giannina Gaslini, Department of Haematology/Oncology, Via Maggio 5, 16148 Genova Quarto (Italy); Klingebiel, T. [University Hospital, Abteilung Hamatologie Oncologie, Ruemelinstrasse 18-23, 7400 Tuebingen (Germany); Pearson, A.D.J. [Royal Victoria Infirmary, Sir J. Spence Institute of Child Health, Children' s Cancer Unit, Queen Victoria Road, Newcastle Upon Tyne (United Kingdom); Pinkerton, R. [The Royal Marsden Hospital, Children' s Department, Downs Road, Sutton (United Kingdom); Zucker, J.M. [Institut Curie, Department of Paediatrics, 3 rue Thuillier, 75231 Paris (France); Hartmann, O. [Institut Gustave Roussy, Department of Paediatrics, rue Camille Desmoulins, 94805 Villejuif (France); Lasset, C. [Centre Leon Berard, Unit of Biostatistics, 28, rue Laennec, 69373 Lyon (France); Ladenstein, R. [St. Anna Kinderhospital, Department of Haematology, Wiener Rotes Kreuz, Kinderspitalgasse 6, 1086 Vienna (Austria); Philip, T. [Centre Leon Berard, Department of Paediatrics, 28, rue Laennec, 69373 Lyon (France)

    1997-10-01

    1070 myeloablative procedures followed by stem cell rescue for neuroblastoma are reviewed. These 1070 procedures are part of the European Group for Blood and Marrow Transplant (EBMTG) registry from the last 17 years (in 4536 patients). In 1070 neuroblastoma patients, survival at 2 years was 49%, at 5 years, 33% and relapses were observed as late as 7 years post-BMT (bone marrow transplant). However, 5-year survivors after megatherapy with BMT for stage 4 disease do have an 80% chance of becoming a long-term survivor. When BMT had been used in first complete response (CR1) no salvage was possible, whereas 15% survivors may be seen if BMT is used for the first time at relapse. Infants with stage 4 neuroblastoma had a 17% toxic death rate and indication in this group is exceptional and not recommended. In a matched cohort (17 allogeneic and 34 autologous), autologous stem cell rescue (SCR) was shown to be at least equal to allogeneic SCR. Multivariate analysis of clinical prognostic factors in children with stage 4 disease over 1 year showed that event-free survival was mainly influenced by two adverse factors before the megatherapy procedure: persisting skeleton lesions ({sup 99}Tc and/or mIBG scan positive) as well as persisting bone marrow (BM) involvement. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  12. The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    International Nuclear Information System (INIS)

    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 [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 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 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

  13. Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Pastorino Fabio

    2010-06-01

    Full Text Available Abstract Background Sharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors. Results In this work, we have demonstrated that the new α,β-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P and D6 vs curcumin P Neuroblastoma: D6 vs both control and curcumin: P . Conclusions Our data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors.

  14. NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest

    International Nuclear Information System (INIS)

    Andries, Vanessa; Vandepoele, Karl; Staes, Katrien; Berx, Geert; Bogaert, Pieter; Van Isterdael, Gert; Ginneberge, Daisy; Parthoens, Eef; Vandenbussche, Jonathan; Gevaert, Kris; Roy, Frans van

    2015-01-01

    NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved. Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21 CIP1/WAF1 were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool. We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21 CIP1/WAF1 in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell

  15. Detection of ciguatoxin in fish tissue using sandwich ELISA and neuroblastoma cell bioassay.

    Science.gov (United States)

    Empey Campora, Cara; Dierking, Jan; Tamaru, Clyde S; Hokama, Yoshitsugi; Vincent, Douglas

    2008-01-01

    The applicability of a new enzyme-linked immunoassay (ELISA) for detecting ciguatoxin (CTX) in fish tissue was evaluated by testing three fish species commonly implicated in ciguatera fish poisoning in Hawaii. A total of 164 individual almaco jack (Seriola rivoliana) and greater amberjack (S. dumerili) and a total of 175 individuals of the blue-spotted grouper (Cephalopholis argus) were caught at various locations in the Hawaiian Islands. Muscle tissue from each individual was assessed for the presence of CTX using two methods: a semi-quantitative ELISA that was recently developed for detecting picogram levels of CTX in fish extract and a neuroblastoma (NB) cell assay commonly used to screen for marine toxins in fish. Results of the tests were highly correlated, with the ELISA indicating the presence of CTX in 9.4% of all fish samples, and the NB assay indicating toxicity in 6.8% of the fish samples. We conclude that the ELISA produces reliable and accurate results that are consistent with those provided by the accepted NB assay and that the ELISA has potential for future applications in screening fish populations for CTX.

  16. Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Pei-Yi Wu

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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 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 μM; (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 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-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 μM to 100 μM.

  18. Characterization of endothelin receptors on a human neuroblastoma cell line: evidence for the ETA subtype.

    Science.gov (United States)

    Wilkes, L C; Boarder, M R

    1991-11-01

    1. Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK-N-MC cell line, derived from a human neuroblastoma. 2. [125I]-ET-1 (2.5 x 10(-11) M) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 +/- 0.04, n = 3) with an apparent Kd of 1.4 +/- 0.3 x 10(-9) M and a Bmax of 3.1 +/- 1.0 pmol mg-1 protein. [125I]-ET-3 (2.5 x 10(-11) M), did not specifically bind to SK-N-MC cells. 3. The binding of [125I]-ET-1 was competitively inhibited by other ET isoforms, the order of potency being ET-1 greater than sarafotoxin S6b greater than ET-3. 4. Association of 1 nM [125I]-ET-1 at 37 degrees C reached apparent equilibrium at 60-80 min, with half-maximal binding being achieved at 12 min. 5. Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]-ET-1 dissociating. The actual amounts of [125I]-ET-1 dissociated were similar in both cases. 6. Incubation of [125I]-ET-3 with SK-N-MC cells at 37 degrees C for 60 min did not result in significant degradation of this peptide. However, [125I]-ET-1 was broken down by incubation with SK-N-MC cells, the pattern of degradation of dissociable [125I]-ET-1 (and that found in the supernatant) being different from that of non-dissociable [125I]-ET-1. 7. ET-1 concentration-dependently induced an increase in total inositol phosphate accumulation in subconfluent (but not in confluent) cultures of SK-N-MC cells (EC50 = 6.43 +/- 1.9 x 1010M). ET-3 was without effect. 8. These results show that ET-1 specifically binds to SK-N-MC cells with the characteristics of an ETA receptor. Our earlier finding that adrenal chromaffin cells express an ETB receptor indicates the existence of multiple ET receptor types on neuronal cells.

  19. Comprehensive DNA methylation analysis of human neuroblastoma cells treated with blonanserin.

    Science.gov (United States)

    Murata, Yui; Nishioka, Masaki; Bundo, Miki; Sunaga, Fumiko; Kasai, Kiyoto; Iwamoto, Kazuya

    2014-03-20

    Blonanserin is a second-generation antipsychotic drug for schizophrenia. The pharmacological actions of blonanserin are shown to be the antagonism of dopamine receptor 2 and serotonin receptors. However, its molecular mechanisms in brain cells have not been fully characterized. Accumulating evidence suggests that antipsychotic drugs and mood stabilizers show epigenetic effects on a wide range of genes in animal and cellular models. We performed genome-wide DNA methylation analysis targeting 479,814 CpG sites of cultured human neuroblastoma cells administered with blonanserin. We found that 3,057 CpG sites showed statistically significant changes in DNA methylation at two different doses of blonanserin (1.36 nM and 13.6 nM). These included hypermethylated CpG sites that were enriched in genes related to axonogenesis and cell morphogenesis involved in neuron differentiation. We also showed that the global effect on DNA methylome depends on the concentration of the drug. With a high dose of blonanserin, the overall methylation levels across all CpG sites significantly increased. These increases in DNA methylation were prominent in the CpG sites distant from promoter regions. We further examined DNA methylation changes in specific genes implicated for the actions of antipsychotic drugs, such as the dopamine receptor 2 (DRD2) gene and the serotonin receptor 2A (HTR2A) gene. We observed that CpG sites that were located within DRD2 and HTR2A genes were significantly hypermethylated by blonanserin. The DNA methylation changes induced by the treatment with blonanserin will be useful for understanding its pharmacological actions at the cellular level. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-12-08

    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.

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

    International Nuclear Information System (INIS)

    Briggs, D.J.

    1989-01-01

    The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-α), beta interferon (IFN-β), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of 3 H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-α, IFN-β, or 1 μg of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of 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-β, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 μg. 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-β produced variable results in non-specific and specific binding while IFN-α caused mainly specific binding to occur. The most significant change brought about by IFN-α was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-α 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-α or IFN-β treated cells

  2. Mechanisms of neuroblastoma cell growth inhibition by CARP-1 functional mimetics.

    Directory of Open Access Journals (Sweden)

    Magesh Muthu

    Full Text Available 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.

  3. Targeted BIRC5 silencing using YM155 causes cell death in neuroblastoma cells with low ABCB1 expression

    NARCIS (Netherlands)

    Lamers, Fieke; Schild, Linda; Koster, Jan; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.

    2012-01-01

    The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing

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

    Science.gov (United States)

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

    2017-05-01

    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. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

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

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    Ross Ferguson

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

    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 deg. 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. (authors)

  7. Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Wang, Ke; Zhou, Fanfan; Zhu, Xue; Zhang, Kai; Huang, Biao; Zhu, Lan; Zhu, Ling

    2014-01-01

    Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor Curcumin (50 μM), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 μM). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.

  8. Knockdown of LRP/LR induces apoptosis in pancreatic cancer and neuroblastoma cells through activation of caspases.

    Science.gov (United States)

    Chetty, Carryn J; Ferreira, Eloise; Jovanovic, Katarina; Weiss, Stefan F T

    2017-11-15

    The 37kDa/67kDa laminin receptor (LRP/LR) serves various physiological and pathological roles such as enhancing tumour-related processes including metastasis, angiogenesis, cellular viability and telomerase activation in cancerous cell lines. The present study investigates the effect of siRNA mediated downregulation of LRP/LR on pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. MTT and BrdU assays revealed that siRNA mediated downregulation of LRP resulted in a significant reduction in cell viability and cell proliferation. In addition, knock-down of LRP resulted in phosphatidylserine externalization, diminished nuclear integrity and significantly enhanced caspase-3 activity, which is indicative of apoptosis. LRP downregulation resulted in a significant increase in caspase-8 activity in IMR-32 cells and enhanced caspase-8 and 9 activity in AsPC-1 cells. These data recommend siRNA mediated knock-down of LRP as a potential therapeutic avenue for the treatment of pancreatic cancer and neuroblastoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines

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    Ingo Lange

    2014-01-01

    Full Text Available Neuroblastoma is an extra-cranial solid cancer in children. MYCN gene amplification is a prognostic indicator of poor outcome in neuroblastoma. Recent studies have shown that the multiple steps involved in cell migration are dependent on the availability of intracellular calcium (Ca2+. Although significant advances have been made in understanding the role of Ca2+ during migration, little has been achieved towards understanding its impact on the progression of diseases such as cancer. Interestingly, previous studies showed that cancer cell migration is regulated by TRPM7, a calcium-permeable ion channel. The objective of the current study was to elucidate the mechanism by which MycN promotes NB cell migration and the mechanism regulating TRPM7 expression. The results showed that MycN increased TRPM7 expression, induced TRPM7 channel activity, increased intracellular Ca2+ signaling, and promoted cell migration in NB cells. The results also showed that inhibition or down-regulation of ornithine decarboxylase (ODC inhibited TRPM7 expression, a process that was reversed by spermidine. Overall, this study provides evidence that MycN promotes TRPM7 expression and cell migration through a mechanism that involves ODC synthesis of polyamines.

  10. 5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.

    Science.gov (United States)

    Garcia-Gil, M; Pesi, R; Perna, S; Allegrini, S; Giannecchini, M; Camici, M; Tozzi, M G

    2003-01-01

    5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology

  11. Antiproliferative effects of mitraphylline, a pentacyclic oxindole alkaloid of Uncaria tomentosa on human glioma and neuroblastoma cell lines.

    Science.gov (United States)

    García Prado, E; García Gimenez, M D; De la Puerta Vázquez, R; Espartero Sánchez, J L; Sáenz Rodríguez, M T

    2007-04-01

    Uncaria tomentosa inner bark extract is a popular plant remedy used in folk medicine to treat tumor and inflammatory processes. In this study, the anti-tumoral effects of its pentacyclic alkaloid mitraphylline were investigated. Furthermore, its growth-inhibitory and cytotoxic effects on glioma GAMG and neuroblastoma SKN-BE(2) cell lines were studied using cyclophosphamide and vincristine as controls. A colter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. Micromolar concentrations of mitraphylline (from 5 to 40 microM) inhibited the growth of both cell lines. It inhibited the growth of the two cell lines studied in a dose-dependent manner. The IC(50) values were 12.3 microM (30h) for SKN-BE(2) and 20 microM (48 h) for GAMG, respectively. This action suggests that mitraphylline is a new and promising agent in the treatment of human neuroblastoma and glioma.

  12. Cocaine and ethanol target 26S proteasome activity and gene expression in neuroblastoma cells.

    Science.gov (United States)

    Caputi, Francesca Felicia; Carboni, Lucia; Mazza, Daria; Candeletti, Sanzio; Romualdi, Patrizia

    2016-04-01

    Ethanol and cocaine are widely abused drugs triggering long-lasting changes in neuronal circuits and synaptic transmission through the regulation of enzyme activity and gene expression. Compelling evidence indicates that the ubiquitin-proteasome system plays a role in the molecular changes induced by addictive substances, impacting on several mechanisms implicated in abuse. The goal of these studies was to evaluate the effects of cocaine or ethanol on proteasome activity in neuroblastoma cells. Moreover, the gene expression of specific subunits was assessed. Chymotrypsin-like activity was measured after 2 h, 24 h, and 48 h exposure to 5 μM cocaine or 40 mM ethanol. Proteasome subunit transcripts were evaluated by qPCR at the same time-points. Treatments modified proteasome function in opposite directions, since cocaine increased and ethanol reduced chymotrypsin-like activity. Interestingly, we observed gene expression alterations induced by these drugs. In the core particle, the β1 and α5 subunits were mainly up-regulated by cocaine, whereas α6 transcripts were mostly decreased. β2 and β5 did not change. Similarly, ethanol exposure generally increased β1 and α5 mRNAs. Moreover, the β2 subunit was significantly up-regulated by ethanol only. The β5 and α6 subunits were not altered. In the regulatory particle, Rpt3 was increased by cocaine exposure, whereas it was reduced by ethanol. No significant Rpn9 alterations were observed. These findings support the notion that addictive substances regulate proteasome function, contributing to the dysregulations related to drug abuse since the availability of adequate subunit amounts is necessary for proper complex assembly and function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Genetics Home Reference: neuroblastoma

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Neuroblastoma Neuroblastoma Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Neuroblastoma is a type of cancer that most often ...

  14. Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

    OpenAIRE

    Huang, L.Y.; Moran, N.; Ehrenstein, G.

    1984-01-01

    We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of b...

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

    Science.gov (United States)

    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.

  16. Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

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    Inman Gareth J

    2010-11-01

    Full Text Available Abstract Background Coiled-coil domain containing 115 (Ccdc115 or coiled coil protein-1 (ccp1 was previously identified as a downstream gene of Fibroblast Growth Factor 2 (FGF2 highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. Methods Forced ccp1 expression in mouse embryonic fibroblast (MEF and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1. Results Ccp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK or ERK Kinase (MEK inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY upon FGF2 treatment was accelerated in ccp1 over-expressing cells. Conclusions All together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

  17. Effects of IKAP/hELP1 deficiency on gene expression in differentiating neuroblastoma cells: implications for familial dysautonomia.

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    Rachel Cohen-Kupiec

    Full Text Available Familial dysautonomia (FD is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype.

  18. Facilitation of 9,10-phenanthrenequinone-elicited neuroblastoma cell apoptosis by NAD(P)H:quinone oxidoreductase 1.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Kamase, Kyoko; Takasawa, Hiroaki; Yamaji, Yukiko; Endo, Satoshi; El-Kabbani, Ossama; Ikari, Akira

    2018-01-05

    9,10-Phenanthrenequinone (PQ), a major quinone component in diesel exhaust particles, is considered to provoke damage of respiratory and vascular cells through highly producing reactive oxygen species (ROS), but little is known about its pathophysiological role in neuronal cell damage. In this study, we found that incubation with 1,2-naphthoquinone, 1,4-naphthoquinone and PQ, major quinone components in diesel exhausts, provokes apoptosis of human neuroblastoma cell lines. SK-N-SH cell treatment with a lethal concentration of PQ facilitated ROS production within 6 h. The treatment also promoted formation of 8-hydroxy-deoxyguanosine, p53 activation, elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, and resultant activation of caspase-9 and caspase-3, inferring that ROS production, DNA damage and mitochondrial dysfunction are crucial processes of the PQ-triggered SK-N-SH cell apoptosis. The PQ treatment of SK-N-SH cells elevated the level of 4-hydroxynonenal (HNE), a cytotoxic reactive aldehyde generated from lipid peroxidation. The treatment with PQ and HNE also decreased cellular levels of total and reduced glutathiones, and the damage elicited by HNE was ameliorated and deteriorated by pretreating with cell-permeable glutathione analog and the depletor, respectively. Moreover, the treatment with PQ and HNE decreased the proteasomal proteolytic activities, suggesting a contribution of decrease in the antioxidant abilities to the ROS-mediated neuroblastoma cell apoptosis. Our comparative analyses of 17 cells showed a positive correlation between the PQ reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) activities. In addition, overexpression and knockdown of NQO1 augmented and lowered, respectively, the ROS production through PQ redox-cycling and the quinone toxicity. Furthermore, the treatment with PQ and HNE up-regulated the NQO1 expression. Taken together, PQ exposure produces large amounts of ROS in neuroblastoma cells via NQO1 up

  19. Adolescent Neuroblastoma of Lower Limb

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    Rajeshwari K

    2013-04-01

    Full Text Available Neuroblastoma is an embryonic tumour of neural crest origin, commonly seen in children with upper abdomen involvement. Rarely neuroblastomas present in adolescents and adults involving lower limb. Histopathologically neuroblastoma of lower limb can be confused with other small round cell tumour especially with Ewing's sarcoma and rhabdomyosarcoma. A 16 year old male presented with 15x11cm swelling, pain and multiple discharging sinuses of right leg since 4 months. Routine haematological and biochemical analysis were within normal limits. Radiology of right leg showed large soft tissue swelling encompassing the pathological fracture of tibia and bowing of fibula. Fine needle aspiration of the swelling revealed malignant small round cell tumour. Histopathology revealed poorly differentiated neuroblastoma of lower limb. The immunohistochemistry of Synaptophysin and Chromogranin were positive and CD 99 was negative. Neuroblastoma diagnosed at unusual site with uncommon age has poor prognosis. Hence, one must keep in mind the differential diagnosis of neuroblastoma as one of the differential diagnosis in evaluating the soft tissue tumours of lower limb.

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

    International Nuclear Information System (INIS)

    Matsushima, H.; Bogenmann, E.

    1990-01-01

    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

  1. A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation.

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    Carla Lucia Esposito

    Full Text Available Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA-treatment of neuroblastoma (NB cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.

  2. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    OpenAIRE

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MP...

  3. Estimation of transition doses for human glioblastoma, neuroblastoma and prostate cell lines using the linear-quadratic formalism

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    John Akudugu

    2015-09-01

    Full Text Available Purpose: The introduction of stereotactic radiotherapy has raised concerns regarding the use of the linear-quadratic (LQ model for predicting radiation response for large fractional doses. To partly address this issue, a transition dose D* below which the LQ model retains its predictive strength has been proposed. Estimates of D* which depends on the a, β, and D0 parameters are much lower than fractional doses typically encountered in stereotactic radiotherapy. D0, often referred to as the final slope of the cell survival curve, is thought to be constant. In vitro cell survival curves generally extend over the first few logs of cell killing, where D0-values derived from the multi-target formalism may be overestimated and can lead to low transition doses. Methods:  D0-values were calculated from first principles for each decade of cell killing, using experimentally-determined a and β parameters for 17 human glioblastoma, neuroblastoma, and prostate cell lines, and corresponding transition doses were derived.Results: D0 was found to decrease exponentially with cell killing. Using D0-values at cell surviving fractions of the order of 10-10 yielded transition doses ~3-fold higher than those obtained from D0-values obtained from conventional approaches. D* was found to increase from 7.84 ± 0.56, 8.91 ± 1.20, and 6.55 ± 0.91 Gy to 26.84 ± 2.83, 23.95 ± 2.03, and 22.49 ± 2.31 Gy for the glioblastoma, neuroblastoma, and prostate cell lines, respectively. Conclusion: These findings suggest that the linear-quadratic formalism might be valid for estimating the effect of stereotactic radiotherapy with fractional doses in excess of 20 Gy.

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

    Science.gov (United States)

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

    2017-06-01

    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.

  5. Retinoic acid postconsolidation therapy for high-risk neuroblastoma patients treated with autologous haematopoietic stem cell transplantation.

    Science.gov (United States)

    Peinemann, Frank; van Dalen, Elvira C; Enk, Heike; Berthold, Frank

    2017-08-25

    Neuroblastoma is a rare malignant disease and mainly affects infants and very young children. The tumours mainly develop in the adrenal medullary tissue, with an abdominal mass as the most common presentation. About 50% of patients have metastatic disease at diagnosis. The high-risk group is characterised by metastasis and other features that increase the risk of an adverse outcome. High-risk patients have a five-year event-free survival of less than 50%. Retinoic acid has been shown to inhibit growth of human neuroblastoma cells and has been considered as a potential candidate for improving the outcome of patients with high-risk neuroblastoma. This review is an update of a previously published Cochrane Review. To evaluate the efficacy and safety of additional retinoic acid as part of a postconsolidation therapy after high-dose chemotherapy (HDCT) followed by autologous haematopoietic stem cell transplantation (HSCT), compared to placebo retinoic acid or to no additional retinoic acid in people with high-risk neuroblastoma (as defined by the International Neuroblastoma Risk Group (INRG) classification system). We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library (2016, Issue 11), MEDLINE in PubMed (1946 to 24 November 2016), and Embase in Ovid (1947 to 24 November 2016). Further searches included trial registries (on 22 December 2016), conference proceedings (on 23 March 2017) and reference lists of recent reviews and relevant studies. We did not apply limits by publication year or languages. Randomised controlled trials (RCTs) evaluating additional retinoic acid after HDCT followed by HSCT for people with high-risk neuroblastoma compared to placebo retinoic acid or to no additional retinoic acid. Primary outcomes were overall survival and treatment-related mortality. Secondary outcomes were progression-free survival, event-free survival, early toxicity, late toxicity, and health-related quality of life. We used standard

  6. Effect of polyunsaturated fatty acids and their metabolites on bleomycin-induced cytotoxic action on human neuroblastoma cells in vitro.

    Directory of Open Access Journals (Sweden)

    Sailaja Polavarapu

    Full Text Available In the present study, we noted that bleomycin induced growth inhibitory action was augmented by all the polyunsaturated fatty acids (PUFAs tested on human neuroblastoma IMR-32 (0.5 × 10(4 cells/100 µl of IMR cells (EPA > DHA > ALA = GLA = AA > DGLA = LA: ∼ 60, 40, 30, 10-20% respectively at the maximum doses used. Of all the prostaglandins (PGE1, PGE2, PGF2α, and PGI2 and leukotrienes (LTD4 and LTE4 tested; PGE1, PGE2 and LTD4 inhibited the growth of IMR-32 cells to a significant degree at the highest doses used. Lipoxin A4 (LXA4, 19,20-dihydroxydocosapentaenoate (19, 20 DiHDPA and 10(S,17(S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (protectin: 10(S,17(SDiHDoHE, metabolites of DHA, significantly inhibited the growth of IMR-32 cells. Pre-treatment with AA, GLA, DGLA and EPA and simultaneous treatment with all PUFAs used in the study augmented growth inhibitory action of bleomycin. Surprisingly, both indomethacin and nordihydroguaiaretic acid (NDGA at 60 and 20 µg/ml respectively enhanced the growth of IMR-32 cells even in the presence of bleomycin. AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. These results suggest that PUFAs suppress growth of human neuroblastoma cells, augment growth inhibitory action of bleomycin by enhancing formation of lipid peroxides and altering the status of anti-oxidants and, in all probability, increase the formation of lipoxins, resolvins and protectins from their respective precursors that possess growth inhibitory actions.

  7. A case of neonatal neuroblastoma

    International Nuclear Information System (INIS)

    Nounaka, Osamu; Gotoh, Toshiaki; Takahashi, Kazuaki; Koyanagi, Tomohiko; Kakizaki, Hidehiro; Nakanishi, Shoichiro.

    1987-01-01

    A two-day-old male infant was referred to us for probable neuroblastoma, because of upper abdominal mass and positive urinary vanillylmandelic acid (VMA). Primary site of neuroblastoma was not found, but clinically IV-S stage neuroblastoma was strongly suspected, so 131 I-metaiodobenzylguanidine (MIBG) scan was performed. RI accumulation was found near the left adrenal region. Thus laparotomy was performed and left adrenal was resected. Liver biopsy was also performed. Microscopically multiple in situ foci of neuroblastoma cells were found in the left adrenal and tumor involvement was also seen in the liver. Skin and bone marrow metastasis were ruled out. Minimal chemotherapy was intended but abandoned soon because of possible spontaneous regression of stage IV-S neuroblastoma. Thereafter liver has been getting smaller and the patient has been doing well. Urinary VMA and homovanillic acid (HVA) per creatinine, which were used for follow-up, have also normalized after 3 months. Treatment of stage IV-S neuroblastoma and early diagnosis by 131 I-MIBG scan were reviewed. (author)

  8. Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state.

    OpenAIRE

    Clark, H F

    1980-01-01

    Each of several strains of fixed rabies virus was found to replicate to high titers in C1300 mouse neuroblastoma (clone NA) cells, without adaptation. Rabies serogroup Lagos bat, Mokola, and Duvenhage viruses also replicated efficiently in NA cells. Kotonkan and Obodhiang viruses replicated efficiently after adaptation, to titers not previously obtained in vitro. Infection in NA cells was frequently more cytopathic than in BHK-21 cells, allowing titration of Kotonkan and Obodhiang viruses by ...

  9. Inhibition of Akt sensitises neuroblastoma cells to gold(III) porphyrin 1a, a novel antitumour drug induced apoptosis and growth inhibition.

    Science.gov (United States)

    Li, W; Xie, Y; Sun, R W-Y; Liu, Q; Young, J; Yu, W-Y; Che, C-M; Tam, P K; Ren, Y

    2009-07-21

    Gold(III) porphyrin 1a is a new class of anticancer drug, which inhibits cell proliferation of wide range of human cancer cell lines and induces apoptosis in human nasopharyngeal carcinoma cells. However, the underlying signalling mechanism by which gold(III) porphyrin 1a modifies the intracellular apoptosis pathways in tumour cells has not been explained in detail in neuroblastoma cells. Cell proliferation and apoptosis were determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V binding, respectively. Western blot assay was used to detect proteins involved in apoptotic and Akt pathways. In vivo tumour growth was assessed by inoculating tumour cells to nude mice subcutaneously, and gold(III) porphyrin 1a was administrated intravenously. This study assessed the antitumour effect and mechanism of gold(III) porphyrin 1a on neuroblastoma in vitro and in vivo. Gold(III) porphyrin 1a displayed a growth inhibition and induction of apoptosis in neuroblastoma cells effectively in vitro, which was accompanied with release of cytochrome c and Smac/DIABLO and caspases activation. Further studies indicated that gold(III) porphyrin 1a inhibited X-linked inhibitor of apoptosis (XIAP). However, we found that gold(III) porphyrin 1a can induce a survival signal, Akt activation within minutes and could last for at least 24 h. To further confirm association between activation of Akt and the effectiveness of gold(III) porphyrin 1a, neuroblastoma cells were treated with API-2, an Akt-specific inhibitor. API-2 sensitised cells to gold(III) porphyrin 1a-induced apoptosis and growth inhibition. These results suggested that Akt may be considered as a molecular 'brake' that neuroblastoma cells rely on to slow down gold(III) porphyrin 1a-induced apoptosis and antiproliferation. Gold(III) porphyrin 1a is a mitochondrial apoptotic stimulus but also activates Akt, suggesting an involvement of Akt in mediating the effectiveness to growth

  10. Mechanisms of neuroblastoma regression

    Science.gov (United States)

    Brodeur, Garrett M.; Bagatell, Rochelle

    2014-01-01

    Recent genomic and biological studies of neuroblastoma have shed light on the dramatic heterogeneity in the clinical behaviour of this disease, which spans from spontaneous regression or differentiation in some patients, to relentless disease progression in others, despite intensive multimodality therapy. This evidence also suggests several possible mechanisms to explain the phenomena of spontaneous regression in neuroblastomas, including neurotrophin deprivation, humoral or cellular immunity, loss of telomerase activity and alterations in epigenetic regulation. A better understanding of the mechanisms of spontaneous regression might help to identify optimal therapeutic approaches for patients with these tumours. Currently, the most druggable mechanism is the delayed activation of developmentally programmed cell death regulated by the tropomyosin receptor kinase A pathway. Indeed, targeted therapy aimed at inhibiting neurotrophin receptors might be used in lieu of conventional chemotherapy or radiation in infants with biologically favourable tumours that require treatment. Alternative approaches consist of breaking immune tolerance to tumour antigens or activating neurotrophin receptor pathways to induce neuronal differentiation. These approaches are likely to be most effective against biologically favourable tumours, but they might also provide insights into treatment of biologically unfavourable tumours. We describe the different mechanisms of spontaneous neuroblastoma regression and the consequent therapeutic approaches. PMID:25331179

  11. The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.

    Science.gov (United States)

    Kollareddy, Madhu; Sherrard, Alice; Park, Ji Hyun; Szemes, Marianna; Gallacher, Kelli; Melegh, Zsombor; Oltean, Sebastian; Michaelis, Martin; Cinatl, Jindrich; Kaidi, Abderrahmane; Malik, Karim

    2017-09-10

    Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  12. Organometallic iron(III-salophene exerts cytotoxic properties in neuroblastoma cells via MAPK activation and ROS generation.

    Directory of Open Access Journals (Sweden)

    Kyu Kwang Kim

    2011-04-01

    Full Text Available The objective of the present study was to investigate the specific effects of Iron(III-salophene (Fe-SP on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as p38 and JNK MAPK activation, DNA degradation, and elevated generation of reactive oxygen species (ROS in SMS-KCNR NB cells. In contrast to Fe-SP, non-complexed salophene or Cu(II-SP did not raise ROS levels in NB or SKOV-3 ovarian cancer control cells. Cytotoxicity of Fe-SP and activation of caspase-3, -7, PARP, pro-apoptotic p38 and JNK MAPK could be prevented by co-treatment with antioxidants suggesting ROS generation is the primary mechanism of cytotoxic action. We report here that Fe-SP is a potent growth-suppressing and cytotoxic agent for in vitro NB cell lines and, due to its high tolerance in previous animal toxicity studies, a potential therapeutic drug to treat NB tumors in vivo.

  13. Role of the GM1 ganglioside oligosaccharide portion in the TrkA-dependent neurite sprouting in neuroblastoma cells.

    Science.gov (United States)

    Chiricozzi, Elena; Pomè, Diego Yuri; Maggioni, Margherita; Di Biase, Erika; Parravicini, Chiara; Palazzolo, Luca; Loberto, Nicoletta; Eberini, Ivano; Sonnino, Sandro

    2017-12-01

    GM1 ganglioside (II 3 NeuAc-Gg 4 Cer) is known to promote neurite formation in neuroblastoma cells by activating TrkA-MAPK pathway. The molecular mechanism by which GM1 is involved in the neurodifferentiation process is still unknown, however, in vitro and in vivo evidences have suggested that the oligosaccharide portion of this ganglioside could be involved. Here, we report that, similarly to the entire GM1 molecule, its oligosaccharide II 3 NeuAc-Gg 4, rather than its ceramide (Cer) portion is responsible for the neurodifferentiation process by augmenting neurite elongation and increasing the neurofilament protein expression in murine neuroblastoma cells, Neuro2a. Conversely, asialo-GM1, GM2 and GM3 oligosaccharides are not effective in neurite elongation on Neuro2a cells, whereas the effect exerted by the Fuc-GM1 oligosaccharide (IV 2 αFucII 3 Neu5Ac-Gg 4 ) is similar to that exerted by GM1 oligosaccharide. The neurotrophic properties of GM1 oligosaccharide are exerted by activating the TrkA receptor and the following phosphorylation cascade. By photolabeling experiments performed with a nitrophenylazide containing GM1 oligosaccharide, labeled with tritium, we showed a direct interaction between the GM1 oligosaccharide and the extracellular domain of TrkA receptor. Moreover, molecular docking analyses confirmed that GM1 oligosaccharide binds the TrkA-nerve growth factor complex leading to a binding free energy of approx. -11.5 kcal/mol, acting as a bridge able to increase and stabilize the TrkA-nerve growth factor molecular interactions. © 2017 International Society for Neurochemistry.

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

    Directory of Open Access Journals (Sweden)

    Angelucci Adriano

    2010-11-01

    Full Text Available Abstract 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

  15. Increasing Antiproliferative Properties of Endocannabinoids in N1E-115 Neuroblastoma Cells through Inhibition of Their Metabolism

    Science.gov (United States)

    Hamtiaux, Laurie; Hansoulle, Laurie; Dauguet, Nicolas; Muccioli, Giulio G.; Gallez, Bernard; Lambert, Didier M.

    2011-01-01

    The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines – N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) - which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis. PMID:22046372

  16. Olfactory neuroblastoma

    International Nuclear Information System (INIS)

    Rashid, D.; Ahmed, B.; Malik, S.M.; Khan, M.

    2000-01-01

    Olfactory neuroblastoma/esthesioneuroblastoma in a rare malignant tumour of the olfactory neuroepithelium. This is a report of 5 cases managed over the last 10 years at Combined Military Hospital, Rawalpindi. Age of the patients at presentation ranged from 27 to 70 years. The main symptoms were unilateral nasal obstruction and intermittent epistaxis. The mean duration of symptoms at presentation was 11 months. Two patients were staged as B and 3 as C at presentation. The stage of the disease correlated with the duration of symptoms. All the cases were diagnosed on histopathology. Three were offered combination of surgery and radiotherapy. One patient received only surgical treatment and one patient received radiotherapy and chemotherapy. Combination of surgery and radiotherapy showed best results. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. 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

    Directory of Open Access Journals (Sweden)

    Ma H

    2016-06-01

    Full Text Available Hongda Ma,1 Yao Yao,2 Changli Wang,1 Liyu Zhang,3 Long Cheng,4 Yiren Wang,5 Tao Wang,6 Erguang Liang,6 Hui Jia,1 Qinong Ye,4 Mingxiao Hou,1 Fan Feng11Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang, 2Department of Pharmacy, Women & Infants Hospital of Zhengzhou, Zhengzhou, 3Shaanxi Institute of Pediatric Disease, Xi’an Children’s Hospital, Xi’an, 4Institute of Biotechnology, Chinese Military Medical Science Academy, Beijing, 5School of Life Science, Shenyang Pharmaceutical University, Shenyang, 6Institute of Toxicology and Pharmacology, Chinese Military Medical Science Academy, Beijing, People’s Republic of ChinaAbstract: 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.Keywords: neuroblastoma, endocrine-disrupting chemicals, environmental estrogens, bisphenol A and nonylphenol, proliferation and metastasis

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

    Directory of Open Access Journals (Sweden)

    Lilia Espada

    Full Text Available 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

  20. Impedance and ac conductivity studies of Ba (Pr1/2Nb1/2) O3 ceramic

    Indian Academy of Sciences (India)

    Home; Journals; Bulletin of Materials Science; Volume 36; Issue 4. Impedance and a.c. conductivity studies of ... Abstract. Impedance and electrical conduction studies of Ba(Pr1/2Nb1/2)O3 ceramic prepared through conventional ceramic fabrication technique are presented. The crystal symmetry, space group and unit cell ...

  1. Effects of exposure to DAMPS and GSM signals on ornithine decarboxylase (ODC) activity: II. SH-SY5Y human neuroblastoma cells.

    Science.gov (United States)

    Billaudel, Bernard; Taxile, Murielle; Poulletier de Gannes, Florence; Ruffie, Gilles; Lagroye, Isabelle; Veyret, Bernard

    2009-06-01

    An increase in Ornithine Decarboxylase (ODC) activity was reported in L929 murine fibroblast cells after exposure to a digital cellular telephone signal. This result was not confirmed by several other studies, including the one reported in a companion paper. As a partner in the Perform-B programme, we extended this study to human neuroblastoma cells (SH-SY5Y), using well-defined waveguide systems to imitate exposure to radiofrequency radiation (RFR): Digital Advanced Mobile Phone System (DAMPS) or Global System for Mobile communications (GSM) signals emitted by mobile phones. Human neuroblastoma cells (SH-SY5Y) were exposed at various Specific Absorption Rates (SAR) to DAMPS or GSM signals using different set-ups. Cell ODC activities were assayed using 14CO2 generation from 14C-labeled L-ornithine. SH-SY5Y cells were incubated for 20 hours, and were blindly exposed to 50 Hz-modulated DAMPS-835 or 217 Hz-modulated GSM-1800 for 8 or 24 h using Information Technologies in Society (IT'IS) waveguides equipped with fans. After cell lysis, ODC activity was determined using 14C-labeled L-ornithine. ODC activity was estimated by the 14CO2 generated from 14C-labeled L-ornithine, as generated d.p.m. 14CO2/h/mg protein. The results showed that, irrespective of the signal used (835 MHz/DAMPS, or 1800 MHz/GSM) and exposure conditions (duration and SAR), human SH-SY5Y neuroblastoma cells did not exhibit any alteration in ODC enzyme activity. This work did not show a significant effect of mobile phone RFR exposure on ODC activity in neuroblastoma cells (SH-SY5Y).

  2. 5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: role of the ERK pathway.

    Science.gov (United States)

    Marinova, Zoya; Walitza, Susanne; Grünblatt, Edna

    2013-07-01

    Disturbances of serotonergic signaling, including the serotonin 2A (5-HT2A) receptor, have been implicated in neuropsychiatric and neurodegenerative disorders. The aim of the present study was to characterize the effect of a 5-HT2A receptor agonist on cytotoxicity in a neuronal cell line and address the involved mechanism. HTR2A mRNA and protein expression in human neuroblastoma SK-N-SH cells was confirmed. Cells were subjected to serum deprivation and cell viability was monitored continuously with xCELLigence. In a dose-response study the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (25 nM to 5 μM) protected against serum deprivation cytotoxicity. The selective 5-HT2A receptor antagonist MDL 11,939, the general protein tyrosine kinase inhibitor genistein, and the extracellular signal-regulated kinase (ERK) pathway MEK inhibitor U0126, all attenuated DOI's protective effect. An antibody array suggested that 1 μM DOI affected phosphorylation of several tyrosine kinases. Western blot further confirmed that DOI transiently increased ERK phosphorylation, indicating its activation. Finally, protective concentrations of DOI increased cellular mitochondrial mass, an effect prevented by pretreatment with U0126. In conclusion, our results suggest that DOI protects SK-N-SH cells against serum deprivation through ERK pathway activation. They imply 5-HT2A receptor modulation as a potential target for neuroprotection. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. SPHK1/sphingosine kinase 1-mediated autophagy differs between neurons and SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Finkbeiner, Steven; Tsvetkov, Andrey S

    2016-08-02

    Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.

  4. Neuroblastoma and Its Zebrafish Model.

    Science.gov (United States)

    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.

  5. Protective effect of Pycnogenol in human neuroblastoma SH-SY5Y cells following acrolein-induced cytotoxicity.

    Science.gov (United States)

    Ansari, Mubeen A; Keller, Jeffrey N; Scheff, Stephen W

    2008-12-01

    Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

  6. [Pt(O,O'-acac)(γ-acac)(DMS)] versus cisplatin: apoptotic effects in B50 neuroblastoma cells.

    Science.gov (United States)

    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.

  7. Bcl-2 and N-Myc Coexpression Increases IGF-IR and Features of Malignant Growth in Neuroblastoma Cell Lines

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    Rama Jasty

    2001-01-01

    Full Text Available The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB2, Bcl2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR.

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

    International Nuclear Information System (INIS)

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C.

    2007-01-01

    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

  9. Neuroprotective Effects of Erucin against 6-Hydroxydopamine-Induced Oxidative Damage in a Dopaminergic-like Neuroblastoma Cell Line

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    Giorgio Cantelli-Forti

    2012-08-01

    Full Text Available Oxidative stress (OS contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson’s disease (PD. A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER, a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA. The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O2•− formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA.

  10. The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells

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    Long Gu

    2015-12-01

    Full Text Available Dysregulated expression of MYC family genes is a hallmark of many malignancies. Unfortunately, these proteins are not amenable to blockade by small molecules or protein-based therapeutic agents. Therefore, we must find alternative approaches to target MYC-driven cancers. Amplification of MYCN, a MYC family member, predicts high-risk neuroblastoma (NB disease. We have shown that R9-caPep blocks the interaction of PCNA with its binding partners and selectively kills human NB cells, especially those with MYCN amplification, and we now show the mechanism. We found elevated levels of DNA replication stress in MYCN-amplified NB cells. R9-caPep exacerbated DNA replication stress in MYCN-amplified NB cells and NB cells with an augmented level of MYC by interfering with DNA replication fork extension, leading to Chk1 dependence and susceptibility to Chk1 inhibition. We describe how these effects may be exploited for treating NB.

  11. Antitumor Effect of Burchellin Derivatives Against Neuroblastoma.

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    Kurita, Masahiro; Takada, Tomomi; Wakabayashi, Noriko; Asami, Satoru; Ono, Shinichi; Uchiyama, Taketo; Suzuki, Takashi

    2018-02-01

    Neuroblastoma is one of the most commonly encountered malignant solid tumors in the pediatric age group. We examined the antitumor effects of five burchellin derivatives against human neuroblastoma cell lines. We evaluated cytotoxicity by the MTT assay for four human neuroblastoma and two normal cell lines. We also performed analysis of the apoptotic induction effect by flow cytometry, and examined the expression levels of apoptosis- and cell growth-related proteins by western blot analysis. We found that one of the burchellin derivatives (compound 4 ) exerted cytotoxicity against the neuroblastoma cell lines. Compound 4 induced caspase-dependent apoptosis via a mitochondrial pathway. The apoptosis mechanisms induced by compound 4 involved caspase-3, -7 and -9 activation and poly (ADP-ribose) polymerase cleavage. In addition, compound 4 induced cell death through inhibition of the cell growth pathway (via extracellular signal-regulated kinase 1 and 2, AKT8 virus oncogene cellular homolog, and signal transducer and activator of transcription 3). Compound 4 exerted cellular cytotoxicity against neuroblastoma cells via induction of caspase-dependent apoptosis, and may offer promise for further development as a useful drug for the treatment of advanced neuroblastoma. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  12. High dose melphalan in the treatment of advanced neuroblastoma: results of a randomised trial (ENSG-1) by the European Neuroblastoma Study Group

    NARCIS (Netherlands)

    Pritchard, Jon; Cotterill, Simon J.; Germond, Shirley M.; Imeson, John; de Kraker, Jan; Jones, David R.

    2005-01-01

    High dose myeloablative chemotherapy ("megatherapy"), with haematopoietic stem cell support, is now widely used to consolidate response to induction chemotherapy in patients with advanced neuroblastoma. In this study (European Neuroblastoma Study Group, ENSG1), the value of melphalan myeloablative

  13. Immunosuppressive microenvironment in neuroblastoma

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    Vito ePistoia

    2013-06-01

    Full Text Available According to the cancer immunoediting model, the interplay between tumor cells and the host immune system is crucial for the control of tumor growth. NB is a pediatric tumor that presents with metastatic disease at diagnosis in about 50% of the cases, the majority of which have poor prognosis. In this Review article, immune escape pathways adopted by human neuroblastoma (NB cells are reviewed. These include intrinsic defects of tumor cells such impaired expression of the HLA class I related antigen processing machinery and functional alterations of the tumor microenvironment induced by NB cell-derived immunosuppressive molecules as MICA and HLA-G. Finally, examples of therapeutic interventions targeting the tumor microenvironment are discussed to emphasize the concept that successful cancer treatment may be achieved using this strategy.

  14. Selegiline increases heme oxygenase-1 expression and the cytotoxicity produced by dopamine treatment of neuroblastoma SK-N-SH cells

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    C.R.M. Rieder

    2004-07-01

    Full Text Available Increased dopamine catabolism may be associated with oxidative stress and neuronal cell death in Parkinson's disease. The present study was carried out to examine the effect of dopamine on the expression of heme oxygenase-1 and -2 (HO-1 and HO-2 in human neuroblastomas (SK-N-SH cell line and the effects of selegiline and antioxidants on this expression. Cells were kept with close control of pH and were incubated with varying concentrations of dopamine (0.1-100 µM for 24 h. HO-1 and HO-2 cDNA probes were prepared by reverse transcription-polymerase chain reaction amplification. The mRNA expression of HO-1 and HO-2 was measured by Northern blot analysis. The levels of HO-1 mRNA increased after dopamine treatment, in a dose-dependent manner, in all cell lines studied, whereas levels of the two HO-2 transcripts did not. The HO-1 and HO-2 protein expression was analyzed by Western blotting. HO-1 protein was undetectable in untreated SK-N-SH cells and increased after treatment with dopamine. In contrast, the HO-2 protein (36 kDa was detected in untreated cells and the levels did not change as a result of treatment. alpha-Tocopherol (10-100 µM and ascorbic acid (100 µM did not attenuate the effects of dopamine. Selegiline (10 µM produced significant increase (P < 0.01 in the induction of HO-1 by dopamine (more than six times the control values. The increased expression of HO-1 following dopamine treatment indicates that dopamine produces oxidative stress in this cell line.

  15. Evidence of vanillin binding to CAMKIV explains the anti-cancer mechanism in human hepatic carcinoma and neuroblastoma cells.

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    Naz, Huma; Tarique, Mohd; Khan, Parvez; Luqman, Suaib; Ahamad, Shahzaib; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2018-01-01

    Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.

  16. Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

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    Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

    2003-05-01

    Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

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

    International Nuclear Information System (INIS)

    Chen Yan; Yu Min; Jones, Dean P.; Greenamyre, J. Timothy; Cai Jiyang

    2006-01-01

    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

  18. Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy.

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    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.

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

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    Alba Garcimartín

    2017-08-01

    Human neuroblastoma SH-SY5Y cells were treated with H2O2 (1–1000 µM for 24 h and AChE activity and AChE and cytochrome c levels were evaluated. AChE activity was strongly increased from 1 µM to 1000 µM of H2O2. The results of the kinetic study showed that H2O2 affected Vmax but not Km; and also that H2O2 changed the sigmoid kinetic observed in control samples to hyperbolic kinetic. Thus, results suggest that H2O2 acts as an allosteric activators. In addition, H2O2, (100–1000 µM reduced the total AChE content and modified its isoform profile (mainly 50-, 70-, and 132-kDa·H2O2 from 100 µM to 1000 µM induced cytochrome c release confirming cell death by apoptosis. All these results together suggest: a the involvement of oxidative stress in the imbalance of AChE; and b treatment with antioxidant agents may be a suitable strategy to protect cholinergic system alterations promoted by oxidative stress.

  20. Graphene Oxide–Silver Nanoparticles Nanocomposite Stimulates Differentiation in Human Neuroblastoma Cancer Cells (SH-SY5Y

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    Sangiliyandi Gurunathan

    2017-11-01

    Full Text Available Recently, graphene and graphene related nanocomposite receive much attention due to high surface-to-volume ratio, and unique physiochemical and biological properties. The combination of metallic nanoparticles with graphene-based materials offers a promising method to fabricate novel graphene–silver hybrid nanomaterials with unique functions in biomedical nanotechnology, and nanomedicine. Therefore, this study was designed to prepare graphene oxide (GO silver nanoparticles (AgNPs nanocomposite (GO-AgNPs containing two different nanomaterials in single platform with distinctive properties using luciferin as reducing agents. In addition, we investigated the effect of GO-AgNPs on differentiation in SH-SY5Y cells. The synthesized GO-AgNPs were characterized by ultraviolet-visible absorption spectroscopy (UV-vis, X-ray diffraction (XRD, scanning electron microscopy (SEM, transmission electron microscopy (TEM and Raman spectroscopy. The differentiation was confirmed by series of cellular and biochemical assays. The AgNPs were distributed uniformly on the surface of graphene oxide with an average size of 25 nm. As prepared GO-AgNPOs induces differentiation by increasing the expression of neuronal differentiation markers and decreasing the expression of stem cell markers. The results indicated that the redox biology involved the expression of various signaling molecules, which play an important role in differentiation. This study suggests that GO-AgNP nanocomposite could stimulate differentiation of SH-SY5Y cells. Furthermore, understanding the mechanisms of differentiation of neuroblastoma cells could provide new strategies for cancer and stem cell therapies. Therefore, these studies suggest that GO-AgNPs could target specific chemotherapy-resistant cells within a tumor.

  1. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA.

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    Mari Gotoh

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

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    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.

  3. Identification of phosphoproteins as possible differentiation markers in all-trans-retinoic acid-treated neuroblastoma cells.

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    Giorgia Mandili

    Full Text Available BACKGROUND: Neuroblastic tumors account for 9-10% of pediatric tumors and neuroblastoma (NB is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special, includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied. CONCLUSIONS/SIGNIFICANCE: Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA and reticulocalbin-1 (RCN1, which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated. Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.

  4. Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a.

    Science.gov (United States)

    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.

  5. Assessing Proteinase K Resistance of Fish Prion Proteins in a Scrapie-Infected Mouse Neuroblastoma Cell Line

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    Evgenia Salta

    2014-11-01

    Full Text Available The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrPC, into an aberrant and partially proteinase K resistant isoform, PrPSc. Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK, as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

  6. DJ1 Expression Downregulates in Neuroblastoma Cells (SK-N-MC Chronically Exposed to HIV-1 and Cocaine.

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    Upal eRoy

    2015-07-01

    Full Text Available Background: HIV-associated neurological disorder (HAND has long been recognized as a consequence of Human Immunodeficiency Virus (HIV infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson’s disease in regulating dopamine transmission and reactive oxygen species (ROS production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder.Methods: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC. Gene expression and protein analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry.Results: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine.Conclusion: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1and/or cocaine indicating oxidative stress level of dopamine neurons.

  7. ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

    Science.gov (United States)

    Bianchi, G; Vuerich, M; Pellegatti, P; Marimpietri, D; Emionite, L; Marigo, I; Bronte, V; Di Virgilio, F; Pistoia, V; Raffaghello, L

    2014-03-20

    Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b(+)/Gr-1(+) cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1(+) population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

  8. Ciliary neurotrophic factor protects SH-SY5Y neuroblastoma cells against Aβ1-42-induced neurotoxicity via activating the JAK2/STAT3 axis.

    Science.gov (United States)

    Wang, Ke; Xie, Minhao; Zhu, Ling; Zhu, Xue; Zhang, Kai; Zhou, Fanfan

    2015-01-01

    The neurotoxicity of aggregated amyloid beta (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD), which leads to neuronal cell damage by inducing oxidative stress and consequently triggering cell apoptosis. Recently, Aβ-dependent inactivation of the Janus tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway was found to play a critical role in the memory impairment related to AD. Previous research indicated that JAK2/STAT3 axis inactivation might be the result of aberrant reactive oxygen species (ROS) generation induced by Aβ in neurons. As the JAK2/STAT3 axis is a major transducer of ciliary neurotrophic factor (CNTF)-mediated neuroprotective activity, this study extensively evaluated whether activation of the JAK2/STAT3 axis by CNTF was responsible for the neuroprotective effect of this protein against Aβ1-42-induced cytotoxicity, oxidative injury and cell apoptosis in human SH-SY5Y neuroblastoma cells. Our data showed that CNTF could attenuate or restore cell injury induced by Aβ1-42 in human SH-SY5Y neuroblastoma cells through activating the JAK2/STAT3 signaling pathway. Furthermore, CNTF strikingly prevented Aβ1-42-induced mitochondrial dysfunction and activation of mitogen-activated protein kinases (MAPKs), an effect that could be potently attenuated by the specific JAK2 inhibitor AG490. In summary, this study confirmed the detailed mechanism accounting for CNTF's protective effect against Aβ1-42-induced cytotoxic events in human SH-SY5Y neuroblastoma cells - information which might significantly contribute to better understanding of the mechanism of action of CNTF as well as providing a novel target in AD therapy.

  9. Large-scale tracking and classification for automatic analysis of cell migration and proliferation, and experimental optimization of high-throughput screens of neuroblastoma cells.

    Science.gov (United States)

    Harder, Nathalie; Batra, Richa; Diessl, Nicolle; Gogolin, Sina; Eils, Roland; Westermann, Frank; König, Rainer; Rohr, Karl

    2015-06-01

    Computational approaches for automatic analysis of image-based high-throughput and high-content screens are gaining increased importance to cope with the large amounts of data generated by automated microscopy systems. Typically, automatic image analysis is used to extract phenotypic information once all images of a screen have been acquired. However, also in earlier stages of large-scale experiments image analysis is important, in particular, to support and accelerate the tedious and time-consuming optimization of the experimental conditions and technical settings. We here present a novel approach for automatic, large-scale analysis and experimental optimization with application to a screen on neuroblastoma cell lines. Our approach consists of cell segmentation, tracking, feature extraction, classification, and model-based error correction. The approach can be used for experimental optimization by extracting quantitative information which allows experimentalists to optimally choose and to verify the experimental parameters. This involves systematically studying the global cell movement and proliferation behavior. Moreover, we performed a comprehensive phenotypic analysis of a large-scale neuroblastoma screen including the detection of rare division events such as multi-polar divisions. Major challenges of the analyzed high-throughput data are the relatively low spatio-temporal resolution in conjunction with densely growing cells as well as the high variability of the data. To account for the data variability we optimized feature extraction and classification, and introduced a gray value normalization technique as well as a novel approach for automatic model-based correction of classification errors. In total, we analyzed 4,400 real image sequences, covering observation periods of around 120 h each. We performed an extensive quantitative evaluation, which showed that our approach yields high accuracies of 92.2% for segmentation, 98.2% for tracking, and 86.5% for

  10. Protective effects of astaxanthin on 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Ikeda, Yasutaka; Tsuji, Shinji; Satoh, Akira; Ishikura, Masaharu; Shirasawa, Takuji; Shimizu, Takahiko

    2008-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although understanding of the pathogenesis of PD remains incomplete, increasing evidence from human and animal studies has suggested that oxidative stress is an important mediator in its pathogenesis. Astaxanthin (Asx), a potent antioxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress-related diseases. This study examined the protective effects of Asx on 6-hydroxydopamine (6-OHDA)-induced apoptosis in the human neuroblastoma cell line SH-SY5Y. Pre-treatment of SH-SY5Y cells with Asx suppressed 6-OHDA-induced apoptosis in a dose-dependent manner. In addition, Asx strikingly inhibited 6-OHDA-induced mitochondrial dysfunctions, including lowered membrane potential and the cleavage of caspase 9, caspase 3, and poly(ADP-ribose) polymerase. In western blot analysis, 6-OHDA activated p38 MAPK, c-jun NH(2)-terminal kinase 1/2, and extracellular signal-regulated kinase 1/2, while Asx blocked the phosphorylation of p38 MAPK but not c-jun NH(2)-terminal kinase 1/2 and extracellular signal-regulated kinase 1/2. Pharmacological approaches showed that the activation of p38 MAPK has a critical role in 6-OHDA-induced mitochondrial dysfunctions and apoptosis. Furthermore, Asx markedly abolished 6-OHDA-induced reactive oxygen species generation, which resulted in the blockade of p38 MAPK activation and apoptosis induced by 6-OHDA treatment. Taken together, the present results indicated that the protective effects of Asx on apoptosis in SH-SY5Y cells may be, at least in part, attributable to the its potent antioxidative ability.

  11. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Claudia Capitini

    Full Text Available 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.

  12. Damage of neuroblastoma cell SH-SY5Y mediated by MPP+ inhibits proliferation of T-cell leukemia Jurkat by co-culture system.

    Science.gov (United States)

    Wang, Fuli; Awan, Umer Farooq; Wang, Yuanyuan; Wang, Luna; Qing, Hong; Ma, Hong; Deng, Yulin

    2014-06-13

    The adaptive immune system has implications in pathology of Parkinson's disease (PD). Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+) for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM) arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2) and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM) induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

  13. Damage of Neuroblastoma Cell SH-SY5Y Mediated by MPP+ Inhibits Proliferation of T-Cell Leukemia Jurkat by Co-Culture System

    Directory of Open Access Journals (Sweden)

    Fuli Wang

    2014-06-01

    Full Text Available The adaptive immune system has implications in pathology of Parkinson’s disease (PD. Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+ for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2 and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

  14. A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ2

    Directory of Open Access Journals (Sweden)

    Dorothy I. Wallace

    2016-01-01

    Full Text Available 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.

  15. The association of congenital neuroblastoma and congenital heart disease

    International Nuclear Information System (INIS)

    Bellah, R.; D'Andrea, A.; Children's Hospital, Boston, MA; Darillis, E.; Fellows, K.E.

    1989-01-01

    Several authors have reported an association between neuroblastoma and congenital heart disease; others contend that, unlike specific wellknown associations between malignancy and congenital defects (Wilm's tumor and aniridia, leukemia and Down's syndrome), no real relationship exists. We present three cases of cyanotic congenital heart disease in which subclinical neuroblastoma was found. We speculate that abnormal neural crest cell migration and development may be a common link between cardiac malformations and congenital neuroblastoma. (orig.)

  16. Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

    Science.gov (United States)

    Grigoryan, Rita S; Yang, Bo; Keshelava, Nino; Barnhart, Jerry R; Reynolds, C Patrick

    2007-11-01

    The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs. Copyright (c) 2007 International Society for Analytical Cytology.

  17. Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

    NARCIS (Netherlands)

    Bate-Eya, Laurel T.; Ebus, Marli E.; Koster, Jan; den Hartog, Ilona J. M.; Zwijnenburg, Danny A.; Schild, Linda; van der Ploeg, Ida; Dolman, M. Emmy M.; Caron, Huib N.; Versteeg, Rogier; Molenaar, Jan J.

    2014-01-01

    Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of

  18. Rapid agonist-induced loss of sup 125 I-. beta. -endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Cone, R.I.; Lameh, J.; Sadee, W. (Univ. of California, San Francisco (United States))

    1991-01-01

    The authors have measured {mu} and {delta} opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of {sup 125}I-{beta}-endorphin ({beta}E) as a tracer, together with {beta}E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface {mu} and {delta} opioid receptor sites. Labeling was at {delta} sites in NG108-15 cells and predominantly at {mu} sites in SK-N-SH cells. Pretreatment with the {mu} and {delta} agonist, DADLE, caused a rapid loss of cell surface {delta} receptor sites in NG108-15 cells, but failed to reduce significantly {mu} receptor density in SK-N-SH cells.

  19. Protection of prenylated flavonoids from Mori Cortex Radicis (Moraceae) against nitric oxide-induced cell death in neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Lee, Hak Ju; Lyu, Da Hyun; Koo, Uk; Nam, Kung-Woo; Hong, Seong Su; Kim, Kem Ok; Kim, Kyeong Ho; Lee, Dongho; Mar, Woongchon

    2012-01-01

    Seven prenylated flavanoids, licoflavone C (1), cyclomulberrin (2), neocyclomorusin (3), sanggenon I (4), morusin (5), kuwanon U (6) and kuwanon E (7), and three 2-arylbenzofurans, moracin P (8), moracin O (9), and mulberrofuran Q (10) were isolated from the MeOH extract of Mori Cortex Radicis. Among these, compounds 2-7 enhanced cell viability in a dose-dependent manner against sodium nitroprusside-induced cell death in neuroblastoma SH-SY5Y cells, which was measured by MTT reduction assay (EC(50) values of 4.4, 5.6, 8.0, 6.4, 8.7, and 11.9 μg/mL, respectively). Among 10 compounds, C-3 prenylated flavones (2, 3, and 5) and prenylated flavanones (4, 6, and 7) showed cell protection. However, compound 1 which lacks the prenyl group at C-3 and three 2-arylbenzofurans (8-10) did not show protective effect. The order of cell protection was as follow: C-3 prenylated flavones (2, 3, and 5) > prenylated flavanones (4, 6, and 7) > 2-arylbenzofurans (8-10) and flavone (1). From this result, we show that some prenylated flavones and flavanones might protect neuronal cells against nitrosative stress-mediated cell death. Even though further evaluations are necessary in vitro and in vivo study, we carefully suggest that some prenylated flavonoids from Mori Cortex Radicis might protect neuronal cells from neurodegenerative diseases.

  20. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Science.gov (United States)

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. PMID:26357513

  1. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Prachya Janhom

    2015-01-01

    Full Text Available In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn. act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD, has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

  2. Neuroprotective Effects of Alpha-Mangostin on MPP(+)-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP(+)-induced apoptosis. The effects of alpha-mangostin on MPP(+)-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP(+) on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP(+). Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP(+). The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP(+) treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP(+)-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

  3. Regulation of Tyrosine Hydroxylase Activity in Cultured Mouse Neuroblastoma Cells: Elevation Induced by Analogs of Adenosine 3′:5′-Cyclic Monophosphate

    Science.gov (United States)

    Waymire, J. C.; Weiner, N.; Prasad, K. N.

    1972-01-01

    Mouse neuroblastoma cells in culture have been used as a model for the study of the mechanism by which activities of tyrosine hydroxylase (EC 1.14.3.a) are regulated in sympathetic tissue. The activity of tyrosine hydroxylase in cultured cells drops to barely detectable activities after 1 week and remains low for months in culture in the uncloned cell line of neuroblastoma. Activity in an adrenergic clone isolated from the uncloned line has about 20% of the activity of the fresh grated tumor cell. N6, O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate causes a concentration and time-dependent increase in enzyme activity in both the cloned and uncloned cell lines. Enzyme activity is elevated by other stable analogs of adenosine 3′:5′-cyclic monophosphate, notably the N6-monobutyryl, 8-aminomethyl, and 8-methylthio derivatives of the cyclic nucleotide; by the inhibitor of cyclic nucleotide phosphodiesterase, papaverine; and by sodium butyrate. Changes in cell morphology and tyrosine hydroxylase activity are shown not to be necessarily related. PMID:4403308

  4. Overview and recent advances in the treatment of neuroblastoma.

    Science.gov (United States)

    Whittle, Sarah B; Smith, Valeria; Doherty, Erin; Zhao, Sibo; McCarty, Scott; Zage, Peter E

    2017-04-01

    Children with neuroblastoma have widely divergent outcomes, ranging from cure in >90% of patients with low risk disease to neuroblastoma, allowing for more accurate risk stratification and treatment reduction in many cases, although newer treatment strategies for children with high-risk and relapsed neuroblastoma are needed to improve outcomes. Areas covered: Neuroblastoma epidemiology, diagnosis, risk stratification, and recent advances in treatment of both newly diagnosed and relapsed neuroblastoma. Expert commentary: The identification of newer tumor targets and of novel cell-mediated immunotherapy agents may lead to novel therapeutic approaches, and clinical trials for regimens designed to target individual genetic aberrations in tumors are underway. A combination of therapeutic modalities will likely be required to improve survival and cure rates for patients with high-risk neuroblastoma.

  5. Treatment of extradural paraspinal neuroblastoma with an intraspinal component

    International Nuclear Information System (INIS)

    Ho, K.S.Y.; Wara, W.M.; Ablin, A.R.

    1982-01-01

    Neuroblastoma originates from neural crest cells and can be found wherever sympathetic neural tissue is normally located. When the tumor arises from a paraspinal sympathetic ganglion, it has a propensity to extend through the intervertebral foramina, producing an extradural paraspinal neuroblastoma with an interspinal component (''dumbell'' neuroblastoma) which may result in spinal cord compression. The records of all children with neuroblastomas referred to the UCSF Department of Radiation Oncology and the Division of Pediatric Oncology from January 1, 1970, to December 31, 1979, are reviewed in this report. Patients who at initial presentation had a ''dumbell'' neuroblastoma were selected for study. Neuroblastoma was diagnosed histologically in all patients except one. Disease-free interval and length of survival was measured from the date of completion of radiotherapy, mostly after surgery. The results of diagnostic X-rays and laboratory studies are shown. Radiotherapeutic doses and results are tabulated. (Auth.)

  6. Impact of plant extracts tested in attention-deficit/hyperactivity disorder treatment on cell survival and energy metabolism in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Schmidt, Andreas Johannes; Krieg, Jürgen-Christian; Hemmeter, Ulrich Michael; Kircher, Tilo; Schulz, Eberhard; Clement, Hans-Willi; Heiser, Philip

    2010-10-01

    Plant extracts such as Hypericum perforatum and Pycnogenol have been tested as alternatives to the classical ADHD drugs. It has been possible to describe neuroprotective effects of such plant extracts. A reduction of ADHD symptoms could be shown in clinical studies after the application of Pycnogenol, which is a pine bark extract. The impacts of the standardized herbal extracts Hypericum perforatum, Pycnogenol and Enzogenol up to a concentration of 5000 ng/mL on cell survival and energy metabolism in human SH-SY5Y neuroblastoma cells has been investigated in the present examination. Hypericum perforatum significantly decreased the survival of cells after treatment with a concentration of 5000 ng/mL, whereas lower concentrations exerted no significant effects. Pycnogenol( induced a significant increase of cell survival after incubation with a concentration of 32.25 ng/mL and a concentration of 250 ng/mL. Other applied concentrations of Pycnogenol failed to exert significant effects. Treatment with Enzogenol did not lead to significant changes in cell survival.Concerning energy metabolism, the treatment of cells with a concentration of 5000 ng/mL Hypericum perforatum led to a significant increase of ATP levels, whereas treatment with a concentration of 500 ng/mL had no significant effect. Incubation of cells with Pycnogenol and Enzogenol exerted no significant effects.None of the tested substances caused any cytotoxic effect when used in therapeutically relevant concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

  7. Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system.

    Science.gov (United States)

    Nakamura, Saki; Nakanishi, Ayumi; Takazawa, Minami; Okihiro, Shunsuke; Urano, Shiro; Fukui, Koji

    2016-01-01

    Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOX TM increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

  8. Pulsed electromagnetic field (PEMF) prevents pro-oxidant effects of H2O2 in SK-N-BE(2) human neuroblastoma cells.

    Science.gov (United States)

    Falone, Stefano; Marchesi, Nicoletta; Osera, Cecilia; Fassina, Lorenzo; Comincini, Sergio; Amadio, Marialaura; Pascale, Alessia

    2016-05-01

    Purpose The redox milieu, together with reactive oxygen species (ROS) accumulation, may play a role in mediating some biological effects of extremely-low-frequency electromagnetic fields (ELF-EMF). Some of us have recently reported that a pulsed EMF (PEMF) improves the antioxidant response of a drug-sensitive human neuroblastoma SH-SY5Y cell line to pro-oxidants. Since drug resistance may affect cell sensitivity to redox-based treatments, we wanted to verify whether drug-resistant human neuroblastoma SK-N-BE(2) cells respond to a PEMF in a similar fashion. Materials and methods SK-N-BE(2) cells were exposed to repeated 2 mT, 75 Hz PEMF (15 min each, repeated 3 times over 5 days), and ROS production, Mn-dependent superoxide dismutase (MnSOD)-based antioxidant protection and viability were assessed after 10 min or 30 min 1 mM hydrogen peroxide. Sham controls were kept at the same time in identical cell culture incubators. Results The PEMF increased the MnSOD-based antioxidant protection and reduced the ROS production in response to a pro-oxidant challenge. Conclusions Our work might lay foundation for the development of non-invasive PEMF-based approaches aimed at elevating endogenous antioxidant properties in cellular or tissue models.

  9. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    Liu, P.-S.; Chiung, Y.-M.; Kao, Y.-Y.

    2006-01-01

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

  12. Comprehensive SNP array study of frequently used neuroblastoma cell lines; copy neutral loss of heterozygosity is common in the cell lines but uncommon in primary tumors

    Directory of Open Access Journals (Sweden)

    Kogner Per

    2011-09-01

    Full Text Available Abstract Background Copy neutral loss of heterozygosity (CN-LOH refers to a special case of LOH occurring without any resulting loss in copy number. These alterations is sometimes seen in tumors as a way to inactivate a tumor suppressor gene and have been found to be important in several types of cancer. Results We have used high density single nucleotide polymorphism arrays in order to investigate the frequency and distribution of CN-LOH and other allelic imbalances in neuroblastoma (NB tumors and cell lines. Our results show that the frequency of these near-CN-LOH events is significantly higher in the cell lines compared to the primary tumors and that the types of CN-LOH differ between the groups. We also show that the low-risk neuroblastomas that are generally considered to have a "triploid karyotype" often present with a complex numerical karyotype (no segmental changes with 2-5 copies of each chromosome. Furthermore a comparison has been made between the three related cell lines SK-N-SH, SH-EP and SH-SY5Y with respect to overall genetic aberrations, and several aberrations unique to each of the cell lines has been found. Conclusions We have shown that the NB tumors analyzed contain several interesting allelic imbalances that would either go unnoticed or be misinterpreted using other genome-wide techniques. These findings indicate that the genetics underlying NB might be even more complex than previously known and that SNP arrays are important analysis tools. We have also showed that these near-CN-LOH events are more frequently seen in NB cell lines compared to NB tumors and that a set of highly related cell lines have continued to evolve secondary to the subcloning event. Taken together our analysis highlights that cell lines in many cases differ substantially from the primary tumors they are thought to represent, and that caution should be taken when drawing conclusions from cell line-based studies.

  13. Intratumoral Delivery of Interferonγ-Secreting Mesenchymal Stromal Cells Repolarizes Tumor-Associated Macrophages and Suppresses Neuroblastoma Proliferation In Vivo.

    Science.gov (United States)

    Relation, Theresa; Yi, Tai; Guess, Adam J; La Perle, Krista; Otsuru, Satoru; Hasgur, Suheyla; Dominici, Massimo; Breuer, Christopher; Horwitz, Edwin M

    2018-02-12

    Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFNγ) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFNγ therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFNγ directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFNγ without increased systemic concentration or toxicity. The TME-specific IFNγ reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model. Stem Cells 2018. © AlphaMed Press 2018.

  14. Alpha-tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: potential therapy of malignancies of the nervous system?

    Czech Academy of Sciences Publication Activity Database

    Swettenham, E.; Witting, P. K.; Salvatore, B.A.; Neužil, Jiří

    2005-01-01

    Roč. 94, č. 5 (2005), s. 1448-1456 ISSN 0022-3042 Institutional research plan: CEZ:AV0Z50520514 Keywords : apoptosis * neuroblastoma * vitamin E analogues Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.604, year: 2005

  15. Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells

    International Nuclear Information System (INIS)

    Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschlaeger, Markus

    2006-01-01

    Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status

  16. Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine.

    Science.gov (United States)

    Boarder, M R; Marriott, D; Adams, M

    1986-12-30

    The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.

  17. Proteomic Profiling of Neuroblastoma Cells Adhesion on Hyaluronic Acid-Based Surface for Neural Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Ming-Hui Yang

    2016-01-01

    Full Text Available The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.

  18. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

    Directory of Open Access Journals (Sweden)

    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  19. Additive effects of vorinostat and MLN8237 in pediatric leukemia, medulloblastoma, and neuroblastoma cell lines.

    Science.gov (United States)

    Muscal, Jodi A; Scorsone, Kathleen A; Zhang, Linna; Ecsedy, Jeffrey A; Berg, Stacey L

    2013-02-01

    Histone deacetylase (HDAC) inhibitors, such as vorinostat, decrease Aurora kinase activity by a variety of mechanisms. Vorinostat and MLN8237, a selective Aurora A kinase inhibitor, disrupt the spindle assembly and the mitotic checkpoint at different points, suggesting that the combination could have increased antitumor activity. The purpose of this study was to determine the cytotoxicity of vorinostat and MLN8237 in pediatric tumor cell lines. Cell survival was measured after 72 h of drug treatment using a modified methyl tetrazolium assay. For drug combination experiments, cells were exposed to medium alone (controls), single drug alone, or to different concentrations of the combination of the two drugs, for a total of 36 concentration pairs per plate. The interaction of the drug combination was analyzed using the universal response surface approach. The cells express the target of MLN8237, Aurora A. For each cell line, the single agent IC(50) for MLN8237 and for vorinostat was in the clinically relevant range. Both drugs inhibited cell survival in a concentration-dependent fashion. At concentrations of MLN8237 exceeding approximately 1 μM, there was a paradoxical increase in viability signal in all three lines that may be explained by inhibition of Aurora B kinase. The combination of MLN8237 and vorinostat showed additive cytotoxicity in all three cell lines and nearly abrogated the paradoxical increase in survival noted at high single-agent MLN8237 concentrations. MLN8237 and vorinostat are active in vitro against cancer cell lines. These results provide important preclinical support for the development of future clinical studies of MLN8237and vorinostat.

  20. Intrarenal neuroblastoma mimics Wilms' tumor

    International Nuclear Information System (INIS)

    Muniz, Maria T. Cartaxo; Soares, Andrezza B.; Freitas, Elizabete M.; Araujo, Marcela; Pureza, Leda M.M.; Morais, Adriana; Antunes, Consuelo; Salles, Terezinha de J. Marques; Borges, Josenilda C.; Morais, Vera L.L. de; Romualdo Filho, Jose; Magalhaes, Mario H.

    2005-01-01

    This work reports the case history of a child with intrarenal neuroblastoma, initially diagnosed as Wilms' tumor. The patient, a one year and three months old girl, presented a hard abdominal mass on the left flank that extended to the meso gastric region, plus fever and paleness. The ultrasound of the entire abdomen revealed an intrarenal mass. Biopsy with fine needle in many points of the tumor revealed Wilms' tumor. The scarcely of the material, however, made immunohistoquemistry impossible at that moment. Because of the child's severe condition the SIOP protocol was started. As no clinical response was observed, an exploratory laparotomy was indicated with partial resection of the tumor and bone marrow aspiration (MO). The histopathologic study revealed a malignant neoplasia of small cells, poorly differentiated. IHQ was negative for WT-1 and positive for NB-84, synaptofisin, cromogranine. N-myc amplification was observed by molecular biology. The bone marrow aspiration identified metastatic small round cells infiltration. Intrarenal neuroblastoma is a rare entity that clinically and radiographically resembles Wilms' tumor. The objective of this case report is to show the importance of immunohistochemical and molecular analysis in the diagnosis of intrarenal neuroblastoma. (author)

  1. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

    Science.gov (United States)

    Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

    2017-05-01

    β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

  2. Neuroblastoma treatment in the post-genomic era.

    Science.gov (United States)

    Esposito, Maria Rosaria; Aveic, Sanja; Seydel, Anke; Tonini, Gian Paolo

    2017-02-08

    Neuroblastoma is an embryonic malignancy of early childhood originating from neural crest cells and showing heterogeneous biological, morphological, genetic and clinical characteristics. The correct stratification of neuroblastoma patients within risk groups (low, intermediate, high and ultra-high) is critical for the adequate treatment of the patients.High-throughput technologies in the Omics disciplines are leading to significant insights into the molecular pathogenesis of neuroblastoma. Nonetheless, further study of Omics data is necessary to better characterise neuroblastoma tumour biology. In the present review, we report an update of compounds that are used in preclinical tests and/or in Phase I-II trials for neuroblastoma. Furthermore, we recapitulate a number of compounds targeting proteins associated to neuroblastoma: MYCN (direct and indirect inhibitors) and downstream targets, Trk, ALK and its downstream signalling pathways. In particular, for the latter, given the frequency of ALK gene deregulation in neuroblastoma patients, we discuss on second-generation ALK inhibitors in preclinical or clinical phases developed for the treatment of neuroblastoma patients resistant to crizotinib.We summarise how Omics drive clinical trials for neuroblastoma treatment and how much the research of biological targets is useful for personalised medicine. Finally, we give an overview of the most recent druggable targets selected by Omics investigation and discuss how the Omics results can provide us additional advantages for overcoming tumour drug resistance.

  3. Bee venom protects SH-SY5Y human neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptotic cell death.

    Science.gov (United States)

    Doo, Ah-Reum; Kim, Seung-Nam; Kim, Seung-Tae; Park, Ji-Yeun; Chung, Sung-Hyun; Choe, Bo-Young; Chae, Younbyoung; Lee, Hyejung; Yin, Chang-Shik; Park, Hi-Joon

    2012-01-06

    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by progressive selective loss of dopaminergic neurons in the substantia nigra. Recently, bee venom was reported to protect dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced mice PD model, however, the underlying mechanism is not fully understood. The objective of the present study is to investigate the neuroprotective mechanism of bee venom against Parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP(+)), in SH-SY5Y human neuroblastoma cells. Our results revealed that bee venom pretreatment (1-100 ng/ml) increased the cell viability and decreased apoptosis assessed by DNA fragmentation and caspase-3 activity assays in MPP(+)-induced cytotoxicity in SH-SY5Y cells. Bee venom increased the anti-apoptotic Bcl-2 expression and decreased the pro-apoptotic Bax, cleaved PARP expressions. In addition, bee venom prevented the MPP(+)-induced suppression of Akt phosphorylation, and the neuroprotective effect of bee venom against MPP(+)-induced cytotoxicity was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. These results suggest that the anti-apoptotic effect of bee venom is mediated by the cell survival signaling, the PI3K/Akt pathway. These results provide new evidence for elucidating the mechanism of neuroprotection of bee venom against PD. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Curcumin Regulates Low-Linear Energy Transfer γ-Radiation-Induced NFκB-Dependent Telomerase Activity in Human Neuroblastoma Cells

    International Nuclear Information System (INIS)

    Aravindan, Natarajan; Veeraraghavan, Jamunarani; Madhusoodhanan, Rakhesh; Herman, Terence S.; Natarajan, Mohan

    2011-01-01

    Purpose: We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. Methods and Materials: SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. Results: Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival. Conclusions: These results

  5. Effects of formaldehyde on mitochondrial dysfunction and apoptosis in SK-N-SH neuroblastoma cells.

    Science.gov (United States)

    Zerin, Tamanna; Kim, Jin-Sun; Gil, Hyo-Wook; Song, Ho-Yeon; Hong, Sae-Yong

    2015-12-01

    Methanol ingestion is neurotoxic in humans due to its metabolites, formaldehyde and formic acid. Here, we compared the cytotoxicity of methanol and its metabolites on different types of cells. While methanol and formic acid did not affect the viability of the cells, formaldehyde (200-800 μg/mL) was strongly cytotoxic in all cell types tested. We investigated the effects of formaldehyde on oxidative stress, mitochondrial respiratory functions, and apoptosis on the sensitive neuronal SK-N-SH cells. Oxidative stress was induced after 2 h of formaldehyde exposure. Formaldehyde at a concentration of 400 μg/mL for 12 h of treatment greatly reduced cellular adenosine triphosphate (ATP) levels. Confocal microscopy indicated that the mitochondrial membrane potential (MMP) was dose-dependently reduced by formaldehyde. A marked and dose-dependent inhibition of mitochondrial respiratory enzymes, viz., NADH dehydrogenase (complex I), cytochrome c oxidase (complex IV), and oxidative stress-sensitive aconitase was also detected following treatment with formaldehyde. Furthermore, formaldehyde caused a concentration-dependent increase in nuclear fragmentation and in the activities of the apoptosis-initiator caspase-9 and apoptosis-effector caspase-3/-7, indicating apoptosis progression. Our data suggests that formaldehyde exerts strong cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial respiratory function and oxidative stress by formaldehyde may therefore be critical in methanol-induced toxicity.

  6. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  7. Infection of neuroblastoma cells with Semliki Forest virus. Incorporation of 35S or 32P

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Phosphate-free medium is used for the incorporation of 32 P and methionine-free medium for 35 S-methionine labelling. After virus replication, the culture shows a clear CPE all of the cells appearing round and dead. Materials used are presented and experimental procedure is described

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

    Czech Academy of Sciences Publication Activity Database

    Khalil, M.A.; Hraběta, J.; Groh, T.; Procházka, Pavel; Doktorová, H.; Eckschlager, T.

    2016-01-01

    Roč. 11, č. 9 (2016), e0162916 E-ISSN 1932-6203 R&D Projects: GA ČR GAP301/12/1734 Institutional support: RVO:68378041 Keywords : cancer stem-cells * histone deacetylase * colon-cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.806, year: 2016

  9. Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2

    Directory of Open Access Journals (Sweden)

    Lin Ming-Yi

    2009-12-01

    Full Text Available Abstract Background The multifunctional protein phosphatase 2A (PP2A is a heterotrimeric serine/threonine protein phosphatase composed of a scaffolding, catalytic and regulatory subunits. By modifying various downstream signal transducers, the aberrant expression of the brain-targeted regulatory subunit PPP2R2B is associated with the onset of a panel of neuronal disorders. The alternatively splicing of PPP2R2B encodes two regulatory subunit isoforms that determine cellular distribution of the neuron-specific holoenzyme to mitochondria (Bβ2 and cytoplasm (Bβ1, respectively. Results Human neuroblastoma cells were transfected with PPP2R2B constructs encoding the complete sequences of Bβ2 and Bβ1, respectively. The colonies with antibiotic resistance were selected as stable cell lines. Both ectopic Bβ1 and Bβ2 clones exhibited characteristics of autophagy. To test how cells respond to reactive oxygen species generators, the cells were treated with either hydrogen peroxide or t-butyl hydroperoxide and Bβ2 clones induced cell death. Suppression of autophagy using either RNA interference of the essential autophagy gene or pharmacological inhibitor rescued cell death caused by oxidative stress. Conclusions Cells with ectopically expressed mitochondria-targeted regulatory subunit PPP2R2B of the holoenzyme PP2A were shown predisposed to autophagy and oxidative stress induced cell death that is related to apoptosis. The results promised a model for studying the mechanism and function of aberrant PPP2R2B expression in neuronal cells. The work provided a new target for understanding and prevention of neuropathogenesis.

  10. 5 '-Amino-4-imidazolecarboxamide riboside induces apoptosis in human neuroblastoma cells via the mitochondrial pathway.

    Science.gov (United States)

    Garcia-Gil, M; Bertini, F; Pesi, R; Voccoli, V; Tozzi, M G; Camici, M

    2006-01-01

    5'-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5'-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

  11. Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration

    Science.gov (United States)

    Fang, Yuqiang; Iu, Catherine Y. Y.; Lui, Cathy N. P.; Zou, Yukai; Fung, Carmen K. M.; Li, Hung Wing; Xi, Ning; Yung, Ken K. L.; Lai, King W. C.

    2014-11-01

    Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.

  12. Human neuroblastoma cell growth in xenogeneic hosts: comparison of T cell-deficient and NK-deficient hosts, and subcutaneous or intravenous injection routes.

    Science.gov (United States)

    Turner, W J; Chatten, J; Lampson, L A

    1990-04-01

    We have examined two features of neuroblastoma cells that had not been well-characterized in a xenogeneic model: The cells display unusual immunologic properties in other experimental systems, and the original tumors display widespread and characteristic patterns of metastasis. To determine the most appropriate immunodeficient host for primary tumor growth, T cell-deficient nude mice, NK-deficient beige mice, beige-nudes, and controls were injected with the well-characterized line CHP-100. To define the pattern of tumor spread, complete autopsies were performed following subcutaneous, intraperitoneal and intravenous injections. CHP-100 consistently formed subcutaneous tumors in T cell-deficient mice (nude and beige-nude), but not in T cell-competent mice (beige, heterozygous nu/+ and bg/+, or wild-type). The growth rate and final size of the subcutaneous tumors were not greater in beige-nudes than in nudes. All mice showed early CHP-100 cell death after subcutaneous injection; the nature of the immunodeficiency was more relevant for the surviving subpopulation. Widespread dissemination was seen following intravenous injection, particularly in beige-nudes. Aspects of the growth patterns were appropriate to the tumor of origin. The behavior in immunodeficient mice suggests that T cells can play a role in controlling the growth of these cells; the next steps will be to define the effector mechanisms, and to determine if they can be exploited for human patients. The hematogenous spread following intravenous injection suggests that insights into the control of blood-borne tumor may also come from further study of this model.

  13. Oleuropein shows copper complexing properties and noxious effect on cultured SH-SY5Y neuroblastoma cells depending on cell copper content.

    Science.gov (United States)

    Capo, Concetta R; Pedersen, Jens Z; Falconi, Mattia; Rossi, Luisa

    2017-12-01

    The secoiridoid oleuropein is a non-flavonoid polyphenol, found in the fruit, leaves and food derivatives from Olea europea. Like other polyphenols it shows a very low toxicity towards healthy tissues and a protective action against cancer or neurodegeneration, but its mechanism of action is not yet understood. In the present report we have used optical and ESR spectroscopy as well as molecular modelling to demonstrate that oleuropein forms a complex with the transition metal copper; the dysmetabolism of this metal is suspected to be involved in both cancer and neurodegeneration. Experiments carried out with the aglycon derivative of oleuropein, produced by β-glycosidase treatment of oleuropein glycoside, showed that also the aglycon forms copper-complexes, but with different spectroscopic features than the glycosidic form. Molecular modelling analysis confirmed that two oleuropein molecules (glycosidic or aglycon forms) can easily coordinate one copper ion. The relationship between oleuropein and copper was investigated in SH-SY5Y human neuroblastoma cells. When cells were depleted of copper by treatment with the copper chelator triethylenetetramine (Trien), that binds copper with higher affinity than oleuropein, oleuropein was less toxic than to copper-adequate cells. Conversely, incubation of SH-SY5Y cells with exogenous copper sulphate increased cell susceptibility to oleuropein. Furthermore SH-SY5Y cells differentiated by retinoic acid pre-treatment showed a lower level of copper, and were more resistant to oleuropein treatment. The oleuropein aglycon was not toxic towards SH-SY5Y cells. In conclusion, the copper-oleuropein complex may be involved in the toxicity of oleuropein towards tumour cells, depending on their copper level. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Dinutuximab in the Treatment of High-Risk Neuroblastoma in Children

    Directory of Open Access Journals (Sweden)

    Hazal Gur

    2017-06-01

    Full Text Available Neuroblastoma is the most common extracranial tumor derived from neural crest cells in childhood, and treatment of high-risk neuroblastoma is a difficulty in oncology field. The discovery of new treatment strategies to treat pediatric patients with high-risk neuroblastoma is important. Dinutuximab (ch14.18; Unituxin, a chimeric human-mouse monoclonal antibody, is approved by Food and Drug Administration in 2015 to be used specifically in the treatment of high-risk neuroblastoma. It binds the disialoganglioside (GD2 antigen on the surface of neuroblastoma cells and induces lysis of GD2-expressed neuroblastoma cells via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. To enhance its activity, it is used with a combination of granulocyte-macrophage colony-stimulating factor, interleukin 2, and 13- cis -retinoic acid. In this review, we discuss the use of dinutuximab in the treatment of high-risk neuroblastoma.

  15. Cytoprotective response induced by electromagnetic stimulation on SH-SY5Y human neuroblastoma cell line.

    Science.gov (United States)

    Osera, Cecilia; Fassina, Lorenzo; Amadio, Marialaura; Venturini, Letizia; Buoso, Erica; Magenes, Giovanni; Govoni, Stefano; Ricevuti, Giovanni; Pascale, Alessia

    2011-10-01

    It is well known that physiological functions and pathological conditions of cells and tissues can be influenced not only by chemical molecules, but also by physical stimuli such as electromagnetic waves. In particular, epidemiological studies suggest possible associations between exposure to electromagnetic fields and an increased risk of tumors and neurodegenerative disorders, such as Alzheimer's disease. However, depending on the dose and on the length of treatment, the electromagnetic stimuli can be harmful or induce a cytoprotective cellular response, suggesting a possible application in medical therapy. In this study, under a tissue engineering viewpoint, we investigated the effects of an electromagnetic wave (magnetic field intensity, 2 mT; frequency, 75 Hz) on a neuronal cellular model characterized by the overexpression of the amyloid precursor protein (APP). After a prolonged electromagnetic treatment, lower mitochondrial activity and proliferation rate, resulting in a higher cellular quiescence, were observed. Focusing on the stress and oxidative pathways, we detected an overall increase of two fundamental proteins, the chaperone heat shock protein HSP70 and the free radical scavenger superoxide dismutase-1 enzyme (SOD-1). Interestingly, we found that the electromagnetic stimulation promotes the nonamyloidogenic processing of APP through an increased expression of the α-secretase ADAM10 and an enhanced release of the soluble neurotrophic factor sAPPα (a product of the ADAM10-mediated cleavage of APP). In conclusion, these findings suggest that the electromagnetic stimulus, if properly administered in terms of dose and timing, is able to induce a cytoprotective response in the cell. Moreover, these results suggest a possible use of this particular physical stimulation to improve the functional capability of the cells to face noxae. © Mary Ann Liebert, Inc.

  16. Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

    Science.gov (United States)

    Lau, Agnes; Westaway, David; McKenzie, Debbie; Aiken, Judd; Kim, Yong-Sun

    2011-01-01

    Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrPC) is required for replication of abnormal prion (PrPSc), which results in accumulation of PrPSc. Although there have been extensive studies using Prnp knockout systems, the normal function of PrPC remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrPC expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Prnp simultaneously, more effectively suppressed PrPC expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrPC depletion, gene expression analysis of PrPC interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrPC in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future. PMID:21494092

  17. 18α-Glycyrrhetinic acid lethality for neuroblastoma cells via de-regulating the Beclin-1/Bcl-2 complex and inducing apoptosis.

    Science.gov (United States)

    Rahman, Md Ataur; Bishayee, Kausik; Habib, Khadija; Sadra, Ali; Huh, Sung-Oh

    2016-10-01

    18α-Glycyrrhetinic acid (18-GA) is a known gap-junction inhibitor with demonstrated anticancer effects. However, the different modes of cell cytotoxicity for 18-GA remain to be characterized. In this study, 18-GA reduced the expression of cell-cell interaction proteins (N- and VE-cadherin), and led to a dose-dependent increase in cytotoxicity of the neuroblastoma cells tested, but was less toxic toward actively dividing human embryonic kidney cells. We found that 18-GA could induce both autophagy and apoptosis. 18-GA mediated autophagy was due to accumulation of Atg5, Atg7 and LC3II and degradation of p62. Individual siRNAs against Atg5 and Atg7 prevented autophagy and resulted in a further loss of viability with 18-GA. In addition, combination of 18-GA with autophagy inhibitor chloroquine produced a more significant cell death. This implied a pro-survival function for autophagy induction with 18-GA. 18-GA also led to the destabilization of Bcl-2/Beclin-1 interaction and cleavage of Beclin-1, a protein known to play role in apoptosis and autophagy induction. Treatment of cells by a pan-caspase inhibitor or a caspase-3 siRNA prevented a large portion of 18-GA mediated cytotoxicity, demonstrating that caspase-dependent apoptosis induction was responsible for most of the observed cytotoxicity. In terms of signaling, 18-GA led to reduced phosphorylation of all three classes of MAP kinases. Taken together, 18-GA or its pathways may lead to more effective, targeted therapeutics against neuroblastoma. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The l1-l2 regularization framework unmasks the hypoxia signature hidden in the transcriptome of a set of heterogeneous neuroblastoma cell lines.

    Science.gov (United States)

    Fardin, Paolo; Barla, Annalisa; Mosci, Sofia; Rosasco, Lorenzo; Verri, Alessandro; Varesio, Luigi

    2009-10-15

    Gene expression signatures are clusters of genes discriminating different statuses of the cells and their definition is critical for understanding the molecular bases of diseases. The identification of a gene signature is complicated by the high dimensional nature of the data and by the genetic heterogeneity of the responding cells. The l1-l2 regularization is an embedded feature selection technique that fulfills all the desirable properties of a variable selection algorithm and has the potential to generate a specific signature even in biologically complex settings. We studied the application of this algorithm to detect the signature characterizing the transcriptional response of neuroblastoma tumor cell lines to hypoxia, a condition of low oxygen tension that occurs in the tumor microenvironment. We determined the gene expression profile of 9 neuroblastoma cell lines cultured under normoxic and hypoxic conditions. We studied a heterogeneous set of neuroblastoma cell lines to mimic the in vivo situation and to test the robustness and validity of the l1-l2 regularization with double optimization. Analysis by hierarchical, spectral, and k-means clustering or supervised approach based on t-test analysis divided the cell lines on the bases of genetic differences. However, the disturbance of this strong transcriptional response completely masked the detection of the more subtle response to hypoxia. Different results were obtained when we applied the l1-l2 regularization framework. The algorithm distinguished the normoxic and hypoxic statuses defining signatures comprising 3 to 38 probesets, with a leave-one-out error of 17%. A consensus hypoxia signature was established setting the frequency score at 50% and the correlation parameter epsilon equal to 100. This signature is composed by 11 probesets representing 8 well characterized genes known to be modulated by hypoxia. We demonstrate that l1-l2 regularization outperforms more conventional approaches allowing the

  19. Drugs Approved for Neuroblastoma

    Science.gov (United States)

    This page lists cancer drugs approved by the Food and Drug Administration (FDA) for neuroblastoma. The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

  20. Apoptosis and necroptosis induced by stenodactylin in neuroblastoma cells can be completely prevented through caspase inhibition plus catalase or necrostatin-1.

    Science.gov (United States)

    Polito, Letizia; Bortolotti, Massimo; Pedrazzi, Manuela; Mercatelli, Daniele; Battelli, Maria Giulia; Bolognesi, Andrea

    2016-01-15

    Stenodactylin is a highly toxic plant lectin purified from the caudex of Adenia stenodactyla, with molecular structure, intracellular routing and enzyme activity similar to those of ricin, a well-known type 2 ribosome-inactivating protein. However, in contrast with ricin, stenodactylin is retrogradely transported not only in peripheral nerves but also in the central nervous system. Stenodactylin properties make it a potential candidate for application in neurobiology and in experimental therapies against cancer. Thus, it is necessary to better clarify the toxic activity of this compound. We investigated the mechanism of stenodactylin-induced cell death in the neuroblastoma-derived cell line, NB100, evaluating the implications of different death pathways and the involvement of oxidative stress. Stenodactylin cytotoxicity was determined by evaluating protein synthesis and other viability parameters. Cell death pathways and oxidative stress were analysed through flow cytometry and microscopy. Inhibitors of apoptosis, oxidative stress and necroptosis were tested to evaluate their protective effect against stenodactylin cytotoxicity. Stenodactylin efficiently blocked protein synthesis and reduced the viability of neuroblastoma cells at an extremely low concentration and over a short time (1 pM, 24 h). Stenodactylin induced the strong and rapid activation of apoptosis and the production of free radicals. Here, for the first time, a complete and long lasting protection from the lethal effect induced by a toxic type 2 ribosome-inactivating protein has been obtained by combining the caspase inhibitor Z-VAD-fmk, to either the hydrogen peroxide scavenger catalase or the necroptotic inhibitor necrostatin-1. In respect to stenodactylin cytotoxicity, our results: (i) confirm the high toxicity to nervous cells, (ii) indicate that multiple cell death pathways can be induced, (iii) show that apoptosis is the main death pathway, (iv) demonstrate the involvement of necroptosis and (v

  1. Herpes simplex virus type I induces an incomplete autophagic response in human neuroblastoma cells.

    Science.gov (United States)

    Santana, Soraya; Bullido, Maria Jesús; Recuero, Maria; Valdivieso, Fernando; Aldudo, Jesus

    2012-01-01

    Autophagy is a homeostatic process involved in the turnover or elimination of cytoplasmic components, damaged organelles, and protein aggregates via a lysosomal degradation mechanism. Autophagy also provides a mechanism of innate immunity, known as xenophagy, designed to protect cells from intracellular pathogens, but it may unfortunately be subverted to act as a pro-viral pathway facilitating the replication of certain viruses. Herpes simplex virus type I (HSV-1) is a neurotropic virus that remains latent in host neurons; it is the most common cause of sporadic viral encephalitis. Moreover, HSV-1 has been related to the pathogenesis of Alzheimer's disease. HSV-1 can modulate the autophagic process through a mechanism mediated by the viral protein ICP34.5. Here we report that HSV-1 induces a strong increase in GFP-LC3 and endogenous LC3 lipidation, and triggers the accumulation of intracellular autophagic compartments (mainly autophagosomes) without enhancing autophagic long-lived protein degradation in the late stages of infection. Autophagy inhibition mediated by ATG5 gene silencing had no effect on viral growth. The present results suggest that HSV-1 infection activates the host autophagic machinery and strongly controls the autophagic process, blocking the fusion of autophagosomes with lysosomes. These events might be important in the neurodegenerative process associated with HSV-1 infection.

  2. Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.

    Science.gov (United States)

    Catterall, W A

    1975-06-10

    The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent

  3. Cytoprotective effects of 12-oxo phytodienoic acid, a plant-derived oxylipin jasmonate, on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Taki-Nakano, Nozomi; Ohzeki, Hiromitsu; Kotera, Jun; Ohta, Hiroyuki

    2014-12-01

    Jasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity. The cells were pretreated with individual jasmonates for 24h and exposed to hydrogen peroxide (H2O2) for 24h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA). Among the jasmonates, only OPDA suppressed H2O2-induced cytotoxicity. OPDA pretreatment also inhibited the H2O2-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2. These results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway. Plant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  4. TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

    Science.gov (United States)

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

    2013-01-01

    The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC. PMID:23841091

  5. TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

    Directory of Open Access Journals (Sweden)

    Antonietta R. Farina

    2013-01-01

    Full Text Available The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.

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

    Directory of Open Access Journals (Sweden)

    Tingguo Kang

    2013-09-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  8. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  9. Aluminium induced endoplasmic reticulum stress mediated cell death in SH-SY5Y neuroblastoma cell line is independent of p53.

    Directory of Open Access Journals (Sweden)

    Syed Husain Mustafa Rizvi

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

  10. Neonatal acute megakaryoblastic leukemia mimicking congenital neuroblastoma

    OpenAIRE

    Kawasaki, Yukako; Makimoto, Masami; Nomura, Keiko; Hoshino, Akihiro; Hamashima, Takeru; Hiwatari, Mitsuteru; Nakazawa, Atsuko; Takita, Junko; Yoshida, Taketoshi; Kanegane, Hirokazu

    2014-01-01

    Key Clinical Message We describe a neonate with abdominal distension, massive hepatomegaly, and high serum neuron-specific enolase level suggestive of congenital neuroblastoma. The patient died of pulmonary hemorrhage after therapy. Autopsy revealed that the tumor cells in the liver indicated acute megakaryocytic leukemia with the RBM15-MKL1 fusion gene.

  11. Molecular events during tumor progression in neuroblastoma

    NARCIS (Netherlands)

    Bernards, R.A.; Lenardo, M.

    1990-01-01

    The N-myc oncogene was first identified as an amplified DNA element with homology to c-myc in human neuroblastoma. Since this initial observation, amplification of N-myc has also been observed in other types of human cancer, predominantly in retinoblastoma and small cell lung cancer. The

  12. Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2-M17 Neuroblastoma Cell Lines upon Differentiation.

    Directory of Open Access Journals (Sweden)

    Roberta Filograna

    Full Text Available Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate on the human neuroblastoma SH-SY5Y and BE(2-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2-M17 represent two alternative cell models for the neuroscience field.

  13. Neuroblastoma in children

    International Nuclear Information System (INIS)

    Hoermann, M.

    2008-01-01

    Neuroblastomas have a very heterogenic clinical presentation, ranging from relatively benign forms with the potential of spontaneous regression, to foudroyant malignant forms. Over half of neuroblastomas must be classified as high-risk tumors with a markedly high rate of recurrence. Despite multidisciplinary therapeutic approaches, the survival rate of children with this type of tumor is still below 40%. An ever-growing amount of data from international studies dating from the early 1970s onwards, points to the necessity of re-evaluating the medical approach in establishing the diagnosis and staging, understanding tumor biology and pathology, as well as therapy planning. (orig.) [de

  14. MicroRNA-184-mediated inhibition of tumour growth in an orthotopic murine model of neuroblastoma.

    Science.gov (United States)

    Tivnan, Amanda; Foley, Niamh H; Tracey, Lorraine; Davidoff, Andrew M; Stallings, Raymond L

    2010-11-01

    Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system. Previous studies have shown that miR-184 expression has anti-proliferative effects in neuroblastoma cells grown in culture. Therefore, it was of interest to evaluate this effect in vivo. Neuroblastoma cells overexpressing miR-184 were injected retroperitoneally into CB17-SCID mice and tumour burden was assessed by measuring bioluminescence. Overall survival was also evaluated. Ectopic overexpression of miR-184 in neuroblastoma cell lines is anti-proliferative. In addition, overexpression of miR-184 led to a significant reduction in tumour growth relative to negative control-treated cohorts in a xenograft model of neuroblastoma. This study demonstrated for the first time that miR-184 significantly reduces tumour growth and increases overall survival in an orthotopic murine model of neuroblastoma through assessment of tumour growth and moribundity relative to control miRNA-treated cohorts.

  15. Environment-Mediated Drug Resistance in Neuroblastoma

    Science.gov (United States)

    2013-10-01

    neuroblastoma. Five specimens were classified as positive for tumor involvement in themarrow (as assessed by the presence of tyrosine hydroxylase –positive cells ...presence of tyrosine hydroxylase – positive tumor cells (black arrow; bar, 50 mm). Right, the data represent the mean percentage (SD) of positive...H, et al. Tyrosine hydroxylase indicates cell differentiation of catechol- amine biosynthesis in neuroendocrine tumors. J Endocrinol Invest 1994;17

  16. Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells

    DEFF Research Database (Denmark)

    Warpman, U; Friberg, L; Gillespie, A

    1998-01-01

    The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick alpha4beta2 nACh......R binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the Ki values for binding inhibition and EC50 values for agonist-induced alpha4beta2 nAChR up-regulation. The effects of epibatidine and nicotine were further...... investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines...

  17. YAP promotes tumorigenesis and cisplatin resistance in neuroblastoma.

    Science.gov (United States)

    Yang, Chao; Tan, Juan; Zhu, Jun; Wang, Shan; Wei, Guanghui

    2017-06-06

    The transcriptional co-activator Yes-associated protein (YAP) is essential for Hippo pathway-driven tumorigenesis in various cancers. However, the expression and function of YAP in neuroblastoma remains elusive. Here, we show that YAP was highly expressed in Neuroblastoma (NB) and expression levels correlated with advanced tumor staging. Knockdown of YAP significantly impaired neuroblastoma proliferation, tumorigenesis, and invasion in vitro. Injection of the YAP inhibitor, Peptide 17, dramatically prevented neuroblastoma subcutaneous tumor growth by efficiently downregulating YAP expression in tumors. Additionally, less proliferative and more apoptotic cells were found in the Peptide 17 treatment group. Furthermore, YAP inhibition significantly inhibited cisplatin-resistant neuroblastoma proliferation, tumorigenesis, and invasion in vitro. The combination of Peptide 17 with low-dose cisplatin efficiently impaired cisplatin-resistant NB subcutaneous tumor growth, being as effective as high-dose cisplatin. Notably, the combination therapy caused lesser liver toxicity in mice compared to the high-dose cisplatin treatment group. Collectively, this work identifies YAP as a novel regulator of neuroblastoma proliferation, tumorigenesis, and invasion and indicates that YAP is a potential therapeutic target for cisplatin-resistant neuroblastoma.

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

    Science.gov (United States)

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

    2017-02-01

    Oxidative stress is involved in cell death in neurodegenerative diseases. Dietary polyphenols can exert health benefits, but their direct effects on neuronal cells are debatable because most phenolics are metabolized and do not reach the brain as they occur in the dietary sources. Herein, we evaluate the effects of a panel of bioavailable polyphenols and derived metabolites at physiologically relevant conditions against H 2 O 2 -induced apoptosis in human neuroblastoma SH-SY5Y cells. Among the 19 metabolites tested, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, gallic acid, ellagic acid, and urolithins prevented neuronal apoptosis via attenuation of ROS levels, increased REDOX activity, and decreased oxidative stress-induced apoptosis by preventing the caspase-3 activation via the mitochondrial apoptotic pathway in SH-SY5Y cells. This suggests that dietary sources containing the polyphenol precursors of these molecules such as cocoa, berries, walnuts, and tea could be potential functional foods to reduce oxidative stress associated with the onset and progress of neurodegenerative diseases.

  19. Pre-exposure of neuroblastoma cell line to pulsed electromagnetic field prevents H2 O2 -induced ROS production by increasing MnSOD activity.

    Science.gov (United States)

    Osera, Cecilia; Amadio, Marialaura; Falone, Stefano; Fassina, Lorenzo; Magenes, Giovanni; Amicarelli, Fernanda; Ricevuti, Giovanni; Govoni, Stefano; Pascale, Alessia

    2015-04-01

    Electromagnetic fields (EMFs) have been linked to increased risk of cancers and neurodegenerative diseases; however, EMFs can also elicit positive effects on biological systems, and redox status seems crucially involved in EMF biological effects. This study aimed to assess whether a short and repeated pulsed EMF (PEMF) could trigger adaptive responses against an oxidative insult in a neuronal cellular model. We found that a 40 min overall (four times a week, 10 min each) pre-exposure to PEMF did not affect major physiological parameters and led to a significant increase of Mn-dependent superoxide dismutase activity in the human neuroblastoma SH-SY5Y cell line. In addition, we found PEMF-pre-exposed cells exhibited decreased reactive oxygen species production following a 30 min H2 O2 challenge, with respect to non pre-exposed cells. Our findings might provide new insights on the role played by short and repeated PEMF stimulations in the enhancement of cellular defenses against oxidative insults. Although studies in normal neuronal cells would be useful to further confirm our hypothesis, we suggest that specific PEMF treatments may have potential biological repercussions in diseases where oxidative stress is implicated. © 2015 Wiley Periodicals, Inc.

  20. Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.

    Science.gov (United States)

    Sontag, Jean-Marie; Nunbhakdi-Craig, Viyada; Mitterhuber, Martina; Ogris, Egon; Sontag, Estelle

    2010-12-01

    Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes. © 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.

  1. Tanshinone IIA Inhibits Glutamate-Induced Oxidative Toxicity through Prevention of Mitochondrial Dysfunction and Suppression of MAPK Activation in SH-SY5Y Human Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Haifeng Li

    2017-01-01

    Full Text Available Glutamate excitotoxicity is associated with many neurological diseases, including cerebral ischemia and neurodegenerative diseases. Tanshinone IIA, a diterpenoid naphthoquinone from Salvia miltiorrhiza, has been shown to suppress presynaptic glutamate release, but its protective mechanism against glutamate-induced neurotoxicity is lacking. Using SH-SY5Y human neuroblastoma cells, we show here that excessive glutamate exposure decreases cell viability and proliferation and increases LDH release. Pretreatment with tanshinone IIA, however, prevents the decrease in cell viability and proliferation and the increase in LDH release induced by glutamate. Tanshinone IIA also attenuates glutamate-induced oxidative stress by reducing reactive oxygen species level and malondialdehyde and protein carbonyl contents and by enhancing activities and protein levels of superoxide dismutase and catalase. We then show that tanshinone IIA prevents glutamate-induced mitochondrial dysfunction by increasing mitochondrial membrane potential and ATP content and by reducing mitochondrial protein carbonyl content. Moreover, tanshinone IIA can inhibit glutamate-induced apoptosis through regulation of apoptosis-related protein expression and MAPK activation, including elevation of Bcl-2 protein level, decrease in Bax and cleaved caspase-3 levels, and suppression of JNK and p38 MAPK activation. Collectively, our findings demonstrate that tanshinone IIA protects SH-SY5Y cells against glutamate toxicity by reducing oxidative stress and regulating apoptosis and MAPK pathways.

  2. Preparation of Ginsenoside Rg3 and Protection against H2O2-Induced Oxidative Stress in Human Neuroblastoma SK-N-SH Cells

    Directory of Open Access Journals (Sweden)

    Gang Li

    2014-01-01

    Full Text Available The aim of this study is to evaluate the protection of ginsenoside Rg3 against oxidative stress in human neuroblastoma SK-N-SH cells. 20(R-ginsenoside Rg3 (20(R-Rg3 and 20(S-ginsenoside Rg3 (20(S-Rg3 were prepared by the method of chemical degradation and column chromatography, and the structure of the two compounds was characterized by 1H-NMR and 13C-NMR spectroscopy. MTT assay and LDH leakage assay were used to determine the cell viability and the oxidative stress cellular model was established by means of H2O2 (600 μM for 4 h. We also investigated the changes of intracellular MDA content, SOD activity, and ROS formation after the treatment of ginsenoside Rg3 for 20 h. The results indicated that both 20 (R-Rg3 and 20 (S-Rg3 had obvious protection against H2O2-induced oxidative stress in SK-N-SH cells. Moreover, 20(R-Rg3 exhibited better antioxidant activity than 20(S-Rg3 in vitro. These findings are expected to provide some implication for further research and application of ginsenoside Rg3 in neuroprotection.

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

    Science.gov (United States)

    Seoposengwe, Keabetswe; van Tonder, Jacob John; Steenkamp, Vanessa

    2013-12-12

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

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

    Directory of Open Access Journals (Sweden)

    Mostafa Waly

    2016-01-01

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

  5. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: Identification of novel biomarkers of effect

    International Nuclear Information System (INIS)

    Harris, W.; Sachana, M.; Flaskos, J.; Hargreaves, A.J.

    2009-01-01

    In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

  6. Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

    Science.gov (United States)

    Bosse, Kristopher R; Raman, Pichai; Zhu, Zhongyu; Lane, Maria; Martinez, Daniel; Heitzeneder, Sabine; Rathi, Komal S; Kendsersky, Nathan M; Randall, Michael; Donovan, Laura; Morrissy, Sorana; Sussman, Robyn T; Zhelev, Doncho V; Feng, Yang; Wang, Yanping; Hwang, Jennifer; Lopez, Gonzalo; Harenza, Jo Lynne; Wei, Jun S; Pawel, Bruce; Bhatti, Tricia; Santi, Mariarita; Ganguly, Arupa; Khan, Javed; Marra, Marco A; Taylor, Michael D; Dimitrov, Dimiter S; Mackall, Crystal L; Maris, John M

    2017-09-11

    We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. High-temperature strength of Nb-1%Zr alloy for irradiation-capsules inner-shell

    International Nuclear Information System (INIS)

    Nomura, Yasushi; Nakata, Hirokatsu; Tanaka, Mitsuo; Fukaya, Kiyoshi.

    1978-04-01

    Coated fuel particles in capsules will be irradiated at about 1600 0 C in JMTR. Nb-1%Zr alloy was chosen for inner shell material of the capsules because of its sufficient strength at 1000 0 C and low induced radioactivity. Nb-1%Zr ingot produced by electron beam melting was formed into seamless tubes by hollowing and swaging, followed by annealing. Creep test in helium flow and tensile test in vacuum were made to examine mechanical strength of the Nb-1%Zr tubes at 1000 0 C. Following are the results; 1) 0.2% yield stress at 1000 0 C is about 6 kg/mm 2 . 2) 3000 hr creep rupture stress at 1000 0 C is about 6 kg/mm 2 . (auth.)

  8. Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

    Science.gov (United States)

    Ivanov, Vladimir N.; Hei, Tom K.

    2015-01-01

    Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20–40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5–20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation. PMID:21910007

  9. Analysis of 1;17 translocation breakpoints in neuroblastoma: implications for mapping of neuroblastoma genes

    NARCIS (Netherlands)

    van Roy, N.; Laureys, G.; van Gele, M.; Opdenakker, G.; Miura, R.; van der Drift, P.; Chan, A.; Versteeg, R.; Speleman, F.

    1997-01-01

    Deletions and translocations resulting in loss of distal 1p-material are known to occur frequently in advanced neuroblastomas. Fluorescence in situ hybridisation (FISH) showed that 17q was most frequently involved in chromosome 1p translocations. A review of the literature shows that 10 of 27 cell

  10. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

    International Nuclear Information System (INIS)

    Crosswell, Hal E; Dasgupta, Anindya; Alvarado, Carlos S; Watt, Tanya; Christensen, James G; De, Pradip; Durden, Donald L; Findley, Harry W

    2009-01-01

    c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. c-Met is highly expressed in most tumors from

  11. Proteome and Acetylome Analysis Identifies Novel Pathways and Targets Regulated by Perifosine in Neuroblastoma

    OpenAIRE

    Gu, Xiao; Hua, Zhongyan; Dong, Yudi; Zhan, Yue; Zhang, Xiaowen; Tian, Wei; Liu, Zhihui; Thiele, Carol J.; Li, Zhijie

    2017-01-01

    Perifosine, an Akt inhibitor, has been shown to be effective in controlling neuroblastoma tumor growth. However, studies indicate that in addition to the ability to inhibit Akt, other mechanisms contribute to perifosine?s anti-tumor activity. To gain insight into perifosine anti-tumor activity in neuroblastoma we have studied changes in the proteome and acetylome after perifosine treatment in SK-N-AS neuroblastoma cells using SILAC labeling, affinity enrichment, high-resolution and LC-MS/MS a...

  12. Facile synthesis of mPEG-luteolin-capped silver nanoparticles with antimicrobial activity and cytotoxicity to neuroblastoma SK-N-SH cells.

    Science.gov (United States)

    Qing, Weixia; Wang, Yong; Li, Xiao; Lu, Minghua; Liu, Xiuhua

    2017-12-01

    We firstly report a facile route for the green synthesis of mPEG-luteolin-capped silver nanoparticles (mPEG-luteolin-AgNPs) using mPEG-luteolin as both the reducer and stabilizer. The reaction was carried out in a stirred aqueous solution at 50°C without additional poisonous reagents. The prepared mPEG-luteolin-AgNPs was characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), zeta potential and UV-vis (UV-vis) spectrum, respectively. The proportions of mPEG-luteolin capped silver nanoparticles is about 89.9%, and the content of silver is 6.65%. The mPEG-luteolin-AgNPs was evaluated the antimicrobial effects on Staphlococcus aureus, Extended spectrum β-Lactamases Staphlococcus aureus, Escherichia Coli and Extended spectrum β-Lactamases Escherichia Coli using drilling hole method. The results showed that both gram-positive and gram-negative bacteria were killed by the mPEG-luteolin-AgNPs at low concentration. Meanwhile, the cell viability assay demonstrated that mPEG-luteolin-AgNPs had toxic effects on human neuroblastoma SK-N-SH cells. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  14. Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

    Science.gov (United States)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte K; Wassélius, Johan; Ekström, Ulf; Abrahamson, Magnus

    2010-11-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the

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

    Science.gov (United States)

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

    2017-01-01

    Tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP, also known as TDCPP), an extensively used flame retardant, is frequently detected in the environment and biota. Recent studies have shown that TDCIPP has neurotoxic effects. In this study, we determined the mechanisms of TDCIPP-induced neurotoxicity in human neuroblastoma (SH-SY5Y) cells. By using morphological examination, flow cytometry, and mitochondrial membrane potential (ΔYm) measurement, we confirmed that exposure to TDCIPP caused apoptosis accompanied by the activation of apoptosis-related genes (e.g. Bax and Bcl-2) and caspase 3 protein in SH-SY5Y cells. Increased reactive oxygen species (ROS) formation and intracellular calcium ions ([Ca 2+ ] i ) were also observed in TDCIPP-treated SH-SY5Y cells. Exposure to TDCIPP led to the activation of protein markers of endoplasmic reticulum (ER) stress, including eukaryotic translation initiation factor 2a subunit (p-EIF2a), activation transcription factor (ATF4), glucose-regulated protein (GRP78), and the proapoptotic factor C/EBP homologous protein (CHOP). To determine the role of the ER in apoptosis, phenyl butyric acid (PBA), an ER stress inhibitor, was applied. Treatment with PBA effectively attenuated TDCIPP-induced ER stress and protected against apoptotic death in SH-SY5Y cells by inhibition of Bax expression and promotion of Bcl-2 expression. Furthermore, we found that pretreatment of the cells with the ROS scavenger N-acetyl cysteine (NAC) inhibited the ER stress response and prevented apoptosis. The combination of PBA and NAC pretreatment could further prevent TDCIPP induced ER-stress and apoptotic death compared with PBA or NAC pretreatment alone. Thus, in the present study, we demonstrated that TDCIPP induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathways in SH-SY5Y cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  17. Neuroblastoma: morphological pattern, molecular genetic features, and prognostic factors

    Directory of Open Access Journals (Sweden)

    A. M. Stroganova

    2016-01-01

    Full Text Available Neuroblastoma, the most common extracranial tumor of childhood, arises from the developing neurons of the sympathetic nervous system (neural cress stem cells and has various biological and clinical characteristics. The mean age at disease onset is 18 months. Neuroblastoma has a number of unique characteristics: a capacity for spontaneous regression in babies younger than 12 months even in the presence of distant metastases, for differentiation (maturation into ganglioneuroma in infants after the first year of life, and for swift aggressive development and rapid metastasis. There are 2 clinical classifications of neuroblastoma: the International neuroblastoma staging system that is based on surgical results and the International Neuroblastoma Risk Group Staging System. One of the fundamentally important problems for the clinical picture of neuroblastoma is difficulties making its prognosis. Along with clinical parameters (a patient’s age, tumor extent and site, some histological, molecular biochemical (ploidy and genetic (chromosomal aberrations, MYCN gene status, deletion of the locus 1p36 and 11q, the longer arm of chromosome 17, etc. characteristics of tumor cells are of considerable promise. MYCN gene amplification is observed in 20–30 % of primary neuroblastomas and it is one of the major indicators of disease aggressiveness, early chemotherapy resistance, and a poor prognosis. There are 2 types of MYCN gene amplification: extrachromosomal (double acentric chromosomes and intrachromosomal (homogenically painted regions. Examination of double acentric chromosomes revealed an interesting fact that it may be eliminated (removed from the nucleus through the formation of micronuclei. MYCN oncogene amplification is accompanied frequently by 1p36 locus deletion and longer 17q arm and less frequently by 11q23 deletion; these are poor prognostic factors for the disease. The paper considers in detail the specific, unique characteristics of the

  18. Effect of fraxetin on antioxidant defense and stress proteins in human neuroblastoma cell model of rotenone neurotoxicity. Comparative study with myricetin and N-acetylcysteine

    International Nuclear Information System (INIS)

    Molina-Jimenez, Maria Francisca; Sanchez-Reus, Maria Isabel; Cascales, Maria; Andres, David; Benedi, Juana

    2005-01-01

    Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. Recently, it has been shown that fraxetin (coumarin) and myricetin (flavonoid) have significant neuroprotective effects against apoptosis induced by rotenone, increase the total glutathione levels in vitro, and inhibit lipid peroxidation. Thus, these considerations prompted us to investigate the way in which fraxetin and myricetin affect the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD), catalase, glutathione reductase (GR), and glutathione peroxidase (GPx) on rotenone neurotoxicity in neuroblastoma cells. N-acetylcysteine (NAC), a potent antioxidant, was employed as a comparative agent. Also, the expression and protein levels of HSP70 by Northern and Western blot analysis were assayed in SH-SY5Y cells. After incubation for 16 h, rotenone significantly increased the expression and activity of MnSOD, GPx, and catalase. When cells were preincubated with fraxetin, there was a decrease in the protein levels and activity of both MnSOD and catalase, in comparison with the rotenone treatment. The myricetin effect was less pronounced. Activity and expression of GPx were increased by rotenone and pre-treatment with fraxetin did not modify significantly these levels. The significant enhancement in HSP70 expression at mRNA and protein levels induced by fraxetin was observed by pre-treatment of cells 0.5 h before rotenone insult. These data suggest that major features of rotenone-induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that fraxetin partially protects against rotenone toxicity affecting the main protection system of the cells against oxidative injury

  19. Silencing Intersectin 1 Slows Orthotopic Neuroblastoma Growth in Mice.

    Science.gov (United States)

    Harris, Jamie; Herrero-Garcia, Erika; Russo, Angela; Kajdacsy-Balla, Andre; O'Bryan, John P; Chiu, Bill

    2017-11-01

    Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1), a scaffold protein involved in phosphoinositide 3-kinase (PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector (pSR), vectors expressing scrambled shRNA (pSCR), or shRNAs targeting ITSN1 (sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000 mm. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6±247.7 mm, pSCR was 1180±159.9 mm, sh#1 was 526.3±212.8 mm, and sh#2 was 589.2±74.91 mm. sh#1-tumors and sh#2-tumors were smaller than pSCR (P=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors (P=0.02), trended towards improved survival in sh#1-tumors (P=0.09), compared with pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared with pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.

  20. The signaling cascades of Ganoderma lucidum extracts in stimulating non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y cell lines.

    Science.gov (United States)

    Pinweha, Sirinthorn; Wanikiat, Payong; Sanvarinda, Yupin; Supavilai, Porntip

    2008-12-19

    Ganoderma lucidum (GL) is a medicinal mushroom that possesses various pharmacological properties which are also documented in the ancient reports where GL is praised for its effects on the promotion of health and longevity. In this study, we have investigated the effect of GL mycelia extracts on the non-amyloidogenic protein secretion (sAPPalpha) and the amyloid precursor protein (APP) expression in SH-SY5Y neuroblastoma cells. In order to characterize the signaling pathway which mediates GL-enhanced sAPPalpha secretion, we used inhibitors of nerve growth factor (NGF) signaling pathways, phosphatidylinositol 3 kinase (PI3K), phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK1/2), to block GL-mediated sAPPalpha secretion as well as ERK1/2 and PKC activation by using Western blot analysis. Our results provided for the first time evidence that GL mycelia extracts increased APP expression and promoted sAPPalpha secretion. In addition, GL extracts activated ERK1/2 and PKC phosphorylation. The complex signaling cascades of PI3K and ERK may be responsible for GL-mediated sAPPalpha secretion.

  1. Anti-cancer stemness and anti-invasive activity of bitter taste receptors, TAS2R8 and TAS2R10, in human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Yoona Seo

    Full Text Available Neuroblastoma (NB originates from immature neuronal cells and currently has a poor clinical outcome. NB cells possess cancer stem cells (CSCs characteristics that facilitate the initiation of a tumor, as well as its metastasis. Human bitter taste receptors, referred to as TAS2Rs, are one of five types of basic taste receptors and they belong to a family of G-protein coupled receptors. The recent finding that taste receptors are expressed in non-gustatory tissues suggest that they mediate additional functions distinct from taste perception. While it is generally admitted that the recognition of bitter tastes may be associated with a self-defense system to prevent the ingestion of poisonous food compounds, this recognition may also serve as a disease-related function in the human body. In particular, the anti-cancer stemness and invasion effects of TAS2Rs on NB cells remain poorly understood. In the present study, endogenous expression of TAS2R8 and TAS2R10 in SK-N-BE(2C and SH-SY5Y cells was examined. In addition, higher levels of TAS2R8 and TAS2R10 expression were investigated in more differentiated SY5Y cells. Both TAS2Rs were up-regulated following the induction of neuronal cell differentiation by retinoic acid. In addition, ectopic transfection of the two TAS2Rs induced neurite elongation in the BE(2C cells, and down-regulated CSCs markers (including DLK1, CD133, Notch1, and Sox2, and suppressed self-renewal characteristics. In particular, TAS2RS inhibited tumorigenicity. Furthermore, when TAS2Rs was over-expressed, cell migration, cell invasion, and matrix metalloproteinases activity were inhibited. Expression levels of hypoxia-inducible factor-1α, a well-known regulator of tumor metastasis, as well as its downstream targets, vascular endothelial growth factor and glucose transporter-1, were also suppressed by TAS2Rs. Taken together, these novel findings suggest that TAS2Rs targets CSCs by suppressing cancer stemness characteristics and NB

  2. A Hybrid Robotic Control System Using Neuroblastoma Cultures

    Science.gov (United States)

    Ferrández, J. M.; Lorente, V.; Cuadra, J. M.; Delapaz, F.; Álvarez-Sánchez, José Ramón; Fernández, E.

    The main objective of this work is to analyze the computing capabilities of human neuroblastoma cultured cells and to define connection schemes for controlling a robot behavior. Multielectrode Array (MEA) setups have been designed for direct culturing neural cells over silicon or glass substrates, providing the capability to stimulate and record simultaneously populations of neural cells. This paper describes the process of growing human neuroblastoma cells over MEA substrates and tries to modulate the natural physiologic responses of these cells by tetanic stimulation of the culture. We show that the large neuroblastoma networks developed in cultured MEAs are capable of learning: establishing numerous and dynamic connections, with modifiability induced by external stimuli and we propose an hybrid system for controlling a robot to avoid obstacles.

  3. mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells.

    Science.gov (United States)

    Kuo, Chung-Wen; Tsai, Meng-Han; Lin, Tsu-Kung; Tiao, Mao-Meng; Wang, Pei-Wen; Chuang, Jiin-Haur; Chen, Shang-Der; Liou, Chia-Wei

    2017-06-07

    Mitochondria consume O₂ to produce ATP and are critical for adaption of hypoxia, but the role of mitochondria in HIF-1α pathway is as yet unclear. In this study, mitochondrial DNA (mtDNA) enriched (SK-N-AS) and depleted (ρ⁰) cells of neuroblastoma were cultured in a hypoxic chamber to simulate a hypoxic condition and then the major components involved in mitochondrial related pathways, hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) were measured. The results showed that hypoxia-stimulated exposure elevated expression of HIF-1α, which was additionally influenced by level of generated ROS within the cytosol. Moreover, elevation of HIF-1α also resulted in increases of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase kinase 1 (PDK1) in both hypoxic cells. The expression of mitochondrial biogenesis related proteins and metabolic components were noted to increase significantly in hypoxic SK-N-AS cells, indicating that mtDNA was involved in mitochondrial retrograde signaling and metabolic pathways. An analysis of dynamic proteins found elevated levels of HIF-1α causing an increased expression of dynamin-related protein 1 (DRP1) during hypoxia; further, the existence of mtDNA also resulted in higher expression of DRP1 during hypoxia. By using siRNA of HIF-1α or DRP1, expression of DRP1 decreased after suppression of HIF-1α; moreover, the expression of HIF-1α was also affected by the suppression of DRP1. In this study, we demonstrated that mtDNA is a mediator of HIF-1α in eliciting metabolic reprogramming, and mitochondrial biogenesis. Identification of a mutual relationship between HIF-1α and DRP1 may be a critical tool in the future development of clinical applications.

  4. A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y).

    Science.gov (United States)

    Santillo, Michael F; Liu, Yitong

    2015-01-01

    Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells. Published by Elsevier Inc.

  5. Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line

    Science.gov (United States)

    Slack, B. E.; Richardson, U. I.; Nitsch, R. M.; Wurtman, R. J.

    1992-01-01

    Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS).

  6. The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells.

    Science.gov (United States)

    Caputi, Francesca Felicia; Acquas, Elio; Kasture, Sanjay; Ruiu, Stefania; Candeletti, Sanzio; Romualdi, Patrizia

    2018-01-10

    Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine's analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of μ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells. A range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated. Data analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation. Present in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

  7. Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements

    International Nuclear Information System (INIS)

    Del Re, Brunella; Marcantonio, Pamela; Capri, Miriam; Giorgi, Gianfranco

    2010-01-01

    Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1 RP element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1 RP integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

  8. Integrative analysis of neuroblastoma and pheochromocytoma genomics data

    Directory of Open Access Journals (Sweden)

    Szabó Peter M

    2012-10-01

    Full Text Available Abstract Background Pheochromocytoma and neuroblastoma are the most common neural crest-derived tumors in adults and children, respectively. We have performed a large-scale in silico analysis of altogether 1784 neuroblastoma and 531 pheochromocytoma samples to establish similarities and differences using analysis of mRNA and microRNA expression, chromosome aberrations and a novel bioinformatics analysis based on cooperative game theory. Methods Datasets obtained from Gene Expression Omnibus and ArrayExpress have been subjected to a complex bioinformatics analysis using GeneSpring, Gene Set Enrichment Analysis, Ingenuity Pathway Analysis and own software. Results Comparison of neuroblastoma and pheochromocytoma with other tumors revealed the overexpression of genes involved in development of noradrenergic cells. Among these, the significance of paired-like homeobox 2b in pheochromocytoma has not been reported previously. The analysis of similar expression patterns in neuroblastoma and pheochromocytoma revealed the same anti-apoptotic strategies in these tumors. Cancer regulation by stathmin turned out to be the major difference between pheochromocytoma and neuroblastoma. Underexpression of genes involved in neuronal cell-cell interactions was observed in unfavorable neuroblastoma. By the comparison of hypoxia- and Ras-associated pheochromocytoma, we have found that enhanced insulin like growth factor 1 signaling may be responsible for the activation of Src homology 2 domain containing transforming protein 1, the main co-factor of RET. Hypoxia induced factor 1α and vascular endothelial growth factor signaling included the most prominent gene expression changes between von Hippel-Lindau- and multiple endocrine neoplasia type 2A-associated pheochromocytoma. Conclusions These pathways include previously undescribed pathomechanisms of neuroblastoma and pheochromocytoma and associated gene products may serve as diagnostic markers and therapeutic

  9. Activation of Nrf2 attenuates carbonyl stress induced by methylglyoxal in human neuroblastoma cells: Increase in GSH levels is a critical event for the detoxification mechanism.

    Science.gov (United States)

    Nishimoto, Shoichi; Koike, Shin; Inoue, Naho; Suzuki, Toshihiro; Ogasawara, Yuki

    2017-02-05

    The present study focused on the methylglyoxal (MG) detoxification mechanism in neuroblastoma cells. The involvement of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway as a defense response against the formation of MG-modified proteins, which is well-known evidence of carbonyl stress, was also examined. We found that MG treatment resulted in accumulation of modified proteins bearing the structure of advanced glycation end products (AGEs) derived from MG in SH-SY5Y cells. This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator, carnosic acid and CDDO-Im, confirming the involvement of the Nrf2 pathway in MG detoxification. Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, AKR1B1, and AKR7A2, the expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels. Furthermore, we demonstrated that a GSH synthesis inhibitor eliminated the MG detoxification effect derived from pretreatment with the Nrf2 activator. These results indicated that increase in GSH levels, induced by pre-treatment with carnosic acid, promoted the formation of the GLO1 substrate, hemithioacetal, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. It was, therefore, determined that promotion of GSH synthesis via the Nrf2/Keap1pathway is important in the MG detoxification mechanism against neuronal MG-induced carbonyl stress, and Nrf2 activators contribute to reduction in the accumulation and toxic expression of carbonyl proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma

    NARCIS (Netherlands)

    Bernards, R.A.; Dessain, S.K.; Weinberg, R.A.

    1986-01-01

    Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases

  11. Mutations in PIK3CA are infrequent in neuroblastoma

    International Nuclear Information System (INIS)

    Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

    2006-01-01

    Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain 'hot spots' where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. These data suggest that activating

  12. Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway

    International Nuclear Information System (INIS)

    Alleaume, Celine; Eychene, Alain; Harnois, Thomas; Bourmeyster, Nicolas; Constantin, Bruno; Caigneaux, Evelyne; Muller, Jean-Marc; Philippe, Michel

    2004-01-01

    Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells

  13. TRAIL Activates a Caspase 9/7-Dependent Pathway in Caspase 8/10-Defective SK-N-SH Neuroblastoma Cells with Two Functional End Points: Induction of Apoptosis and PGE2 Release

    Directory of Open Access Journals (Sweden)

    Giorgio Zauli

    2003-09-01

    Full Text Available Most neuroblastoma cell lines do not express apical caspases 8 and 10, which play a key role in mediating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL cytotoxicity in a variety of malignant cell types. In this study, we demonstrated that TRAIL induced a moderate but significant increase of apoptosis in the caspase 8/10-deficient SK-N-SH neuroblastoma cell line, through activation of a novel caspase 9/7 pathway. Concomitant to the induction of apoptosis, TRAIL also promoted a significant increase of prostaglandin E2 (PGE2 release by SKN-SH cells. Moreover, coadministration of TRAIL plus indomethacin, a pharmacological inhibitor of cyclooxygenase (COX, showed an additive effect on SKN-SH cell death. In spite of the ability of TRAIL to promote the phosphorylation of both ERKi/2 and p38/MAPK, which have been involved in the control of COX expression/activity, neither PD98059 nor SB203580, pharmacological inhibitors of the ERKi/2 and p38/MAPK pathways, respectively, affected either PGE2 production or apoptosis induced by TRAIL. Finally, both induction of apoptosis and PGE2 release were completely abrogated by the broad caspase inhibitor z-VAD4mk, suggesting that both biologic end points were regulated in SK-N-SH cells through a caspase 9/7-dependent pathway.

  14. Competitive interaction of agonists and antagonists with 5-HT3 recognition sites in membranes of neuroblastoma cells labelled with [3H]ICS 205-930

    International Nuclear Information System (INIS)

    Hoyer, D.; Neijt, H.C.; Karpf, A.

    1989-01-01

    [3H]ICS 205-930 labelled 5-HT3 recognition sites in membranes prepared from murine neuroblastoma N1E-115 cells. Binding was rapid, reversible, saturable and stereoselective to an apparently homogeneous population of sites. Kinetic studies revealed that agonists and antagonists produced a monophasic dissociation reaction of [3H]ICS 205-930 from its recognition sites. The dissociation rate constant of the radioligand was similar whether the dissociation was induced by an agonist or an antagonist. Competition studies carried out with agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. Competition curves were best fit for a 1 site model. [3H]ICS 205-930 binding sites displayed the pharmacological profile of a 5-HT3 receptor. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were apparently competitive in nature, as demonstrated in kinetic and equilibrium experiments. In saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabelled agonists and antagonists, apparent Bmax values were not reduced whereas apparent Kd values were increased in the presence of competing ligands. There was a good agreement between apparent pKB values calculated for the competing ligands in saturation experiments and pKd values calculated from competition experiments. The present data demonstrate that [3H]ICS 205-930 labels a homogeneous population of sites at which agonists and antagonists interact competitively

  15. Comparison of the neurotoxicities between volatile organic compounds and fragrant organic compounds on human neuroblastoma SK-N-SH cells and primary cultured rat neurons

    Directory of Open Access Journals (Sweden)

    Yasue Yamada

    2015-01-01

    Full Text Available These are many volatile organic compounds (VOCs that are synthesized, produced from petroleum or derived from natural compounds, mostly plants. Fragrant and volatile organic compounds from plants have been used as food additives, medicines and aromatherapy. Several clinical and pathological studies have shown that chronic abuse of VOCs, mainly toluene, causes several neuropsychiatric disorders. Little is known about the mechanisms of neurotoxicity of the solvents. n-Octanal, nonanal, and 2-ethyl-1-hexanol, which are used catalyzers or intermediates of chemical reactions, are released into the environment. Essential oils have the functions of self-defense, sterilization, and antibiosis in plants. When volatile organic compounds enter the body, there is the possibility that they will pass through the blood–brain barrier (BBB and affect the central nervous system (CNS. However, the direct effects of volatile organic compounds on neural function and their toxicities are still unclear. We compared the toxicities of n-octanal, nonanal and 2-ethyl-1-hexanol with those of five naturally derived fragrant organic compounds (FOCs, linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and n-phenethyl alcohol. MTT assay of human neuroblastoma SK-N-SH cells showed that the IC50 values of linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and phenethyl alcohol were 1.33, 2.3, >5, >5, and 2.39 mM, respectively, and the IC50 values of toluene, n-octanal, nonanal and 2-ethyl-1-hexanol were 850, 37.2, 8.31 and 15.1 μM, respectively. FOCs showed lower toxicities than those of VOCs. These results indicate that FOCs are safer than other compounds.

  16. Gene expression profiling in response to the histone deacetylase inhibitor BL1521 in neuroblastoma

    International Nuclear Information System (INIS)

    Ruijter, Annemieke J.M. de; Meinsma, Rutger J.; Bosma, Peter; Kemp, Stephan; Caron, Huib N.; Kuilenburg, Andre B.P. van

    2005-01-01

    Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype

  17. microRNA-221 Enhances MYCN via Targeting Nemo-like Kinase and Functions as an Oncogene Related to Poor Prognosis in Neuroblastoma.

    Science.gov (United States)

    He, Xiao-Yan; Tan, Zheng-Lan; Mou, Qin; Liu, Fang-Jie; Liu, Shan; Yu, Chao-Wen; Zhu, Jin; Lv, Lin-Ya; Zhang, Jun; Wang, Shan; Bao, Li-Ming; Peng, Bin; Zhao, Hui; Zou, Lin

    2017-06-01

    Purpose: MYCN is one of the most well-characterized genetic markers of neuroblastoma. However, the mechanisms as to how MYCN mediate neuroblastoma tumorigenesis are not fully clear. Increasing evidence has confirmed that the dysregulation of miRNAs is involved in MYCN-mediated neuroblastoma tumorigenesis, supporting their potential as therapeutic targets for neuroblastoma. Although miR-221 has been reported as one of the upregulated miRNAs, the interplay between miR-221 and MYCN-mediated neuroblastoma progression remains largely elusive. Experimental Design: The expression of miR-221 in the formalin-fixed, paraffin-embedded tissues from 31 confirmed patients with neuroblastoma was detected by locked nucleic acid- in situ hybridization and qRT-PCR. The correlation between miR-221 expression and clinical features in patients with neuroblastoma was assessed. The mechanisms as to how miR-221 regulate MYCN in neuroblastoma were addressed. The effect of miR-221 on cellular proliferation in neuroblastoma was determined both in vitro and in vivo Results: miR-221 was significantly upregulated in neuroblastoma tumor cells and tissues that overexpress MYCN, and high expression of miR-221 was positively associated with poor survival in patients with neuroblastoma. Nemo-like kinase (NLK) as a direct target of miR-221 in neuroblastoma was verified. In addition, overexpression of miR-221 decreased LEF1 phosphorylation but increased the expression of MYCN via targeting of NLK and further regulated cell cycle, particularly in S-phase, promoting the growth of neuroblastoma cells. Conclusions: This study provides a novel insight for miR-221 in the control of neuroblastoma cell proliferation and tumorigenesis, suggesting potentials of miR-221 as a prognosis marker and therapeutic target for patients with MYCN overexpressing neuroblastoma. Clin Cancer Res; 23(11); 2905-18. ©2016 AACR . ©2016 American Association for Cancer Research.

  18. Telomerase activation by genomic rearrangements in high-risk neuroblastoma

    Science.gov (United States)

    Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L.; Sand, Frederik; Heuckmann, Johannes M.; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Glöckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R.; Savelyeva, Larissa; Watkins, Simon C.; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H.; Herrmann, Carl; O’Sullivan, Roderick J.; Westermann, Frank; Thomas, Roman K.; Fischer, Matthias

    2016-01-01

    Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours. PMID:26466568

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Epigenetic regulation of neuroblastoma development.

    Science.gov (United States)

    Durinck, Kaat; Speleman, Frank

    2018-01-19

    In recent years, technological advances have enabled a detailed landscaping of the epigenome and the mechanisms of epigenetic regulation that drive normal cell function, development and cancer. Rather than merely a structural entity to support genome compaction, we now look at chromatin as a very dynamic and essential constellation that is actively participating in the tight orchestration of transcriptional regulation as well as DNA replication and repair. The unique feature of chromatin flexibility enabling fast switches towards more or less restricted epigenetic cellular states is, not surprisingly, intimately connected to cancer development and treatment resistance, and the central role of epigenetic alterations in cancer is illustrated by the finding that up to 50% of all mutations across cancer entities affect proteins controlling the chromatin status. We summarize recent insights into epigenetic rewiring underlying neuroblastoma (NB) tumor formation ranging from changes in DNA methylation patterns and mutations in epigenetic regulators to global effects on transcriptional regulatory circuits that involve key players in NB oncogenesis. Insights into the disruption of the homeostatic epigenetic balance contributing to developmental arrest of sympathetic progenitor cells and subsequent NB oncogenesis are rapidly growing and will be exploited towards the development of novel therapeutic strategies to increase current survival rates of patients with high-risk NB.

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

    Science.gov (United States)

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

    2017-06-01

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

  2. Mouse Prion Protein (PrP) Segment 100 to 104 Regulates Conversion of PrPC to PrPSc in Prion-Infected Neuroblastoma Cells

    Science.gov (United States)

    Hara, Hideyuki; Okemoto-Nakamura, Yuko; Shinkai-Ouchi, Fumiko; Hanada, Kentaro; Yamakawa, Yoshio

    2012-01-01

    Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrPSc; PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrPC) to PrPSc and the subsequent conversion of PrPC to PrPSc. We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrPC and PrPSc. Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrPSc state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrPSc and interfered with the conversion of endogenous MoPrPC. The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrPSc. Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrPC reduced the accumulation of PrPSc after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrPC plays a key role in conversion after binding to MoPrPSc. PMID:22398286

  3. Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Nieto-Alamilla, Gustavo; Escamilla-Sánchez, Juan; López-Méndez, María-Cristina; Molina-Hernández, Anayansi; Guerrero-Hernández, Agustín; Arias-Montaño, José-Antonio

    2018-04-01

    In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H 3 receptor (hH 3 R 445 and hH 3 R 365 ) on [ 35 S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca 2+ ions ([Ca 2+ ]i) and depolarization-evoked [ 3  H]-dopamine release. Maximal specific binding (B max ) of [ 3  H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH 3 R 445 and SH-SY5Y-hH 3 R 365 cells, respectively, with similar dissociation constants (K d , 0.86 nM and 0.81 nM). The mRNA of the hH 3 R 365 isoform was 40.9 ± 7.9% of the hH 3 R 445 isoform. No differences in receptor affinity were found for the H 3 R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [ 35 S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH 3 R 445 cells ([ 35 S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH 3 R 365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [ 3  H]-dopamine release evoked by 100 mM K + (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH 3 R 445 and SH-SY5Y-hH 3 R 365 cells), or the inhibition of depolarization-induced increase in [Ca 2+ ]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH 3 R 445 cells 0.639 ± 0.049, SH-SY5Y-hH 3 R 365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH 3 R 445 and hH 3 R 365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

  4. Effect of Auger electrons internalized as Indium-111 labelled N-MYC phosphorothionate antisense oligonucleotide (In-111-N-MYC-AS) on human neuroblastoma cells: In vitro and in vivo studies

    International Nuclear Information System (INIS)

    Watanabe, N.; Tanada, S.; Sasaki, Y.

    2005-01-01

    Full text: Auger electrons which enter into cells cause biological effects with high-LET short range radiation on the neighborhood. The purpose of this study was to investigate the internalization of In-111-N-myc-AS and suppression of N-MYC in human neuroblastoma cells. Fifteen-mer AS, which was complementary to the region of the mRNA of N-myc beginning with ATG start codon, was derivatized with SCN-Bn-EDTA and labeled with In-111. In-111-N-myc-AS (1.33MBq/nmol) was prepared as naked form and encapsulated in cationic multilamellar liposome (CML). The internalization of In-111-N-myc-AS with or without CML into human neuroblastoma SK-N-DZ cells was determined both in vitro (cell culture) and in vivo (tumor bearing nude mice) studies by Southern blot analysis. Quantity of N-MYC in the tumor cells was also measured by Western blot analysis. In in vitro system 0.69-02pmol of In-111-N-myc-AS (80pmol) with CML was internalized in the cells (5x106) by 12h at 4 deg. C, which increased to 8.05?0.43pmol at 37 deg. C. The internalized naked In-111-N-myc-AS was 0.58?0.01pmol and 0.92?0.03pmol at 4 and 37 0 C, respectively. In vivo study revealed the internalization of In-111-N-myc-AS (8nmol) with CML in tumor cells (5x106) as 6.44?0.71pmol, while none (0pmol) of naked In-111-N-myc-AS was internalized. The effect of Auger electrons was shown by a decrease of N-MYC of the tumor cells by 20.6?2.49% in vitro and 12.9?1.17% in vivo in the case of In-111-N-myc-AS with CML, whereas unlabeled AS with CML or In-111-phosphorothioate sense oligonucleotide did not decrease the quantity of N-MYC of the tumor cells in vitro or in vivo. In conclusion, In-111-N-myc-AS with CML could be internalized into human neuroblastoma cells and suppress the activity of N-myc gene, which may prove useful for targeted Auger electrons radiotherapy. (author)

  5. Effect of Auger electrons internalized as Indium-111-labeled N-MYC phosphorothioate antisense oligonucleotide (In-111-N-MYC-AS) on human neuroblastoma cells: In-vitro and in-vivo studies

    International Nuclear Information System (INIS)

    Watanabe, Naoyuki; Tanada, Shuju; Sasaki, Yasuhito

    2004-01-01

    Full text: Auger electrons, which enter into cells, cause biological effects with high-LET short-range radiation on the neighborhood. The purpose of this study is to investigate the internalization of In-111-N-myc-AS and suppression of NMYC in human neuroblastoma cells. Fifteen-mer AS, which was complementary to the region of the mRNA of N-myc beginning with ATG start codon, was derivated with SCN-Bn-EDTA and labeled with In-111. In-111-Nmyc-AS (1.33MBq/nmol) was prepared as naked form and encapsulated in cationic multilamellar liposome (CML). The internalization of In-111-N-myc-AS with or without CML into human neuroblastoma SK-N-DZ cells was determined both in vitro (cell culture) and in vivo (tumor bearing nude mice) studies by Southern blot analysis. Quantity of N-MYC in the tumor cells was also measured by Western blot analysis. In in-vitro system 0.69∫0.02pmol of In-111-N-myc-AS (80pmol) with CML was internalized in the cells (5x106) by 12h at 4 deg. C, which increased to 8.05±0.43pmol at 37 deg. C. The internalized naked In-111-N-myc-AS was 0.58±0.01pmol and 0.92±0.03pmol at 4 and 37 deg. C, respectively. In-vivo study revealed the internalization of In-111-N-myc-AS (8nmol) with CML in tumor cells (5x106) as 6.44±0.71pmol, while none (0pmol) of naked In-111-Nmyc-AS was internalized. The effect of Auger electrons was shown by a decrease of N-MYC of the tumor cells by 20.6±2.49% in- vitro and 12.9±1.17% in vivo in the case of In-111-N-myc-AS with CML, whereas unlabeled AS with CML or In-111-phosphorothioate sense oligonucleotide did not decrease the quantity of N-MYC of the tumor cells in- vitro or in- vivo. In-111-N-myc-AS with CML could be internalized into human neuroblastoma cells and suppress the activity of N-myc gene, which may prove useful for targeted Auger electrons radiotherapy. (author)

  6. Acute Complications After High-Dose Chemotherapy and Stem-Cell Rescue in Pediatric Patients With High-Risk Neuroblastoma Treated in Countries With Different Resources

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Elzembely

    2018-03-01

    Full Text Available Purpose: High-dose chemotherapy with autologous stem-cell rescue (SCR is a key component of high-risk neuroblastoma (HRNB therapy. Carboplatin, etoposide, and melphalan (CEM or busulfan and melphalan (Bu/Mel are the most evaluated, effective high-dose chemotherapy for HRNB on the basis of results from major cooperative group studies. Toxicity profiles vary between these regimens, and practice variation exists regarding the preferred high-dose therapy (HDT. We sought to evaluate the safety of HDT and autologous SCR for HRNB in a resource-limited country (Egypt compared with the resource-rich United States. Patients and Methods: We performed a retrospective comparative review of single CEM-based HDT/SCR outcomes through day 100 for HRNB at the Fred Hutchinson Cancer Research Center (FH in the United States (2005 to 2015 versus Bu/Mel-based HDT at El-Sheikh Zayed Specialized Hospital (SZ in Egypt (2009 to 2015. Results: Forty-four patients at FH and 77 patients at SZ were reviewed. Pretransplant hepatic comorbidities were significantly higher at SZ (29 of 77 v nine of 44; P = .05, with 19 of 77 patients at SZ having hepatitis infection. Engraftment was delayed after SZ-Bu/Mel therapy compared with FH-CEM therapy for neutrophils (median 12 days v 10 days, respectively; P < .001 and platelets (median 20 days v 18 days, respectively; P < .001. Sinusoidal obstruction syndrome occurred later, after SZ-Bu/Mel therapy (median 19 days v 7 days; P = .033, and four of eight cases were fatal (six of eight patients had underlying hepatitis infection, whereas three of three cases after FH-CEM therapy were moderately severe. Resource utilization associated with the number of days with fever, antibiotic use, and the number of transfusions administered was significantly higher after FH-CEM therapy than after SZ-Bu/Mel therapy. Conclusion: Use of autologous stem-cell transplantation is feasible in the context of a resource-limited country.

  7. Adult Neuroblastoma Complicated by Increased Intracranial Pressure: A Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Patrick L. Stevens

    2014-01-01

    Full Text Available Neuroblastoma is the third most commonly occurring malignancy of the pediatric population, although it is extremely rare in the adult population. In adults, neuroblastoma is often metastatic and portends an extremely poor overall survival. Our case report documents metastatic neuroblastoma occurring in a healthy 29-year-old woman whose course was complicated by an unusual presentation of elevated intracranial pressures. The patient was treated with systemic chemotherapy, I131 metaiodobenzylguanidine (MIBG radiotherapy, and autologous stem cell transplant (SCT. Unfortunately the patient’s response to therapy was limited and she subsequently died. We aim to review neuroblastoma in the context of increased intracranial pressure and the limited data of neuroblastoma occurring in the adult population, along with proposed treatment options.

  8. Therapeutic Innovations for Targeting Childhood Neuroblastoma: Implications of the Neurokinin-1 Receptor System.

    Science.gov (United States)

    Berger, Michael; VON Schweinitz, Dietrich

    2017-11-01

    Neuroblastoma is the most common solid extracranial malignant tumor in children. Despite recent advances in the treatment of this heterogenous tumor with surgery and chemotherapy, the prognosis in advanced stages remains poor. Interestingly, neuroblastoma is one of the few solid tumors, to date, in which an effect for targeted immunotherapy has been proven in controlled clinical trials, giving hope for further advances in the treatment of this and other tumors by targeted therapy. A large array of novel therapeutic options for targeted therapy of neuroblastoma is on the horizon. To this repεrtoirε, the neurokinin-1 receptor (NK1R) system was recently added. The present article explores the most recent developments in targeting neuroblastoma cells via the NK1R and how this new knowledge could be helpful to create new anticancer therapies agains neuroblastoma and other cancers. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  9. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

    Science.gov (United States)

    Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

    2017-05-01

    High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

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

    Science.gov (United States)

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

    2016-05-01

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

  11. Neuroprotective Effects of 1,2-Diarylpropane Type Phenylpropanoid Enantiomers from Red Raspberry against H2O2-Induced Oxidative Stress in Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Zhou, Le; Yao, Guo-Dong; Song, Xiao-Yu; Wang, Jie; Lin, Bin; Wang, Xiao-Bo; Huang, Xiao-Xiao; Song, Shao-Jiang

    2018-01-10

    Red raspberry (Rubus idaeus L.) is an edible fruit-producing species belonging to the Rosaceae family. In our search for the health-promoting constituents from this fruit, four pairs of enantiomeric phenylpropanoids (1a/1b-4a/4b), including three new compounds (1a and 2a/2b), were isolated from red raspberry. Their structures were elucidated by a combination of the extensive NMR spectroscopic data analyses, high-resolution electrospray ionization mass spectrometry and comparison between the experimental measurements of electronic circular dichroism (ECD) and calculated ECD spectra by time-dependent density functional theory (TDDFT). In addition, their neuroprotective effects against H 2 O 2 -induced oxidative stress in human neuroblastoma SH-SY5Y cells were investigated, and the results showed enantioselectivity, in which that 3a exhibited noticeable neuroprotective activity, while its enatiomer 3b exhibited no obvious protective effect. Further study demonstrated that 3a could selectively inhibit the apoptosis induction and reactive oxygen species (ROS) accumulation by enhancing the activity of catalase (CAT) in H 2 O 2 -treated human neuroblastoma SH-SY5Y cells. These findings shed much light on a better understanding of the neuroprotective effects of these enantiomers and provide new insights into developing better treatment of neurodegenerative diseases in the future.

  12. Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

    Science.gov (United States)

    Mussai, Francis; Egan, Sharon; Hunter, Stuart; Webber, Hannah; Fisher, Jonathan; Wheat, Rachel; McConville, Carmel; Sbirkov, Yordan; Wheeler, Kate; Bendle, Gavin; Petrie, Kevin; Anderson, John; Chesler, Louis; De Santo, Carmela

    2015-01-01

    Neuroblastoma is the most common extra cranial solid tumour of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumour cells suppress T cell proliferation, through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34+ progenitor proliferation. Finally we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1 specific TCR and GD2-specific CAR engineered T cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for neuroblastoma patients. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumour and blood that leads to impaired immune surveillance and sub-optimal efficacy of immunotherapeutic approaches. PMID:26054597

  13. Insulin Promotes Survival of Amyloid-Beta Oligomers Neuroblastoma Damaged Cells via Caspase 9 Inhibition and Hsp70 Upregulation

    Directory of Open Access Journals (Sweden)

    M. Di Carlo

    2010-01-01

    Full Text Available Alzheimer's disease (AD and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

  14. Acceleration of gene transfection efficiency in neuroblastoma cells through polyethyleneimine/poly(methyl methacrylate) core-shell magnetic nanoparticles

    Science.gov (United States)

    Tencomnao, Tewin; Klangthong, Kewalin; Pimpha, Nuttaporn; Chaleawlert-umpon, Saowaluk; Saesoo, Somsak; Woramongkolchai, Noppawan; Saengkrit, Nattika

    2012-01-01

    Background The purpose of this study was to demonstrate the potential of magnetic poly(methyl methacrylate) (PMMA) core/polyethyleneimine (PEI) shell (mag-PEI) nanoparticles, which possess high saturation magnetization for gene delivery. By using mag-PEI nanoparticles as a gene carrier, this study focused on evaluation of transfection efficiency under magnetic induction. The potential role of this newly synthesized nanosphere for therapeutic delivery of the tryptophan hydroxylase-2 (TPH-2) gene was also investigated in cultured neuronal LAN-5 cells. Methods The mag-PEI nanoparticles were prepared by one-step emulsifier-free emulsion polymerization, generating highly loaded and monodispersed magnetic polymeric nanoparticles bearing an amine group. The physicochemical properties of the mag-PEI nanoparticles and DNA-bound mag-PEI nanoparticles were investigated using the gel retardation assay, atomic force microscopy, and zeta size measurements. The gene transfection efficiencies of mag-PEI nanoparticles were evaluated at different transfection times. Confocal laser scanning microscopy confirmed intracellular uptake of the magnetoplex. The optimal conditions for transfection of TPH-2 were selected for therapeutic gene transfection. We isolated the TPH-2 gene from the total RNA of the human medulla oblongata and cloned it into an expression vector. The plasmid containing TPH-2 was subsequently bound onto the surfaces of the mag-PEI nanoparticles via electrostatic interaction. Finally, the mag-PEI nanoparticle magnetoplex was delivered into LAN-5 cells. Reverse-transcriptase polymerase chain reaction was performed to evaluate TPH-2 expression in a quantitative manner. Results The study demonstrated the role of newly synthesized high-magnetization mag-PEI nanoparticles for gene transfection in vitro. The expression signals of a model gene, luciferase, and a therapeutic gene, TPH-2, were enhanced under magnetic-assisted transfection. An in vitro study in neuronal cells

  15. La infección por virus dengue induce la disminución de marcadores de diferenciación en células de neuroblastoma = Dengue virus infection down-regulates differentiation markers in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Rincón Forero, Verónica del Pilar

    2011-06-01

    Full Text Available Introducción: cerca del 5% de los pacientes con dengue hemorrágico pueden presentar manifestaciones neurológicas; sin embargo, existe poca información sobre la infección directa por el virus dengue (DENV en neuronas.Objetivo: determinar el papel del fenotipo neuronal en la infección por DENV en células de neuroblastoma SH-SY5Y inducidas o no a la diferenciación con ácido retinoico (AR.Materiales y métodos: células SH-SY5Y fueron inducidas con AR a diferenciarse e infectadas con DENV. Posteriormente se cuantificó la expresión de antígeno viral y de dos marcadores de diferenciación (GAP43 y sinaptofisina. También se evaluó la viabilidad postinfección por la técnica de MTT.Resultados: se encontró que las células diferenciadas son más susceptibles a la infección por DENV, pues se detectó en ellas mayor cantidad de antígeno viral que en las indiferenciadas. A pesar de que el virus indujo muerte celular en ambos tipos de células, la proporción fue mayor en las indiferenciadas (40,3% frente a 21,5%. La infección por DENV en células SH-SY5Y diferenciadas indujo una disminución significativa en la expresión de GAP-43 y sinaptofisina.Conclusiones: los resultados que se presentan permiten sugerir una relación entre la infección viral y la función neuronal, que podría ser importante para esclarecer la patogénesis de las manifestaciones neurológicas durante las formas graves de dengue.

  16. Genomewide analysis of gene expression associated with Tcof1 in mouse neuroblastoma.

    Science.gov (United States)

    Mogass, Michael; York, Timothy P; Li, Lin; Rujirabanjerd, Sinitdhorn; Shiang, Rita

    2004-12-03

    Mutations in the Treacher Collins syndrome gene, TCOF1, cause a disorder of craniofacial development. We manipulated the levels of Tcof1 and its protein treacle in a murine neuroblastoma cell line to identify downstream changes in gene expression using a microarray platform. We identified a set of genes that have similar expression with Tcof1 as well as a set of genes that are negatively correlated with Tcof1 expression. We also showed that the level of Tcof1 and treacle expression is downregulated during differentiation of neuroblastoma cells into neuronal cells. Inhibition of Tcof1 expression by siRNA induced morphological changes in neuroblastoma cells that mimic differentiation. Thus, expression of Tcof1 and treacle synthesis play an important role in the proliferation of neuroblastoma cells and we have identified genes that may be important in this pathway.

  17. Clearance of Damaged Mitochondria Through PINK1 Stabilization by JNK and ERK MAPK Signaling in Chlorpyrifos-Treated Neuroblastoma Cells.

    Science.gov (United States)

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

    2017-04-01

    Mitochondrial quality control and clearance of damaged mitochondria through mitophagy are important cellular activities. Studies have shown that PTEN-induced putative protein kinase 1 (PINK1) and Parkin play central roles in triggering mitophagy; however, little is known regarding the mechanism by which PINK1 modulates mitophagy in response to reactive oxygen species (ROS)-induced stress. In this study, chlorpyrifos (CPF)-induced ROS caused mitochondrial damage and subsequent engulfing of mitochondria in double-membrane autophagic vesicles, indicating that clearance of damaged mitochondria is due to mitophagy. CPF treatment resulted in PINK1 stabilization on the outer mitochondrial membrane and subsequently increased Parkin recruitment from the cytosol to the abnormal mitochondria. We found that PINK1 physically interacts with Parkin in the mitochondria of CPF-treated cells. Furthermore, a knockdown of PINK1 strongly inhibited the LC3-II protein level by blocking Parkin recruitment. This indicates that CPF-induced mitophagy is due to PINK1 stabilization in mitochondria. We observed that PINK1 stabilization was selectively regulated by ROS-mediated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling activation but not p38 signaling. In the mitochondria of CPF-exposed cells, pretreatment with specific inhibitors of JNK and ERK1/2 significantly decreased PINK1 stabilization and Parkin recruitment and blocked the LC3-II protein level. Specifically, JNK and ERK1/2 inhibition also dramatically blocked the interaction between PINK1 and Parkin. Our results demonstrated that PINK1 regulation plays a critical role in CPF-induced mitophagy. The simple interpretation of these results is that JNK and ERK1/2 signaling regulates PINK1/Parkin-dependent mitophagy in the mitochondria of CPF-treated cells. Overall, this study proposes a novel molecular regulatory mechanism of PINK1 stabilization under CPF exposure.

  18. Mitochondrial Effects of PGC-1alpha Silencing in MPP+ Treated Human SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Qinyong Ye

    2017-05-01

    Full Text Available The dopaminergic neuron degeneration and loss that occurs in Parkinson’s disease (PD has been tightly linked to mitochondrial dysfunction. Although the aged-related cause of the mitochondrial defect observed in PD patients remains unclear, nuclear genes are of potential importance to mitochondrial function. Human peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α is a multi-functional transcription factor that tightly regulates mitochondrial biogenesis and oxidative capacity. The goal of the present study was to explore the potential pathogenic effects of interference by the PGC-1α gene on N-methyl-4-phenylpyridinium ion (MPP+-induced SH-SY5Y cells. We utilized RNA interference (RNAi technology to probe the pathogenic consequences of inhibiting PGC-1α in the SH-SY5Y cell line. Remarkably, a reduction in PGC-1α resulted in the reduction of mitochondrial membrane potential, intracellular ATP content and intracellular H2O2 generation, leading to the translocation of cytochrome c (cyt c to the cytoplasm in the MPP+-induced PD cell model. The expression of related proteins in the signaling pathway (e.g., estrogen-related receptor α (ERRα, nuclear respiratory factor 1 (NRF-1, NRF-2 and Peroxisome proliferator-activated receptor γ (PPARγ also decreased. Our finding indicates that small interfering RNA (siRNA interference targeting the PGC-1α gene could inhibit the function of mitochondria in several capacities and that the PGC-1α gene may modulate mitochondrial function by regulating the expression of ERRα, NRF-1, NRF-2 and PPARγ. Thus, PGC-1α can be considered a potential therapeutic target for PD.

  19. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  20. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    Energy Technology Data Exchange (ETDEWEB)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  1. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB.20

    International Nuclear Information System (INIS)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT 1C receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing [ 3 H]serotonin, [ 3 H]lysergic acid diethylamide or [ 3 H]dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor

  2. SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

    Science.gov (United States)

    Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

    2016-05-01

    This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Marrow Derived Antibody Library for the Treatment of Neuroblastoma

    Science.gov (United States)

    2015-12-01

    identified with strong binding to tumor cells. In Figure 1, the positive binding of phage clones to MGT-003 cells were visualized using anti- M13 phage ...neuroblastoma patients using phage display and B cell hybridoma technologies. The scope of this project is to use NB patient-derived materials to...create NB cell lines, xenograft models, NB specific phage display libraries and to identify and amplify functional anti-NB specific antibodies for future

  4. Mesenchymal change and drug resistance in neuroblastoma.

    Science.gov (United States)

    Naiditch, Jessica A; Jie, Chunfa; Lautz, Timothy B; Yu, Songtao; Clark, Sandra; Voronov, Dimitry; Chu, Fei; Madonna, Mary Beth

    2015-01-01

    Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P change via multiple pathways in the transition to a drug-resistant state. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. A Pilot Trial of Humanized Anti-GD2 Monoclonal Antibody (hu14.18K322A) with Chemotherapy and Natural Killer Cells in Children with Recurrent/Refractory Neuroblastoma.

    Science.gov (United States)

    Federico, Sara M; McCarville, M Beth; Shulkin, Barry L; Sondel, Paul M; Hank, Jacquelyn A; Hutson, Paul; Meagher, Michael; Shafer, Aaron; Ng, Catherine Y; Leung, Wing; Janssen, William E; Wu, Jianrong; Mao, Shenghua; Brennan, Rachel C; Santana, Victor M; Pappo, Alberto S; Furman, Wayne L

    2017-11-01

    Purpose: Anti-GD2 mAbs, acting via antibody-dependent cell-mediated cytotoxicity, may enhance the effects of chemotherapy. This pilot trial investigated a fixed dose of a unique anti-GD2 mAb, hu14.18K322A, combined with chemotherapy, cytokines, and haploidentical natural killer (NK) cells. Experimental Design: Children with recurrent/refractory neuroblastoma received up to six courses of hu14.18K322A (40 mg/m 2 /dose, days 2-5), GM-CSF, and IL2 with chemotherapy: cyclophosphamide/topotecan (courses 1,2), irinotecan/temozolomide (courses 3,4), and ifosfamide/carboplatin/etoposide (courses 5,6). Parentally derived NK cells were administered with courses 2, 4, and 6. Serum for pharmacokinetic studies of hu14.18K322A, soluble IL2 receptor alpha (sIL2Rα) levels, and human antihuman antibodies (HAHA) were obtained. Results: Thirteen heavily pretreated patients (9 with prior anti-GD2 therapy) completed 65 courses. One patient developed an unacceptable toxicity (grade 4 thrombocytopenia >35 days). Four patients discontinued treatment for adverse events (hu14.18K322A allergic reaction, viral infection, surgical death, second malignancy). Common toxicities included grade 3/4 myelosuppression (13/13 patients) and grade 1/2 pain (13/13 patients). Eleven patients received 29 NK-cell infusions. The response rate was 61.5% (4 complete responses, 1 very good partial response, 3 partial responses) and five had stable disease. The median time to progression was 274 days (range, 239-568 days); 10 of 13 patients (77%) survived 1 year. Hu14.18K322A pharmacokinetics was not affected by chemotherapy or HAHA. All patients had increased sIL2Rα levels, indicating immune activation. Conclusions: Chemotherapy plus hu14.18K322A, cytokines, and NK cells is feasible and resulted in clinically meaningful responses in patients with refractory/recurrent neuroblastoma. Further studies of this approach are warranted in patients with relapsed and newly diagnosed neuroblastoma. Clin Cancer Res; 23

  6. Genome-wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma.

    Science.gov (United States)

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J; Almutairi, Bader; Etchevers, Heather C; McConville, Carmel; Malik, Karim T A; Brown, Keith W

    2017-04-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

  7. Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

    Science.gov (United States)

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R.; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J.; Almutairi, Bader; Etchevers, Heather C.; McConville, Carmel; Malik, Karim T. A.

    2016-01-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27862318

  8. MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma.

    LENUS (Irish Health Repository)

    Tivnan, Amanda

    2011-01-01

    Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis.

  9. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    International Nuclear Information System (INIS)

    Akter, Jesmin; Takatori, Atsushi; Islam, Md. Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

    2014-01-01

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas

  10. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Akter, Jesmin [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Islam, Md. Sazzadul [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakazawa, Atsuko [Department of Pathology, National Center for Child Health and Development, Tokyo (Japan); Ozaki, Toshinori, E-mail: tozaki@chiba-cc.jp [Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nagase, Hiroki [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakagawara, Akira [Saga Medical Centre, 840-8571 (Japan)

    2014-10-10

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.

  11. LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis.

    Science.gov (United States)

    Zhu, Shizhen; Zhang, Xiaoling; Weichert-Leahey, Nina; Dong, Zhiwei; Zhang, Cheng; Lopez, Gonzalo; Tao, Ting; He, Shuning; Wood, Andrew C; Oldridge, Derek; Ung, Choong Yong; van Ree, Janine H; Khan, Amish; Salazar, Brittany M; Lummertz da Rocha, Edroaldo; Zimmerman, Mark W; Guo, Feng; Cao, Hong; Hou, Xiaonan; Weroha, S John; Perez-Atayde, Antonio R; Neuberg, Donna S; Meves, Alexander; McNiven, Mark A; van Deursen, Jan M; Li, Hu; Maris, John M; Look, A Thomas

    2017-09-11

    A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. An Involvement of PI3-K/Akt Activation and Inhibition of AIF Translocation in Neuroprotective Effects of Undecylenic Acid (UDA) Against Pro-Apoptotic Factors-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Jantas, Danuta; Piotrowski, Marek; Lason, Wladyslaw

    2015-12-01

    Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies. © 2015 Wiley Periodicals, Inc.

  13. Verhalten von unbestrahltem ZrNb 1-Rohr unter tangentialer Zugdehnung in jodhaltiger Atmosphäre

    Science.gov (United States)

    Böhmert, J.; Lübke, L.; Tzscheutschler, W.

    1986-07-01

    The effect of iodine on the mechanical properties of unirradiated recrystallized ZrNb 1 tubing at 350°C was studied by means of the ring tension test. At strain rates < 10 -3/s iodine leads to a substantial decrease in the totale elongation and the reduction of area, but does neither change the 0.2% yield stress, the ultimate tensile stress nor the uniform elongation. The results indicate that iodine induced crack formation in unirradiated recrystallized ZrNb 1 occurs only in the necking region after marked plastic deformation.

  14. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Science.gov (United States)

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies

  15. Identification of BIRC6 as a novel intervention target for neuroblastoma therapy

    Directory of Open Access Journals (Sweden)

    Lamers Fieke

    2012-07-01

    Full Text Available Abstract Background Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. Methods Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform (http://r2.amc.nl. BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. Results We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP, that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines. Conclusion Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.

  16. Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

    Science.gov (United States)

    Burchill, Susan A; Beiske, Klaus; Shimada, Hiroyuki; Ambros, Peter F; Seeger, Robert; Tytgat, Godelieve A M; Brock, Penelope R; Haber, Michelle; Park, Julie R; Berthold, Frank

    2017-04-01

    The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate

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

    Science.gov (United States)

    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 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. Notch ligand Delta-like 1 as a novel molecular target in childhood neuroblastoma.

    Science.gov (United States)

    Bettinsoli, P; Ferrari-Toninelli, G; Bonini, S A; Prandelli, C; Memo, M

    2017-05-19

    Neuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths. It is an heterogeneous disease that does not always respond to classical therapy; so the identification of new and specific molecular targets to improve existing therapy is needed. We have previously demonstrated the involvement of the Notch pathway in the onset and progression of neuroblastoma. In this study we further investigated the role of Notch signaling and identified Delta-like 1 (DLL1) as a novel molecular target in neuroblastoma cells with a high degree of MYCN amplification, which is a major oncogenic driver in neuroblastoma. The possibility to act on DLL1 expression levels by using microRNAs (miRNAs) was assessed. DLL1 mRNA and protein expression levels were measured in three different neuroblastoma cell lines using quantitative real-time PCR and Western Blot analysis, respectively. Activation of the Notch pathway as a result of increased levels of DLL1 was analyzed by Immunofluorescence and Western Blot methods. In silico tools revealed the possibility to act on DLL1 expression levels with miRNAs, in particular with the miRNA-34 family. Neuroblastoma cells were transfected with miRNA-34 family members, and the effect of miRNAs transfection on DLL1 mRNA expression levels, on cell differentiation, proliferation and apoptosis was measured. In this study, the DLL1 ligand was identified as the Notch pathway component highly expressed in neuroblastoma cells with MYCN amplification. In silico analysis demonstrated that DLL1 is one of the targets of miRNA-34 family members that maps on chromosome regions that are frequently deregulated or deleted in neuroblastoma. We studied the possibility to use miRNAs to target DLL1. Among all miRNA-34 family members, miRNA-34b is able to significantly downregulate DLL1 mRNA expression levels, to arrest cell proliferation and to induce neuronal differentiation in malignant neuroblastoma cells

  19. Congenital neuroblastoma with placental involvement

    OpenAIRE

    Kume, Ayako; Morikawa, Teppei; Ogawa, Makiko; Yamashita, Aki; Yamaguchi, Shunichi; Fukayama, Masashi

    2014-01-01

    We describe an extremely rare case of congenital neuroblastoma with placental involvement. A fetus with a left abdominal mass detected during ultrasonography at 23 weeks’ gestation developed hydrops fetalis by 26 weeks’ gestation. The mother developed hypertension at 26 5/7 weeks’ gestation. Based on a clinical diagnosis of pregnancy-induced hypertension, labor was induced at 26 6/7 weeks. However, intrauterine fetal death was diagnosed during delivery. Postmortern examination revealed a soli...

  20. Impedance and ac conductivity studies of Ba (Pr1/2Nb1/2) O3 ceramic

    Indian Academy of Sciences (India)

    Complex impedance as well as electric modulus analyses suggested dielectric relaxation to be of non-Debye type and negative temperature coefficient of resistance character. The correlated barrier hopping model was employed to successfully explain the mechanism of charge transport in Ba(Pr1/2Nb1/2)O3. The a.c. ...

  1. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  2. Iodine-131 Metaiodobenzylguanidine Therapy for Neuroblastoma: Reports So Far and Future Perspective

    Directory of Open Access Journals (Sweden)

    Daiki Kayano

    2015-01-01

    Full Text Available Neuroblastoma, which derives from neural crest, is the most common extracranial solid cancer in childhood. The tumors express the norepinephrine (NE transporters on their cell membrane and take in metaiodobenzylguanidine (MIBG via a NE transporter. Since iodine-131 (I-131 MIBG therapy was firstly reported, many trails of MIBG therapy in patients with neuroblastoma were performed. Though monotherapy with a low dose of I-131 MIBG could achieve high-probability pain reduction, the objective response was poor. In contrast, more than 12 mCi/kg I-131 MIBG administrations with or without hematopoietic cell transplantation (HCT obtain relatively good responses in patients with refractory or relapsed neuroblastoma. The combination therapy with I-131 MIBG and other modalities such as nonmyeloablative chemotherapy and myeloablative chemotherapy with HCT improved the therapeutic response in patients with refractory or relapsed neuroblastoma. In addition, I-131 MIBG therapy incorporated in the induction therapy was proved to be feasible in patients with newly diagnosed neuroblastoma. To expand more the use of MIBG therapy for neuroblastoma, further studies will be needed especially in the use at an earlier stage from diagnosis, in the use with other radionuclide formations of MIBG, and in combined use with other therapeutic agents.

  3. Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

    Science.gov (United States)

    Maugeri, Marco; Barbagallo, Davide; Barbagallo, Cristina; Banelli, Barbara; Di Mauro, Stefania; Purrello, Francesco; Magro, Gaetano; Ragusa, Marco; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

    2016-12-13

    Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5'-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5'-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

  4. Abeta production as consequence of cellular death of a human neuroblastoma overexpressing APP.

    Science.gov (United States)

    Recuero, María; Serrano, Elena; Bullido, María J; Valdivieso, Fernando

    2004-07-16

    In human brain the Abeta peptide is produced mainly by neurons and the overexpression of amyloid precursor protein (APP) that involves an increase in Abeta secretion, has been observed in some areas of the Alzheimer's disease patients brain. We have generated two stably transfected human neuroblastoma lines which overexpress APP; both of them secreted Abeta and showed morphological changes and cell death with apoptotic program characteristics. Interestingly, coculture experiments with the untransfected human neuroblastoma cell line showed that the Abeta peptide was not responsible for the death in those cell lines; additionally, we indicate that upon cell death, Abeta peptide is secreted into cell medium.

  5. Toxicity assessment of organochlorine compounds detected in water environment using cultured human cell lines; Hito yurai saibo baiyokei wo mochiita suikankyo shiryochu no yuki enso kagobutsu no dokusei hyoka

    Energy Technology Data Exchange (ETDEWEB)

    Kunimoto, M.; Yonemoto, J.; Soma, Y.; Nakasugi, O. [National Institute for Environmental Studies, Tsukuba (Japan)

    1997-11-10

    As part of validation processes of in vitro toxicity assays for the risk assessment of environmental hazards, we applied an in vitro toxicity test using two human cell lines, neuroblastoma NB-1 cells and glioblastoma U-87 MG cells, to the assessment of organochlorine compounds detected in the water environment. The in vitro toxicity assay using NB-1 cells was calibrated by testing reference chemicals proposed by MEIC (Multicenter Evaluation of In Vitro Cytotoxicity), an international program for the validation of in vitro cytotoxicity assays. Beforehand, an assay using cells in frozen stock without subcultivation was examined by comparing IC50 values with the ordinary assay using subcultured cells. IC50 values for MEIC reference chemicals from the former assay showed good correlation with those from the latter assay, suggesting that the assay using cells in frozen stock can be used at least for the assessment of basal cytotoxicity. IC50 values for ten organochlorine compounds frequently detected in the sediment samples from contaminated rivers, p-chloroaniline, 3,4-dichloroaniline, p-dichlorobenzene, o-dichlorobenzene, Tris (2-chloroethyl)-phosphate, 2,5-dichlorophenol, 2,5-dichloroanisol, Triclosan and Triclocarban, were obtained with the in vitro assays and compared with their LD50 values in rats. No significant correlation, however, was seen between the IC50 and LD50 values, indicating that further improvement of in vitro toxicity assays is necessary for the application to the risk assessment of environmental hazards. 7 refs., 4 figs., 1 tab.

  6. induced apoptosis of neuroblastoma

    African Journals Online (AJOL)

    SK-N-SH cell pellets were re-suspended in lysis buffer [(20 ... mixtures were incubated with 60 uL TMB substrates for 45 min and then mixed with the stop buffer. The absorbance of each sample was measured at 450 nm. .... Cell lysates were further resolved by SDS-PAGE to verify the changes in protein expression in.

  7. Electrical Properties of Ba3Ca1.18Nb1.82O9-  Proton-Conducting Electrolyte Prepared by a Combustion Method

    KAUST Repository

    Bi, Lei

    2013-10-07

    Ba3Ca1.18Nb1.82O9-δ (BCN18), regarded as a promising proton-conducting electrolyte material for solid oxide fuel cells, is usually synthesized by a solid-state reaction because of the limited choice of Nb precursors. This study presents a wet chemical route for preparing BCN18 powders that were then sintered into pellets. Electrochemical impedance spectroscopy studies indicated that BCN18 pellets show proton conductivity, since their total conductivity in wet air was significantly larger than that in dry air. However, a detailed analysis showed that only the BCN18 bulk behaves as a proton conductor, while its grain boundary conductivity did not increase in wet air.

  8. Processing and microstructure of Nb-1 percent Zr-0.1 percent C alloy sheet

    Science.gov (United States)

    Uz, Mehmet; Titran, Robert H.

    1992-01-01

    A systematic study was carried out to evaluate the effects of processing on the microstructure of Nb-1 wt. pct. Zr-0.1 wt. pct. C alloy sheet. The samples were fabricated by cold rolling different sheet bars that were single-, double- or triple-extruded at 1900 K. Heat treatment consisted on one- or two-step annealing of different samples at temperatures ranging from 1350 to 1850 K. The assessment of the effects of processing on microstructure involved characterization of the precipitates including the type, crystal structure, chemistry and distribution within the material as well as an examination of the grain structure. A combination of various analytical and metallographic techniques were used on both the sheet samples and the residue extracted from them. The results show that the relatively coarse orthorhombic Nb2C carbides in the as-rolled samples transformed to rather fine cubic monocarbides of Nb and Zr with varying Zr/Nb ratios upon subsequent heat treatment. The relative amount of the cubic carbides and the Zr/Nb ratio increased with increasing number of extrusions prior to cold rolling. Furthermore, the size and the aspect ratio of the grains appear to be strong functions of the processing history of the material. These and other results obtained will be presented with the emphasis on a possible relationship between processing and microstructure.

  9. Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

    Science.gov (United States)

    Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

    2017-07-01

    Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  10. MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

    Science.gov (United States)

    Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B.; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M.; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E.; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H.; Deubzer, Hedwig E.

    2016-01-01

    The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma. PMID:27572323

  11. Neuroblastoma : Crossing borders in targeted therapy

    NARCIS (Netherlands)

    Bate-Eya, L.T.

    2017-01-01

    Neuroblastoma is the most commonly diagnosed childhood cancer and accounts for about 15% of all pediatric malignancies deaths. Thus far, the treatment options of neuroblastoma is limited with only a 30-40% long term survival rate in high-risk patients. In this thesis, we describe the isolation and

  12. Advances in the translational genomics of neuroblastoma

    Science.gov (United States)

    Bosse, Kristopher R.; Maris, John M.

    2015-01-01

    Neuroblastoma is an embryonal malignancy that commonly affects young children and is remarkably heterogenous in its malignant potential. Recently, the genetic basis of neuroblastoma has come into focus, which has catalyzed not only a more comprehensive understanding of neuroblastoma tumorigenesis, but has also revealed novel oncogenic vulnerabilities that are being leveraged therapeutically. Neuroblastoma is a model pediatric solid tumor in its use of recurrent genomic alterations, such as high-level MYCN amplification, for risk stratification. Given the relative paucity of recurrent activating somatic point mutations or gene fusions in primary neuroblastoma tumors studied at initial diagnosis, innovative treatment approaches beyond small molecules targeting mutated or dysregulated kinases will be required moving forward to achieve noticeable improvements in overall patient survival. However, the clonally acquired, oncogenic aberrations in relapsed neuroblastomas are currently being defined and may offer an opportunity to improve patient outcomes with molecularly targeted therapy directed towards aberrantly regulated pathways in relapsed disease. This review will summarize the current state of knowledge of neuroblastoma genetics and genomics, highlighting the improved prognostication and potential therapeutic opportunities that have arisen from recent advances in understanding germline predisposition, recurrent segmental chromosomal alterations, somatic point mutations and translocations, and clonal evolution in relapsed neuroblastoma. PMID:26539795

  13. Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

    Science.gov (United States)

    Murray, Jayne; Valli, Emanuele; Yu, Denise M T; Truong, Alan M; Gifford, Andrew J; Eden, Georgina L; Gamble, Laura D; Hanssen, Kimberley M; Flemming, Claudia L; Tan, Alvin; Tivnan, Amanda; Allan, Sophie; Saletta, Federica; Cheung, Leanna; Ruhle, Michelle; Schuetz, John D; Henderson, Michelle J; Byrne, Jennifer A; Norris, Murray D; Haber, Michelle; Fletcher, Jamie I

    2017-09-01

    The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Disseminated peripheral neuroblastoma in a Rhodesian Ridgeback dog.

    Science.gov (United States)

    Cook, R W; Abraham, L A; McCowan, C I

    2017-04-01

    A 4-year-old neutered male Rhodesian Ridgeback dog with right-sided Horner's syndrome, bilateral laryngeal paralysis, neck pain and bilateral hindlimb ataxia was euthanased following deterioration of its neurological status. Necropsy examination revealed an off-white retropharyngeal neoplastic mass (100 × 30 × 30 mm) attached to the base of the skull on the right side and macroscopic nodular metastases in the spleen and three vertebral bodies (C6, C7 and T6), including a nodule attached to the dura at C7. Histological evidence of neuroblastic tumour was detected in these macroscopic lesions, a regional lymph node, bone marrow of a femur and all 15 vertebral bodies (C1-T8) examined, including the three with macroscopic metastases, and in the lumens of small blood vessels in the lungs and liver. Ganglion cell differentiation was detected only in the primary retropharyngeal mass, one splenic nodule and the C7 dural nodule. Neoplastic cells were immunoreactive to neurofilament protein (ganglion cells only), vimentin and synaptophysin, and were negative for S100 protein, GFAP, CD3 and Pax5. The diagnosis was disseminated peripheral neuroblastoma, differentiating subtype (International Neuroblastoma Pathology Classification), with likely primary involvement of the right cranial cervical ganglion. This appears to be the first report of neuroblastoma in a dog with widespread occult haematogenous metastasis to bone marrow. © 2017 Australian Veterinary Association.

  15. Association of telomerase activity with radio- and chemosensitivity of neuroblastomas

    Directory of Open Access Journals (Sweden)

    Willich Normann

    2010-07-01

    Full Text Available Abstract Background Telomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA and telomere length on the outcome of radio- and chemotherapy in neuroblastoma. Methods We studied the effects on dominant negative (DN mutant, wild type (WT of the telomerase catalytic unit (hTERT using neuroblastoma cell lines. The cells were irradiated with 60Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI50 was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay. Results Compared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line. Conclusions Our results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1 or Side population (SP may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.

  16. Intestinal Lymphangiectasia Secondary to Neuroblastoma

    Directory of Open Access Journals (Sweden)

    RM Reifen

    1994-01-01

    Full Text Available An eight month-old infant presented with a 10-day history of vomiting and diarrhea, and a one-week history of swelling of the lower extremities. Laboratory evaluations revealed hypoproteinemia and lymphocytopenia due to protein-losing enteropathy. Peroral small bowel biopsy showed intestinal lymphangiectasia. Subsequent onset of unexplained ecchymosis and obstructive jaundice resulted in additional studies which revealed an omental neuroblastoma as the underlying etiology of the infant’s symptoms. This report emphasizes the importance of considering secondary, obstructive causes for lymphangiectasia and protein-losing enteropathy.

  17. MIBG-treatment in neuroblastoma

    International Nuclear Information System (INIS)

    Treuner, J.; Gerein, V.; Klingebiel, T.; Schwabe, D.; Feine, U; Happ, J.; Niethammer, D.; Maul, F.; Dopfer, R.; Kornhuber, B.; Berthold, F.; Jurgens, H.; Hor, G.

    1988-01-01

    This paper reports the results of 27 children with neuroblastoma treated with 131 I-Metaiodobenzylguanidine (MIBG). They were either refractory to conventional therapy or experienced relapse after initially successful treatment. 7 children revealed stage IV and 20 stage III at the beginning of MIBG-treatment. MIBG was administered by infusion lasting from 30 min to 30 hrs. In most children the dose was split into two portions each infused over a period of 4 hrs with a 24 hrs interval between. Courses were repeated up to 6 times and maximum activity given to one patient cumulatively was 38,221 MBq. 24 patients were valuable for analysis of results

  18. Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan

    Science.gov (United States)

    Chaturvedi, Nagendra K.; McGuire, Timothy R.; Coulter, Don W.; Shukla, Ashima; McIntyre, Erin M.; Sharp, John Graham; Joshi, Shantaram S.

    2016-01-01

    Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic. PMID:26934655

  19. Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

    Science.gov (United States)

    Caillaud, A; Eixarch, H; de la Iglesia, P; Rodriguez, M; Dominguez, L; Andree, K B; Diogène, J

    2012-01-01

    The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

  20. Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

    Science.gov (United States)

    Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J; Reifenberger, Guido; Büsselberg, Dietrich

    2017-04-04

    Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 μM) or TOPO (0.1 nM-1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

  1. Expression of Truncated Neurokinin-1 Receptor in Childhood Neuroblastoma is Independent of Tumor Biology and Stage.

    Science.gov (United States)

    Pohl, Alexandra; Kappler, Roland; Mühling, Jakob; VON Schweinitz, Dietrich; Berger, Michael

    2017-11-01

    Neuroblastoma is an embryonal malignancy arising from the aberrant growth of neural crest progenitor cells of the sympathetic nervous system. The tachykinin receptor 1 (TACR1) - substance P complex is associated with tumoral angiogenesis and cell proliferation in a variety of cancer types. Inhibition of TACR1 was recently described to impede growth of NB cell lines. However, the relevance of TACR1 in clinical settings is unknown. We investigated gene expression levels of full-length and truncated TACR1 in 59 neuroblastomas and correlated these data with the patients' clinical parameters such as outcome, metastasis, International Neuroblastoma Staging System (INSS) status, MYCN proto-oncogene, bHLH transcription factor (MYCN) status, gender and age. Our results indicated that TACR1 is ubiquitously expressed in neuroblastoma but expression levels are independent of clinical parameters. Our data suggest that TACR1 might serve as a potent anticancer target in a large variety of patients with neuroblastoma, independent of tumor biology and clinical stage. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. Activated ALK signals through the ERK-ETV5-RET pathway to drive neuroblastoma oncogenesis.

    Science.gov (United States)

    Lopez-Delisle, Lucille; Pierre-Eugène, Cécile; Louis-Brennetot, Caroline; Surdez, Didier; Raynal, Virginie; Baulande, Sylvain; Boeva, Valentina; Grossetête-Lalami, Sandrine; Combaret, Valérie; Peuchmaur, Michel; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2018-03-01

    Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alk mut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/Ret M919T tumors exhibit histological features and expression profiles close to MYCN/Alk mut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alk mut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and Alk F1178L -driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.

  3. Nuclear medicine and multimodality imaging of pediatric neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Wolfgang Peter; Pfluger, Thomas [Ludwig-Maximilians-University of Munich, Department of Nuclear Medicine, Munich (Germany); Coppenrath, Eva [Ludwig-Maximilians-University of Munich, Department of Radiology, Munich (Germany)

    2013-04-15

    Neuroblastoma is an embryonic tumor of the peripheral sympathetic nervous system and is metastatic or high risk for relapse in nearly 50% of cases. Therefore, exact staging with radiological and nuclear medicine imaging methods is crucial for defining the adequate therapeutic choice. Tumor cells express the norepinephrine transporter, which makes metaiodobenzylguanidine (MIBG), an analogue of norepinephrine, an ideal tumor specific agent for imaging. MIBG imaging has several disadvantages, such as limited spatial resolution, limited sensitivity in small lesions and the need for two or even more acquisition sessions. Most of these limitations can be overcome with positron emission tomography (PET) using [F-18]2-fluoro-2-deoxyglucose [FDG]. Furthermore, new tracers, such as fluorodopa or somatostatin receptor agonists, have been tested for imaging neuroblastoma recently. However, MIBG scintigraphy and PET alone are not sufficient for operative or biopsy planning. In this regard, a combination with morphological imaging is indispensable. This article will discuss strategies for primary and follow-up diagnosis in neuroblastoma using different nuclear medicine and radiological imaging methods as well as multimodality imaging. (orig.)

  4. Optimization of liposomal topotecan for use in treating neuroblastoma.

    Science.gov (United States)

    Chernov, Lina; Deyell, Rebecca J; Anantha, Malathi; Dos Santos, Nancy; Gilabert-Oriol, Roger; Bally, Marcel B

    2017-06-01

    The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK-N-SH, IMR-32 and LAN-1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper-drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug-to-lipid ratio engendered significant increases in drug retention. Dose-range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug-to-lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10-fold increase in plasma half-life and a 1000-fold increase in AUC 0-24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN-1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high-dose radiotherapy such as 131 I-metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  5. Bactericidal and Cytotoxic Activities of Polyphenol Extracts from Solanum tuberosum spp. tuberosum and spp. andigena Cultivars on Escherichia coli and Human Neuroblastoma SH-SY5Y Cells In Vitro

    Directory of Open Access Journals (Sweden)

    María Ximena Silveyra

    2018-01-01

    Full Text Available Potatoes (Solanum tuberosum L. are a good source of dietary antioxidant polyphenols. This study investigated the potential antioxidant, bactericidal, and cytotoxic activities in vitro of the phenolic compounds present in tubers of one S. tuberosum spp. tuberosum (Summerside, and three S. tuberosum spp. andigena (landraces Moradita, Waicha, and Santa María cultivars. Both the content of phenolic acids, chlorogenic acids (CGAs being the most abundant, and the antioxidant activity were higher in extracts from skin than from flesh. Extracts from Moradita flesh and Summerside skin showed bactericidal activity against Escherichia coli ATCC 25922 but failed to inhibit pathogenic E. coli O157. Both extracts lack pigmentation but do contain 5-CGA, caffeic, and ferulic acids. Positive control with gentamicin and commercial 5-CGA resulted in a complete inhibition of bacterial growth. In addition, all potato extracts and commercial 5-CGA diminished dose-dependently human neuroblastoma SH-SY5Y cell viability. Skin extracts were more potent than flesh extracts. Among flesh extracts, Moradita was the most potent. Together, our results suggest that Moradita flesh could provide a desirable source of important health-promoting substances. Findings indicate that the biological activity of potato extracts is a combination of various bioactive compounds and contribute to the revalorization of potato as a functional food.

  6. Withania somnifera water extract as a potential candidate for differentiation based therapy of human neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Hardeep Kataria

    Full Text Available Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Conventional therapy of neuroblastoma includes the differentiation agents. Unlike chemo-radiotherapy, differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. Keeping in view the limited toxicity of Withania somnifera (Ashwagandha, the current study was aimed to investigate the efficacy of Ashwagandha water extract (ASH-WEX for anti-proliferative potential in neuroblastoma and its underlying signalling mechanisms. ASH-WEX significantly reduced cell proliferation and induced cell differentiation as indicated by morphological changes and NF200 expression in human IMR-32 neuroblastoma cells. The induction of differentiation was accompanied by HSP70 and mortalin induction as well as pancytoplasmic translocation of the mortalin in ASH-WEX treated cells. Furthermore, the ASH-WEX treatment lead to induction of neural cell adhesion molecule (NCAM expression and reduction in its polysialylation, thus elucidating its anti-migratory potential, which was also supported by downregulation of MMP 2 and 9 activity. ASH-WEX treatment led to cell cycle arrest at G0/G1 phase and increase in early apoptotic population. Modulation of cell cycle marker Cyclin D1, anti-apoptotic marker bcl-xl and Akt-P provide evidence that ASH-WEX may prove to be a promising phytotherapeutic intervention in neuroblatoma related malignancies.

  7. Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells

    Science.gov (United States)

    Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2014-01-01

    Molecules that inhibit the formation of an abnormal isoform of prion protein (PrPSc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrPSc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrPC) and PrPSc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrPSc levels without altering intracellular distribution of PrPSc. PPS did not change the distribution and levels of PrPC, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrPC to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrPSc formation. In contrast, CPZ and U18666A initiated the redistribution of PrPSc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrPC. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrPSc redistribution by CPZ or U18666A partly antagonized PrPSc degradation, suggesting that the transfer of PrPSc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrPSc degradation. This study revealed that precise analysis of the intracellular dynamics of PrPC and PrPSc provides important information for understanding the mechanism of anti-prion agents. PMID:25181483

  8. The Role of Intracellular Calcium for the Development and Treatment of Neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Satheesh, Noothan Jyothi; Büsselberg, Dietrich, E-mail: dib2015@qatar-med.cornell.edu [Weill Cornell Medical College in Qatar, Qatar Foundation-Education City, POB 24144, Doha (Qatar)

    2015-05-22

    Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca{sup 2+}]{sub i}) is a secondary messenger which regulates numerous cellular processes and, therefore, its concentration is tightly regulated. This review focuses on the role of [Ca{sup 2+}]{sub i} in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca{sup 2+}]{sub i} is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca{sup 2+}]{sub i} for the function of anti-cancer drugs is illuminated in this review as [Ca{sup 2+}]{sub i} could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca{sup 2+}]{sub i} could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma.

  9. Narcolepsy/Cataplexy and Occult Neuroblastoma

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2013-11-01

    Full Text Available Investigators at the University of Chicago and Northwestern University, Chicago, IL; University Hospital Southampton, UK; and Kiev Paediatric Hospital, Ukraine, report three children with narcolepsy and cataplexy subsequently diagnosed with neuroblastoma.

  10. Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

    OpenAIRE

    Morscher, Raphael Johannes; Aminzadeh-Gohari, Sepideh; Feichtinger, Ren? Gunther; Mayr, Johannes Adalbert; Lang, Roland; Neureiter, Daniel; Sperl, Wolfgang; Kofler, Barbara

    2015-01-01

    Introduction Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer?s oxidative phosphorylation system. Methods Xenografts were established in CD-1 nude mice by subcutaneous injection of two ne...

  11. Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism | Office of Cancer Genomics

    Science.gov (United States)

    Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells.

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

    Science.gov (United States)

    Gneo, Luciana; Ruggeri, Pierdomenico; Cappabianca, Lucia; Farina, Antonietta Rosella; Di Ianni, Natalia; Mackay, Andrew Reay

    2016-12-06

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

  13. MEK inhibitors as a novel therapy for neuroblastoma: Their in vitro effects and predicting their efficacy.

    Science.gov (United States)

    Tanaka, Tomoko; Higashi, Mayumi; Kimura, Koseki; Wakao, Junko; Fumino, Shigehisa; Iehara, Tomoko; Hosoi, Hajime; Sakai, Toshiyuki; Tajiri, Tatsuro

    2016-12-01

    A recent study reported that relapsed neuroblastomas had frequent RAS-ERK pathway mutations. We herein investigated the effects and pathways of MEK inhibitors, which inhibit the RAS-ERK pathway, as a new molecular-targeted therapy for refractory neuroblastomas. Five neuroblastoma cell lines were treated with trametinib (MEK inhibitor) or CH5126766 (RAF/MEK inhibitor). Growth inhibition was analyzed using a cell viability assay. ERK phosphorylation and the MYCN expression were analyzed by immunoblotting or immunohistochemistry. RAS/RAF mutations were identified by direct sequencing or through the COSMIC database. Both MEK inhibitors showed growth inhibition effects on cells with ERK phosphorylation, but almost no effect on cells without. In immunoblotting analyses, ERK phosphorylation and MYCN expression were suppressed in ERK active cells by these drugs. Furthermore, phosphorylated-ERK immunohistochemistry corresponded to the drug responses. Regarding the relationship between RAS/Raf mutations and ERK phosphorylation, ERK was phosphorylated in one cell line (NLF) without RAS/Raf mutations. MEK inhibitors are a promising molecular-targeted therapeutic option for ERK active neuroblastomas. The efficacy of MEK inhibitors corresponds to ERK phosphorylation, while RAS/RAF mutations are not always detected in drug-sensitive cells. Phosphorylated-ERK immunohistochemistry is thus a useful method to analyze ERK activity and predict the therapeutic effects of MEK inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Neuroblastoma

    Science.gov (United States)

    ... the eyes Profuse sweating Rapid heart rate ( tachycardia ) Brain and nervous system problems may include: Inability to empty the bladder Loss of movement ( paralysis ) of the hips, legs, or feet (lower extremities) ...

  15. Neuroblastoma

    Science.gov (United States)

    ... the cancer has spread to the bones or bone marrow) weakness, numbness, inability to move a body part, or difficulty walking (if the cancer presses on the spinal cord) drooping eyelid, unequal pupils, sweating, and red ...

  16. MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma

    LENUS (Irish Health Repository)

    Tivnan, Amanda

    2011-01-25

    ABSTRACT Background Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis. Methods A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons\\/sec\\/cm2). Results Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to

  17. Vincristine resistance in relapsed neuroblastoma can be efficiently overcome by Smac mimetic LCL161 treatment.

    Science.gov (United States)

    Frommann, Kristin; Appl, Birgit; Hundsdoerfer, Patrick; Reinshagen, Konrad; Eschenburg, Georg

    2018-01-31

    In spite of good initial therapy response neuroblastomas often spread to distant organs or relapse after periods of remission. Dysregulation of apoptosis, a hallmark of cancer, is often effected by elevated levels of antiapoptotic signals leading to resistance against chemotherapeutic drugs. Inhibitors of apoptosis proteins (IAPs) are crucial cellular apoptosis regulators. Targeting IAPs with Smac mimetics has been demonstrated as a promising strategy for treatment of neuroblastoma and other tumors. In paired neuroblastoma cell lines, obtained from the same patient at time of diagnosis (CHLA-15) and postchemotherapy during progressive disease (CHLA-20), expression of crucial IAPs was determined. Furthermore, effects of vincristine on viability, cytotoxicity, apoptosis induction and caspase-3/7 activation were determined. Cellular IAP-1 (cIAP-1) and X-linked IAP (XIAP) expression was increased in cell line CHLA-20. Moreover, biological effects of vincristine were significantly lower in these cells. Treatment of cells with Smac mimetic LCL161 increased the effects of vincristine in CHLA-15 cells and more importantly was able to overcome vincristine resistance in CHLA-20 cells. These findings demonstrate the potential of Smac mimetics for the development of novel therapeutic approaches for the treatment of relapsed/resistant neuroblastoma. Copyright © 2018. Published by Elsevier Inc.

  18. The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription.

    Science.gov (United States)

    Wong, Matthew; Tee, Andrew E L; Milazzo, Giorgio; Bell, Jessica L; Poulos, Rebecca C; Atmadibrata, Bernard; Sun, Yuting; Jing, Duohui; Ho, Nicholas; Ling, Dora; Liu, Pei Yan; Zhang, Xu Dong; Hüttelmaier, Stefan; Wong, Jason W H; Wang, Jenny; Polly, Patsie; Perini, Giovanni; Scarlett, Christopher J; Liu, Tao

    2017-05-01

    Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1 , and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR . ©2017 American Association for Cancer Research.

  19. Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

    Science.gov (United States)

    Hart, Lori S; Rader, JulieAnn; Raman, Pichai; Batra, Vandana; Russell, Mike R; Tsang, Matthew; Gagliardi, Maria; Chen, Lucy; Martinez, Daniel; Li, Yimei; Wood, Andrew; Kim, Sunkyu; Parasuraman, Sudha; Delach, Scott; Cole, Kristina A; Krupa, Shiva; Boehm, Markus; Peters, Malte; Caponigro, Giordano; Maris, John M

    2017-04-01

    Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Results: Sensitivity to binimetinib and ribociclib was inversely related ( r = -0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling. Clin Cancer Res; 23(7); 1785-96. ©2016 AACR . ©2016 American Association for Cancer Research.

  20. Higgs amplitude mode in the BCS superconductors Nb1-xTi(x)N induced by terahertz pulse excitation.

    Science.gov (United States)

    Matsunaga, Ryusuke; Hamada, Yuki I; Makise, Kazumasa; Uzawa, Yoshinori; Terai, Hirotaka; Wang, Zhen; Shimano, Ryo

    2013-08-02

    Ultrafast responses of BCS superconductor Nb(1-x)Ti(x)N films in a nonadiabatic excitation regime were investigated by using terahertz (THz) pump-THz probe spectroscopy. After an instantaneous excitation with the monocycle THz pump pulse, a transient oscillation emerges in the electromagnetic response in the BCS gap energy region. The oscillation frequency coincides with the asymptotic value of the BCS gap energy, indicating the appearance of the theoretically anticipated collective amplitude mode of the order parameter, namely the Higgs amplitude mode. Our result opens a new pathway to the ultrafast manipulation of the superconducting order parameter by optical means.

  1. Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin

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    Marcela Pinedo

    2017-01-01

    Full Text Available According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx. The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

  2. Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Berliocchi, Laura; Chiappini, Carlotta; Adornetto, Annagrazia; Gentile, Debora; Cerri, Silvia; Russo, Rossella; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2018-02-01

    d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX ® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and

  3. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

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    J. Guan

    2016-09-01

    Full Text Available The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori, has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

  4. Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model.

    Science.gov (United States)

    Morscher, Raphael Johannes; Aminzadeh-Gohari, Sepideh; Feichtinger, René Gunther; Mayr, Johannes Adalbert; Lang, Roland; Neureiter, Daniel; Sperl, Wolfgang; Kofler, Barbara

    2015-01-01

    Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer's oxidative phosphorylation system. Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content). Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention. Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose

  5. Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model.

    Directory of Open Access Journals (Sweden)

    Raphael Johannes Morscher

    Full Text Available Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer's oxidative phosphorylation system.Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content.Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens

  6. A primary sellar neuroblastoma mimicking a pituitary adenoma: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Gun; Heo, Young Jin; Kim, Eun Kyoung; Baek, Jin Wook; Jeong, Hae Woong; Jung, Hyun Seok [Busan Paik Hospital, Inje University College of Medicine, Busan (Korea, Republic of)

    2016-12-15

    Intracranial neuroblastomas are uncommon malignant tumors that usually arise in the supratentorial parenchymal or paraventricular location. A primary neuroblastoma arising in the sella turcica is extremely rare. We report a case of a 76-year-old man who presented with progressive bitemporal hemianopsia. His pituitary hormone levels were within the normal range, except for slightly increased prolactin. Pituitary magnetic resonance imaging revealed a solitary sellar mass with supra- and parasellar extension that mimicked a non-functioning pituitary adenoma or meningioma. The tumor was excised by transsphenoidal resection. Histopathologic analysis revealed small cells surrounded by a dense fibrillary stroma as well as strong expression of neural markers. Hence, the patient was diagnosed with sellar neuroblastoma. Prolactin levels normalized in the immediate postoperative period, although visual disturbances persisted. Herein, we describe the clinical manifestations, MRI characteristics, and histopathologic findings of this case.

  7. Long-term control of olfactory neuroblastoma in a dog treated with surgery and radiation therapy.

    Science.gov (United States)

    Gumpel, E; Moore, A S; Simpson, D J; Hoffmann, K L; Taylor, D P

    2017-07-01

    Olfactory neuroblastoma is a rare malignancy of the nasal cavity in dogs that is thought to arise from specialised sensory neuroendocrine olfactory cells derived from the neural crest. An 8-year-old dog was presented for reclusiveness and pacing. On CT and MRI, a contract-enhancing mass was disclosed within the rostral fossa, extending caudally from the cribriform plate into the left nasal sinus. Surgical excision was performed and the diagnosis was histological grade III (Hyams grading scheme) olfactory neuroblastoma. Based on human CT criteria this was high stage (modified Kadish stage C). Surgical excision was incomplete and was followed by curative-intent radiation therapy using a linear accelerator to a total dose of 48 Gy. The dog survived 20 months after diagnosis. Although olfactory neuroblastoma is a rare tumour in dogs, aggressive local therapy may allow for prolonged survival, even when the tumour is advanced. © 2017 Australian Veterinary Association.

  8. Iodine 131 labeled GD2 monoclonal antibody in the diagnosis and therapy of human neuroblastoma

    International Nuclear Information System (INIS)

    Cheung, N.K.V.; Miraldi, F.D.

    1988-01-01

    High dose marrow ablative therapy followed by autologous bone marrow transplantation (ABMT) has prolonged survival in patients with neuroblastoma. Total body and focal irradiation play an integral role in the overall treatment of this disease. The biological basis for radiation is the radiosensitivity and the lack of sublethal repair in neuroblastoma cells. However, radiation therapy has not by itself been adequate because of the usual widespread nature of neuroblastoma and the inability to achieve selective tumor versus normal tissue delivery, especially at multiple tumor sites. Monoclonal antibodies are agents selected for their specificity for human tumors. In vivo they have the ability of targeting selectively to occult metastases. This paper discusses how the availability of radioisotopes and the development of conjugation chemistries have greatly expanded the potentials of these antibodies

  9. NF1 Is a Tumor Suppressor in Neuroblastoma that Determines Retinoic Acid Response and Disease Outcome

    NARCIS (Netherlands)

    Hölzel, Michael; Huang, Sidong; Koster, Jan; Ora, Ingrid; Lakeman, Arjan; Caron, Huib; Nijkamp, Wouter; Xie, Jing; Callens, Tom; Asgharzadeh, Shahab; Seeger, Robert C.; Messiaen, Ludwine; Versteeg, Rogier; Bernards, René

    2010-01-01

    Retinoic acid (RA) induces differentiation of neuroblastoma cells in vitro and is used with variable success to treat aggressive forms of this disease. This variability in clinical response to RA is enigmatic, as no mutations in components of the RA signaling cascade have been found. Using a

  10. Neuroblastoma management in Chinese children.

    Science.gov (United States)

    Li, Kai; Dong, Kuiran; Gao, Jiechun; Yao, Wei; Xiao, Xianmin; Zheng, Shan

    2012-04-01

    This study assesses the clinical features of neuroblastoma and survival. Data for 98 patients between January 2000 and December 2006 at Children's Hospital of Fudan University, Shanghai, China, were retrospectively analyzed. Diagnostic methods included imaging, 24-hr urine catecholamines, bone marrow biopsies, and histopathology analyses. Treatment followed the modified Japanese Study Group Protocol. Clinical characteristics, treatment, and outcome were depicted, and difficulties encountered were analyzed. The median age of patients was 48 months. There were 3, 13, 31, 49, and 2 patients in stages 1, 2, 3, 4, and 4s disease, respectively. Positive urinary vanillylmandelic acid (VMA) prevalence was low in localized disease (51.1%) and high in disseminated disease (70.6%, p = .03). Gross total resection rate was 60.8%. The five-year overall survival (OS) rate was 80% for stages 1 and 2, 48.3% for stage 3, and 20% for stage 4. The five-year OS rates significantly decreased in children older than 18 months (p indigence were the main causes for poorer outcome in late stages.

  11. Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

    NARCIS (Netherlands)

    Michaelis, M.; Rothweiler, F.; Agha, B.; Barth, S.; Voges, Y.; Loeschmann, N.; von Deimling, A.; Breitling, R.; Doerr, H. Wilhelm; Roedel, F.; Speidel, D.; Cinatl, J.; Cinatl Jr., J.; Stephanou, A.

    Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3,

  12. BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models.

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    Anastasia Wyce

    Full Text Available BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726, and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

  13. Neutron powder diffraction and magnetic study of perovskites Pb(Mn1/2Nb1/2)O3 and Pb(Mn1/4Fe1/4Nb1/2)O3

    International Nuclear Information System (INIS)

    Ivanov, S.A.; Nordblad, P.; Tellgren, R.; Rundlof, H.; Andre, G.; Bouree, F.

    2008-01-01

    The structural and magnetic properties of the complex metal oxides Pb(Mn 1/2 Nb 1/2 )O 3 (PMNO) and Pb(Mn 1/4 Fe 1/4 Nb 1/2 )O 3 (PMFNO), which belong to a class of disordered perovskites have been studied. The magnetic susceptibilities of PMNO showed hysteresis between field cooled and zero-field cooled conditions below the transition of 15 K, suggesting that the material has a spin-glass feature. Neutron diffraction patterns of PMNO showed no evidence of a long-range magnetic ordering at 1.5 K, which is consistent with spin-glass behavior. Rietveld refinements of neutron powder diffraction data collected at different temperatures between 1.5 and 700 K have been carried out in order to extract structural information. The crystal structure of this compound is cubic (space group Pm3-barm) within the whole temperature interval. The Mn and Nb ions were found to be disordered over the perovskite B-sites. The main feature of this structure is the positional disorder at the Pb site, the importance of which in connection with the ferroic transitions is briefly discussed. The Pb cations show a positional disorder shifting from their high-symmetry positions along the [1 1 1] direction. The effect of Fe-doping on PMNO has been studied. The substitution of Fe at the Mn site in PMFNO results in a small changes of the magnetic properties without significant differences in the crystal structures. The factors governing the observed structural and magnetic properties of PMNO and PMFNO are discussed and compared with those of other quaternary Mn-containing perovskites. For the PbB 3+ 1/2 Nb 1/2 O 3 series with the isomorphous substitution B 3+ , graphs of average lattice parameters of the perovskite phase and the temperatures of ferroelectric and magnetic phase transitions as functions of the B 3+ cation radius were constructed and are discussed. Influence of A-cation sublattice on magnetic properties is also considered

  14. Assessment of Cellular Responses after Short- and Long-Term Exposure to Silver Nanoparticles in Human Neuroblastoma (SH-SY5Y and Astrocytoma (D384 Cells

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    Teresa Coccini

    2014-01-01

    Full Text Available Silver nanoparticle (AgNP, 20 nm neurotoxicity was evaluated by an integrated in vitro testing protocol employing human cerebral (SH-SY5Y and D384 cell lines. Cellular response after short-term (4–48 h, 1–100 μg/ml and prolonged exposure (up to 10 days, 0.5–50 μg/ml to AgNP was assessed by MTT, calcein-AM/PI, clonogenic tests. Pulmonary A549 cells were employed for data comparison along with silver nitrate as metal ionic form. Short-term data: (i AgNP produced dose- and time-dependent mitochondrial metabolism changes and cell membrane damage (effects starting at 25 μg/ml after 4 h: EC50s were 40.7 ± 2.0 and 49.5 ± 2.1 μg/ml for SH-SY5Y and D384, respectively. A549 were less vulnerable; (ii AgNP doses of ≤ 18 μg/ml were noncytotoxic; (iii AgNO3 induced more pronounced effects compared to AgNP on cerebral cells. Long-term data: (i low AgNP doses (≤1 μg/ml compromised proliferative capacity of all cell types (cell sensibility: SHSY5Y > A549 > D384. Colony number decrease in SH-SY5Y and D384 was 50% and 25%, respectively, at 1 μg/ml, and lower dose (0.5 μg/ml was significantly effective towards SH-SY5Y and pulmonary cells; (ii cell proliferation activity was more affected by AgNO3 than AgNPs. In summary, AgNP-induced cytotoxic effects after short-term and prolonged exposure (even at low doses were evidenced regardless of cell model types.

  15. Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway.

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    Bhushan Vijay Nagpure

    Full Text Available Alzheimer's disease (AD is the leading cause of senile dementia in today's society. Its debilitating symptoms are manifested by disturbances in many important brain functions, which are influenced by adenosine. Hence, adenosinergic system is considered as a potential therapeutic target in AD treatment. In the present study, we found that sodium hydrosulfide (NaHS, an H2S donor, 100 µM attenuated HENECA (a selective A2A receptor agonist, 10-200 nM induced β-amyloid (1-42 (Aβ42 production in SH-SY5Y cells. NaHS also interfered with HENECA-stimulated production and post-translational modification of amyloid precursor protein (APP by inhibiting its maturation. Measurement of the C-terminal APP fragments generated from its enzymatic cleavage by β-site amyloid precursor protein cleaving enzyme 1 (BACE1 showed that NaHS did not have any significant effect on β-secretase activity. However, the direct measurements of HENECA-elevated γ-secretase activity and mRNA expressions of presenilins suggested that the suppression of Aβ42 production in NaHS pretreated cells was mediated by inhibiting γ-secretase. NaHS induced reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB. NaHS significantly reduced the elevated cAMP and Aβ42 production caused by forskolin (an adenylyl cyclase, AC agonist alone or forskolin in combination with IBMX (a phosphodiesterase inhibitor, but had no effect on those caused by IBMX alone. Moreover, pretreatment with NaHS significantly attenuated HENECA-elevated AC activity and mRNA expressions of various AC isoforms. These data suggest that NaHS may preferentially suppress AC activity when it was stimulated. In conclusion, H2S attenuated HENECA induced Aβ42 production in SH-SY5Y neuroblastoma cells through inhibiting γ-secretase via a cAMP dependent pathway.

  16. Mdm2 Deficiency Suppresses MYCN-Driven Neuroblastoma Tumorigenesis In Vivo

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    Zaowen Chen

    2009-08-01

    Full Text Available Neuroblastoma is derived from neural crest precursor components of the peripheral sympathetic nervous system and accounts for more than 15% of all pediatric cancer deaths. A clearer understanding of the molecular basis of neuroblastoma is required for novel therapeutic approaches to improve morbidity and mortality. Neuroblastoma is uniformly p53 wild type at diagnosis and must overcome p53-mediated tumor suppression during pathogenesis. Amplification of the MYCN oncogene correlates with the most clinically aggressive form of the cancer, and MDM2, a primary inhibitor of the p53 tumor suppressor, is a direct transcriptional target of, and positively regulated by, both MYCN and MYCC. We hypothesize that MDM2 contributes to MYCN-driven tumorigenesis helping to ameliorate p53-dependent apoptotic oncogenic stress during tumor initiation and progression. To study the interaction of MYCN and MDM2, we generated an Mdm2 haploinsufficient transgenic animal model of neuroblastoma. In Mdm2+/-MYCN transgenics, tumor latency and animal survival are remarkably extended, whereas tumor incidence and growth are reduced. Analysis of the Mdm2/p53 pathway reveals remarkable p53 stabilization counterbalanced by epigenetic silencing of the p19Arf gene in the Mdm2 haploinsufficient tumors. In human neuroblastoma xenograft models, conditional small interfering RNA-mediated knockdown of MDM2 in cells expressing wild-type p53 dramatically suppresses tumor growth in a p53-dependent manner. In summary, we provided evidence for a crucial role for direct inhibition of p53 by MDM2 and suppression of the p19ARF/p53 axis in neuroblastoma tumorigenesis, supporting the development of therapies targeting these pathways.

  17. Chemotherapy-Induced Apoptosis in a Transgenic Model of Neuroblastoma Proceeds Through p53 Induction

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    Louis Chesler

    2008-11-01

    Full Text Available Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apoptosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM induced a complete remission in p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induction of the Bcl-2 homology domain-3-only proteins PUMA and Bim, leading to the activation of Bax and cleavage of caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53. Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors. This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apoptosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.

  18. Bilateral cystic neuroblastoma: imaging features and differential diagnoses

    International Nuclear Information System (INIS)

    Cassady, C.; Winters, W.D.

    1997-01-01

    Neuroblastoma is one of the most common malignant tumors of childhood, with 40 % arising in the adrenal glands. Bilateral adrenal involvement from synchronous development or metastatic spread of the tumor is seen in less than 10 % of children with neuroblastoma [1[. Neuroblastoma rarely presents as a cystic suprarenal mass that is difficult to differentiate from adrenal hemorrhage, extralobar sequestration, or dilated upper-pole renal calyces. To our knowledge, bilateral cystic neuroblastoma has not been previously reported. We present a case of bilateral cystic adrenal neuroblastoma to demonstrate the imaging features of this unusual entity, and to expand the differential diagnosis of bilateral cystic suprarenal masses in an infant. (orig.). With 2 figs

  19. Proteome and Acetylome Analysis Identifies Novel Pathways and Targets Regulated by Perifosine in Neuroblastoma

    Science.gov (United States)

    Gu, Xiao; Hua, Zhongyan; Dong, Yudi; Zhan, Yue; Zhang, Xiaowen; Tian, Wei; Liu, Zhihui; Thiele, Carol J.; Li, Zhijie

    2017-01-01

    Perifosine, an Akt inhibitor, has been shown to be effective in controlling neuroblastoma tumor growth. However, studies indicate that in addition to the ability to inhibit Akt, other mechanisms contribute to perifosine’s anti-tumor activity. To gain insight into perifosine anti-tumor activity in neuroblastoma we have studied changes in the proteome and acetylome after perifosine treatment in SK-N-AS neuroblastoma cells using SILAC labeling, affinity enrichment, high-resolution and LC-MS/MS analysis. Bioinformatic analysis indicates that, a total of 5,880 proteins and 3,415 lysine acetylation sites were quantified in SK-N-AS cells and 216 differentially expressed proteins and 115 differentially expressed lysine acetylation sites were obtained. These differentially expressed proteins and lysine acetylated proteins were involved in a number of different biological functions, metabolic pathways and pathophysiological processes. This study details the impact of perifosine on proteome and lysine acetylome in SK-N-AS cells and expands our understanding of the mechanisms of perifosine action in neuroblastoma. PMID:28165023

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

    Science.gov (United States)

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

    2017-06-01

    Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H 2 O 2 )-induced inflammaging state in SH-SY5Y cells. Our data showed that p21 Cip1 and p16 INK4a , cell cycle arrest markers, and the number of senescence-associated β-galactosidase (SA-βgal) staining increased significantly in H 2 O 2 -treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H 2 O 2 treated cells. Cells pretreated with H 2 O 2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H 2 O 2 , could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H 2 O 2 . The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Neuroblastoma presenting with the nephrotic syndrome.

    Science.gov (United States)

    Zheng, H L; Maruyama, T; Matsuda, S; Satomura, K

    1979-08-01

    A case of neuroblastoma in a 4-yr-old boy presenting with the nephrotic syndrome is reported. Nether thrombosis nor stenosis of the renal veins and the inferior vena cava was present. Electron microscopy revealed lumps of subepithelial deposits as well as thickening and tortuosity of the glomerular basement membrane compatible with membranous nephropathy. It is postulated that deposition of neuroblastoma-associated immune complexes on the glomerular basement membrane-was responsible for the development of the nephrotic syndrome in this patient. Five pediatric cases of nephrotic syndrome associated with extrarenal neoplasia were collected from the world literature and are briefly discussed.

  2. Development of Nb-1%Zr-0.1%C alloy as structural components for high temperature reactors

    Energy Technology Data Exchange (ETDEWEB)

    Vishwanadh, B. [Materials Science Division, Bhabha Atomic Research Center, Trombay, Mumbai (India); Vaibhav, K.; Jha, S.K.; Mirji, K.V. [Nuclear Fuel Complex, Hyderabad (India); Samajdar, I. [Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology-Bombay, Powai, Mumbai (India); Srivastava, D. [Materials Science Division, Bhabha Atomic Research Center, Trombay, Mumbai (India); Tewari, R., E-mail: rtewaribarc@yahoo.co.in [Materials Science Division, Bhabha Atomic Research Center, Trombay, Mumbai (India); Saibaba, N. [Nuclear Fuel Complex, Hyderabad (India); Dey, G.K. [Materials Science Division, Bhabha Atomic Research Center, Trombay, Mumbai (India)

    2012-08-15

    The Nb-1Zr-0.1C (wt.%) alloy is being considered for structural components in the proposed Compact High-Temperature-Reactors (HTR). The present work reports on the development of 30-50 kg ingots of the alloy in correct composition as well as technology for forming the material in various shapes. The work deals with the deformation behavior of as-cast material at different temperatures and strain rates, recrystallization behavior at different temperature and time and evolution of microstructures at different processing conditions (as-cast, deformed and recrystallized). The as-cast Nb alloys were deformed up to 35% at different temperatures. The deformation results showed that the flow stress of the as-cast Nb alloy increases with increasing temperature from 800 Degree-Sign C to 1000 Degree-Sign C. Beyond 1200 Degree-Sign C, substantial decrease in the strength of the alloy was noticed. To determine the optimum recrystallization temperature and time for the alloy, several heat treatments were conducted by systematically varying temperature and time. It was found that the deformed Nb alloy could be recrystallized by annealing at 1300 Degree-Sign C for 3 h. The microstructures of the as-cast, deformed and recrystallized samples of Nb-1%Zr-0.1%C alloy were systematically characterized by optical, electron back scattered diffraction (EBSD) and transmission electron microscopy techniques. The Nb-1Zr-0.1C alloy showed significant differences in the microstructure after different thermo-mechanical treatments. Microstructures of the Nb alloy showed two phases: the matrix (bcc) phase and the carbide phase. Electron Microscopy and energy dispersive spectroscopic analyses revealed that the carbide precipitation undergoes various phase transformations. The as-cast structure of Nb alloy had hexagonal Nb{sub 2}C precipitates in the Nb matrix and after extrusion, the deformed microstructure had two types of carbide precipitates: needle and rectangular morphology precipitates. The

  3. Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma.

    Science.gov (United States)

    Maser, Tyler; Rich, Maria; Hayes, David; Zhao, Ping; Nagulapally, Abhinav B; Bond, Jeffrey; Saulnier Sholler, Giselle

    2017-06-01

    Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  4. Differentiation in neuroblastoma: diffusion-limited hypoxia induces neuro-endocrine secretory protein 55 and other markers of a chromaffin phenotype.

    Directory of Open Access Journals (Sweden)

    Fredrik Hedborg

    2010-09-01

    Full Text Available Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen (hypoxia plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 (NESP55, a novel member of the chromogranin family, as a marker for this process.Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2α. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors.

  5. Inhibition ofSTAT3with the Generation 2.5 Antisense Oligonucleotide, AZD9150, Decreases Neuroblastoma Tumorigenicity and Increases Chemosensitivity.

    Science.gov (United States)

    Odate, Seiichi; Veschi, Veronica; Yan, Shuang; Lam, Norris; Woessner, Richard; Thiele, Carol J

    2017-04-01

    Purpose: Neuroblastoma is a pediatric tumor of peripheral sympathoadrenal neuroblasts. The long-term event-free survival of children with high-risk neuroblastoma is still poor despite the improvements with current multimodality treatment protocols. Activated JAK/STAT3 pathway plays an important role in many human cancers, suggesting that targeting STAT3 is a promising strategy for treating high-risk neuroblastoma. Experimental Design: To evaluate the biologic consequences of specific targeting of STAT3 in neuroblastoma, we assessed the effect of tetracycline (Tet)-inducible STAT3 shRNA and the generation 2.5 antisense oligonucleotide AZD9150 which targets STAT3 in three representative neuroblastoma cell line models (AS, NGP, and IMR32). Results: Our data indicated that Tet-inducible STAT3 shRNA and AZD9150 inhibited endogenous STAT3 and STAT3 target genes. Tet-inducible STAT3 shRNA and AZD9150 decreased