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Sample records for neuroblastoma cell proliferation

  1. 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.

  2. Enhancer of zeste homolog 2 regulates cell differentiation and proliferation in neuroblastoma

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    Amallia N. Setyawati

    2014-12-01

    Full Text Available Background Neuroblastoma (NB is one of the most common extracranial solid tumors occurring in infancy and childhood with highly variable outcomes. Polycomb group (PcG proteins are epigenetic gene silencers. Enhancer of zeste homolog 2 (EZH2 is a member of the polycomb repressor complex 2 (PRC2 group, with the main function to catalyze the polycomb repressor complex by methylating lysine 9 and 27 of histone H3. This study aimed to investigate the biological functionality of EZH2 in NB. Methods This was an experimental study with an analysis of correlation initially of the known prognostic factors of NB patients’ outcomes, by comparing the expression of v-myc avian myelocytomatosis viral oncogene neuroblastoma (MYCN with that of EZH2, on the basis of the patients’ overall and relapse free survival rates. This was followed with a biological functional study to assess the role of EZH2 expression in NB. Results EZH2 knockdown induces neurite extension and differentiation marker growth associated protein 43 (GAP43 in NB cells, although it does not affect cell cycle. By ectopic expression of EZH2, all-trans retinoic acid (ATRA induced neurite extension was suppressed and GAP43 was decreased. Overall, EZH2 seems to have an important role in NB cell differentiation. Although EZH2 did not alter cell proliferation, in the soft agar colony formation assay there was a significant increase in total colony number and number of large colonies. Conclusion Our result clarified the potential role of EZH2 in the regulation of cell differentiation and proliferation, which subsequently may play an important role in the poor prognosis of NB patients.

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

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    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.

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

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    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. Enhancer of zeste homolog 2 regulates cell differentiation and proliferation in neuroblastoma

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    Amallia N. Setyawati

    2015-12-01

    Full Text Available BACKGROUND Neuroblastoma  (NB  is  one  of  the  most  common  extracranial  solid  tumors occurring in infancy and childhood with highly variable outcomes. Polycomb group (PcG proteins are epigenetic gene silencers. Enhancer of zeste homolog 2 (EZH2 is a member of the polycomb repressor complex 2 (PRC2 group, with  the  main  function  to  catalyze  the  polycomb  repressor  complex  by methylating lysine 9 and 27 of histone H3. This study aimed to investigate the biological functionality of EZH2 in NB.   METHODS This was an experimental study with an analysis of correlation initially of the known prognostic factors of NB patients’ outcomes, by comparing the expression of v-myc avian myelocytomatosis viral oncogene neuroblastoma (MYCN with that of EZH2, on the basis of the patients’ overall and relapse free survival rates. This was followed with a biological functional study to assess the role of EZH2 expression in NB.   RESULTS EZH2 knockdown induces neurite extension and differentiation marker growth associated  protein  43  (GAP43  in  NB  cells,  although  it  does  not  affect  cell cycle. By ectopic expression of EZH2, all-trans retinoic acid (ATRA inducedneurite extension was suppressed and GAP43 was decreased. Overall, EZH2 seems to have an important role in NB cell differentiation. Although EZH2 did not alter cell proliferation, in the soft agar colony formation assay there was a significant increase in total colony number and number of large colonies.   CONCLUSION Our  result  clarified  the  potential  role  of  EZH2  in  the  regulation  of  cell differentiation and proliferation, which subsequently may play an important role in the poor prognosis of NB patients.

  6. 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.

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

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    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.

  8. Scavenger receptor class B type 1 regulates neuroblastoma cell proliferation, migration and invasion.

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    Panchoo, Marlyn; Lacko, Andras

    2018-01-01

    Neuroblastoma (NB) is an extra cranial pediatric embryonal tumor most prevalent in children less than 1 year of age. NB accounts for 7% of all pediatric cancers but accounts for 15% of all childhood cancer deaths. Scavenger receptor class B type 1 (SR-B1), a mediator of cellular cholesterol uptake, is overexpressed in and have been linked to the aggressiveness of many cancers. Nevertheless, no studies have so far investigated the relationship between SR-B1 and NB. Elucidation of receptors that promote NB may pave the way for discovery of new therapeutic targets. Here we show that inhibition of SR-B1 reduced cell survival, migration and invasion, and cholesterol content in NB cell lines. Additionally analysis of SR-B1 levels in NB patient biopsies using the R2: Genomics Analysis and Visualization Platform showed that high SR-B1 expression correlated with decreased overall and event-free survival. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286

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    Pizarro, Javier G; Folch, Jaume; Esparza, Jos? Luis; Jordan, J.; Pall?s, Merc?; Camins, Antoni

    2008-01-01

    Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G2/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-...

  10. Anaplastic large cell neuroblastoma.

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    Abramowsky, Carlos R; Katzenstein, Howard M; Alvarado, Carlos S; Shehata, Bahig M

    2009-01-01

    Anaplastic large cell neuroblastomas (ALCNB) are a subset of undifferentiated neuroblastomas with marked pleomorphic and anaplastic features that render them diagnostically challenging. We reviewed the records of all patients diagnosed with ALCNB at Children's Healthcare of Atlanta (Egleston Children's Hospital) for their clinical, biologic, and pathologic characteristics and their treatment outcomes. From 1998 to 2006, 7 patients were diagnosed with ALCNB. All patients presented with abdominal-pelvic masses, 3 of them of adrenal origin and 2 with thoracic extension, with clinical stages 3 or 4, and were considered to have high-risk disease. The N-MYC oncogene was amplified in 3 cases and catecholamines were elevated in 5 of 6 patients tested. All pretreatment tumors demonstrate pleomorphic, anaplastic morphology with bizarre mitoses admixed with undifferentiated but monomorphic cells with minimal if any neuropil or neuro-ganglionic differentiation. Immunohistochemical markers for neuron specific enolase (NSE) and synaptophysin were strongly positive in all specimens and chromogranin in 4 of 5. Interestingly, all tumors showed strong Fli-1 nuclear positivity despite a negative CD-99 stain. However, reverse transcription polymerase chain reaction or fluorescent in-situ hybridization testing for Ewing sarcoma transcripts was negative in 4 available specimens. This same Fli-1 antibody had tested negative on 30 conventional neuroblastomas, indicating a peculiar cross reactivity with this subset of ALCNB. Posttreatment biopsies showed maturation changes to more conventional neuroblastoma histology in 5 of the 7 cases. Follow-up ranged from 9 months to 4 years from diagnosis (median: 25 months). Five patients are still alive after treatment, 1 died 9 months after diagnosis, and another patient refused high-risk therapy and progressed and died 9 months from diagnosis. Anaplastic large cell neuroblastomas are a subset of undifferentiated neuroblastomas characterized by the

  11. Role of trace metals in cell proliferation in the human neuroblastoma: relations with the oncogene N-myc; Le role des metaux trace dans la proliferation des cellules de neuroblastome humaine: relations avec l`oncogene N-myc

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    Moretto, Ph.; Michelet, C. [Centre d`Etudes Nucleaires, Bordeaux-1 Univ., 33 Gradignan (France); Gouget, B.; Ortega, R.; Sergiant, C.; Llabador, Y.; Simonoff, M. [URA 451, Gradignan (France); Benard, J. [Laboratoire de Pharmacologie Clinique et Moleculaire, Institut Gustave Roussy, 94 - Villejuif (France)

    1997-06-01

    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) 8 refs.

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

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

    2016-09-01

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

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

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    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

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

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

    2014-01-01

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

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

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

    2014-01-01

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

  16. Involvement of GTA protein NC2β in Neuroblastoma pathogenesis suggests that it physiologically participates in the regulation of cell proliferation

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    Ferro Alfredo

    2008-06-01

    Full Text Available Abstract Background The General Transcription Apparatus (GTA comprises more than one hundred proteins, including RNA Polymerases, GTFs, TAFs, Mediator, and cofactors such as heterodimeric NC2. This complexity contrasts with the simple mechanical role that these proteins are believed to perform and suggests a still uncharacterized participation to important biological functions, such as the control of cell proliferation. Results To verify our hypothesis, we analyzed the involvement in Neuroblastoma (NB pathogenesis of GTA genes localized at 1p, one of NB critical regions: through RT-PCR of fifty eight NB biopsies, we demonstrated the statistically significant reduction of the mRNA for NC2β (localized at 1p22.1 in 74% of samples (p = 0.0039. Transcripts from TAF13 and TAF12 (mapping at 1p13.3 and 1p35.3, respectively were also reduced, whereas we didn't detect any quantitative alteration of the mRNAs from GTF2B and NC2α (localized at 1p22-p21 and 11q13.3, respectively. We confirmed these data by comparing tumour and constitutional DNA: most NB samples with diminished levels of NC2β mRNA had also genomic deletions at the corresponding locus. Conclusion Our data show that NC2β is specifically involved in NB pathogenesis and may be considered a new NB biomarker: accordingly, we suggest that NC2β, and possibly other GTA members, are physiologically involved in the control of cell proliferation. Finally, our studies unearth complex selective mechanisms within NB cells.

  17. 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.

  18. 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...

  19. Neuroblastoma

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    Davidoff, Andrew M.

    2012-01-01

    Neuroblastoma is a heterogeneous disease; tumors can spontaneously regress or mature, or display an aggressive, therapy-resistant phenotype. Increasing evidence indicates that the biologic and molecular features of neuroblastoma significantly influence and are highly predictive of clinical behavior. Because of this, neuroblastoma has served as a paradigm for biological risk assessment and treatment assignment. Most current clinical studies of neuroblastoma base therapy and its intensity on a ...

  20. 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.

  1. Neuroblastoma

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    ... also may be at higher risk for other cancers. Caring for Your Child Being told your child has neuroblastoma can be ... Cancer Center Use Finn's Story to Talk About Cancer Preparing Your Child for Surgery Late Effects of Cancer and Cancer ...

  2. Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression.

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    Lam, Wilbur A; Cao, Lizhi; Umesh, Vaibhavi; Keung, Albert J; Sen, Shamik; Kumar, Sanjay

    2010-02-10

    Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that

  3. Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

    Science.gov (United States)

    Bhaskara, Vasantha Kumar; Mohanam, Indra; Gujrati, Meena; Mohanam, Sanjeeva

    2014-01-01

    Background Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions. Methods We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone. Results Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells. Conclusions Intermittent hypoxic

  4. Nifurtimox induces apoptosis of neuroblastoma cells in vitro and in vivo.

    Science.gov (United States)

    Saulnier Sholler, Giselle L; Brard, Laurent; Straub, Jennifer A; Dorf, Lee; Illeyne, Sharon; Koto, Karen; Kalkunte, Satyan; Bosenberg, Marcus; Ashikaga, Taka; Nishi, Rae

    2009-03-01

    Neuroblastoma is the most common extracranial solid tumor in children and, when disseminated, carries a poor prognosis. Even with aggressive combinations of chemotherapy, surgery, autologous bone marrow transplant, and radiation, long-term survival remains at 30% and new therapies are needed. Recently, a patient with neuroblastoma who acquired Chagas disease was treated with nifurtimox with subsequent reduction in tumor size. The effect of nifurtimox on the neuroblastoma cell lines CHLA-90, LA1-55n, LA-N2, SMS-KCNR, and SY5Y was examined. Nifurtimox decreased cell viability in a concentration-dependent manner. Cell morphology, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay, and caspase-3 activation indicate that cell death was primarily due to apoptosis. Nifurtimox also suppressed basal and TrkB-mediated Akt phosphorylation, and the cytotoxicity of nifurtimox was attenuated by a tyrosine hydroxylase inhibitor (alpha-methyl-tyrosine). Nifurtimox killed catecholaminergic, but not cholinergic, autonomic neurons in culture. In vivo xenograft models showed inhibition of tumor growth with a histologic decrease in proliferation and increase in apoptosis. These results suggest that nifurtimox induces cell death in neuroblastoma. Therefore, further studies are warranted to develop nifurtimox as a promising new treatment for neuroblastoma.

  5. 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.

  6. 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.

  7. 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.

  8. Generation of reactive oxygen species mediates butein-induced apoptosis in neuroblastoma cells.

    Science.gov (United States)

    Chen, Ya-Hui; Yeh, Chi-Wei; Lo, Hui-Chen; Su, Shih-Li; Hseu, You-Cheng; Hsu, Li-Sung

    2012-04-01

    Flavonoids exhibit chemopreventive and chemotherapeutic effects. Butein, a bioactive flavonoid isolated from numerous native plants, has been shown to induce apoptosis in human cancer cells. In the current study, the molecular mechanisms of butein action on cell proliferation and apoptosis of neuroblastoma cells were evaluated. Treatment with butein decreased the viability of Neuro-2A neuroblastoma cells in a dose- and time-dependent manner. The dose-dependent nature of butein-induced apoptosis was characterized by an increase in the sub-G1 phase population. Treatment with butein significantly increased intracellular reactive oxygen species (ROS)levels and reduced the Bcl-2/Bax ratio, triggering the cleavage of pro-caspase 3 and poly-(ADP-ribose) polymerase (PARP). Pre-treatment with the antioxidant agent, N-acetyl cysteine (NAC), blocks butein-induced ROS generation and cell death. NAC also recovers butein-induced apoptosis-related protein alteration. In conclusion, butein-triggered neuroblastoma cells undergo apoptosis via generation of ROS, alteration of the Bcl‑2/Bax ratio, and cleavage of pro-caspase 3 and PARP. Our results suggest that butein may serve as a potential therapeutic agent for the treatment of neuroblastoma.

  9. Evaluation of potential prognostic value of Bmi-1 gene product and selected markers of proliferation (Ki-67 and apoptosis (p53 in the neuroblastoma group of tumors

    Directory of Open Access Journals (Sweden)

    Katarzyna Taran

    2016-02-01

    Full Text Available Introduction: Cancer in children is a very important issue in pediatrics. The least satisfactory treatment outcome occurs among patients with clinically advanced neuroblastomas. Despite much research, the biology of this tumor still remains unclear, and new prognostic factors are sought. The Bmi-1 gene product is a currently highly investigated protein which belongs to the Polycomb group (PcG and has been identified as a regulator of primary neural crest cells. It is believed that Bmi‑1 and N-myc act together and are both involved in the pathogenesis of neuroblastoma. The aim of the study was to assess the potential prognostic value of Bmi-1 protein and its relations with mechanisms of proliferation and apoptosis in the neuroblastoma group of tumors.Material/Methods: 29 formalin-fixed and paraffin-embedded neuroblastoma tissue sections were examined using mouse monoclonal antibodies anti-Bmi-1, anti-p53 and anti-Ki-67 according to the manufacturer’s instructions.Results: There were found statistically significant correlations between Bmi-1 expression and tumor histology and age of patients.Conclusions: Bmi-1 seems to be a promising marker in the neuroblastoma group of tumors whose expression correlates with widely accepted prognostic parameters. The pattern of BMI-1 expression may indicate that the examined protein is also involved in maturation processes in tumor tissue.

  10. ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 activation.

    Science.gov (United States)

    Ren, Ping; Yue, Ming; Xiao, Daibiao; Xiu, Ruijuan; Gan, Lei; Liu, Hudan; Qing, Guoliang

    2015-01-01

    Amplification of the MYCN gene in human neuroblastoma predicts poor prognosis and resistance to therapy. We previously showed that MYCN-amplified neuroblastoma cells constantly require large amounts of glutamine to support their unabated growth. However, the identity and regulation of the transporter(s) that capture glutamine in MYCN-amplified neuroblastoma cells and the clinical significance of the transporter(s) in neuroblastoma diagnosis remain largely unknown. Here, we performed a systemic glutamine influx analysis and identified that MYCN-amplified neuroblastoma cells predominantly rely on activation of ASCT2 (solute carrier family 1 member 5, SLC1A5) to maintain sufficient levels of glutamine essential for the TCA cycle anaplerosis. Consequently, ASCT2 depletion profoundly inhibited glutaminolysis, concomitant with a substantial decrease in cell proliferation and viability in vitro and inhibition of tumourigenesis in vivo. Mechanistically, we identified ATF4 as a novel regulator which coordinates with N-Myc to directly activate ASCT2 expression. Of note, ASCT2 expression, which correlates with that of N-Myc and ATF4, is markedly elevated in high-stage neuroblastoma tumour samples compared with low-stage ones. More importantly, high ASCT2 expression is significantly associated with poor prognosis and survival of neuroblastoma patients. In aggregate, these findings elucidate a novel mechanism depicting how cell autonomous insults (MYCN amplification) and microenvironmental stresses (ATF4 induction) in concert coordinate ASCT2 activation to promote aggressive neuroblastoma progression, and establish ASCT2 as a novel biomarker in patient prognosis and stratification. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas.

    Science.gov (United States)

    Ikegaki, Naohiko; Shimada, Hiroyuki; Fox, Autumn M; Regan, Paul L; Jacobs, Joshua R; Hicks, Sakeenah L; Rappaport, Eric F; Tang, Xao X

    2013-04-09

    Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines [SKNAS, SKNBE(2)C, CHP134, and SY5Y] with epigenetic modifiers for a short time, followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for more than a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSCs (iCSCs). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected s.c. into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSCs metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated large-cell NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, whereas the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest both that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells and that epigenetic changes may have contributed to the development of these most malignant NB cells.

  12. 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.

  13. Simultaneous Measurement of Neural Spike Recordings and Multi-Photon Calcium Imaging in Neuroblastoma Cells

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    Jeehyun Kim

    2012-11-01

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

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

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    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.

  15. 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.

  16. 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.

  17. Cell proliferation in carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, S.M.; Ellwein, L.B. (Univ. of Nebraska Medical Center, Omaha (USA))

    1990-08-31

    Chemicals that induce cancer at high doses in animal bioassays often fail to fit the traditional characterization of genotoxins. Many of these nongenotoxic compounds (such as sodium saccharin) have in common the property that they increase cell proliferation in the target organ. A biologically based, computerized description of carcinogenesis was used to show that the increase in cell proliferation can account for the carcinogenicity of nongenotoxic compounds. The carcinogenic dose-response relationship for genotoxic chemicals (such as 2-acetylaminofluorene) was also due in part to increased cell proliferation. Mechanistic information is required for determination of the existence of a threshold for the proliferative (and carcinogenic) response of nongenotoxic chemicals and the estimation of risk for human exposure.

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

    Directory of Open Access Journals (Sweden)

    Keiichi Tabata

    2015-04-01

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

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

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    Salwen Helen R

    2010-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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

  20. 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

  1. 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.

  2. Nifurtimox Induces Apoptosis of Neuroblastoma Cells in vitro and in vivo

    OpenAIRE

    Sholler, Giselle L Saulnier; Brard, Laurent; Straub, Jennifer A; Dorf, Lee; Illyene, Sharon; Koto, Karen; Kalkunte, Satyan; Bosenberg, Marcus; Ashikaga, Taka; Nishi, Rae

    2009-01-01

    Neuroblastoma is the most common extracranial solid tumor in children and, when disseminated, carries a poor prognosis. Even with aggressive combinations of chemotherapy, surgery, autologous bone marrow transplant and radiation, long-term survival remains at 30% and new therapies are needed. Recently, a patient with neuroblastoma who acquired Chagas disease was treated with nifurtimox with subsequent reduction in tumor size. The effect of nifurtimox on the neuroblastoma cell lines CHLA-90, LA...

  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. Polysulfide promotes neuroblastoma cell differentiation by accelerating calcium influx.

    Science.gov (United States)

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

    2015-04-10

    Polysulfides are a typical type of bound sulfur, which is physiologically stable form of sulfur species, derived from the hydrogen sulfide (H2S) that is generated endogenously in cells. We previously reported that bound sulfur protects neuronal cells from oxidative injury. In the present study, we demonstrated that polysulfides inhibited cell growth and promoted neurite outgrowth in mouse neuroblastoma Neuro2A (N2A) cells. However, Na2S showed no effect on neurite outgrowth in N2A cells. Furthermore, 2-APB and SKF96365, which are typical transient receptor potential (TRP) channel inhibitors, suppressed the neurite outgrowth induced by Na2S4. These new findings suggest that bound sulfur could induce neurite outgrowth and cell differentiation of N2A cells by accelerating calcium influx. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2015-09-01

    down regulation in LTE-T cells is not caused by specific culture conditions. T lymphocytes were activated with immobilized OKT3 (1 μg ml) and...a lethal acute respiratory distress syndrome and severe eosinophilia were reported in a patient vaccinated with irradiated autologous myeloblasts...condition ‘day 15’ indicates HPSE expression in LTE-T cells cultured for 14 d and re-stimulated with immobilized OKT3 and CD28-specific antibodies

  7. Synthesis of New Quinoxalines Containing an Oxirane Ring by the TDAE Strategy and in Vitro Evaluation in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marc Montana

    2014-09-01

    Full Text Available Neuroblastoma is an aggressive pediatric malignancy with significant chemotherapeutic resistance. In order to obtain new compounds active on neuroblastoma cell lines, we investigated the reactivity of carbanion formed via TDAE in quinoxaline series. The new synthesized compounds were tested for their anti-proliferative activity on two neuroblastoma cell lines, and seven oxirane derivatives obtained interesting activities.

  8. 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

  9. Synergistic interaction between cisplatin and gemcitabine in neuroblastoma cell lines and multicellular tumor spheroids

    NARCIS (Netherlands)

    Besançon, Odette G.; Tytgat, Godelieve A. M.; Meinsma, Rutger; Leen, René; Hoebink, Jerry; Kalayda, Ganna V.; Jaehde, Ulrich; Caron, Huib N.; van Kuilenburg, André B. P.

    2012-01-01

    The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A

  10. Anti-cancer effects of artesunate in a panel of chemoresistant neuroblastoma cell lines

    NARCIS (Netherlands)

    Michaelis, Martin; Kleinschmidt, Malte C.; Barth, Susanne; Rothweiler, Florian; Geiler, Janina; Breitling, Rainer; Mayer, Bernd; Deubzer, Hedwig; Witte, Olaf; Kreuter, Joerg; Doerr, Hans Wilhelm; Cinatl, Jaroslav; Cinatl, Jindrich; Witt, Olaf; Cinatl Jr., Jindrich

    2010-01-01

    Artemisinin derivatives are well-tolerated anti-malaria drugs that also exert anti-cancer activity. Here, we investigated artemisinin and its derivatives dihydroartemisinin and artesunate in a panel of chemosensitive and chemoresistant human neuroblastoma cells as well as in primary neuroblastoma

  11. 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

  12. Intermittent Hypoxia Regulates Stem-like Characteristics and Differentiation of Neuroblastoma Cells

    Science.gov (United States)

    Bhaskara, Vasantha Kumar; Mohanam, Indra; Rao, Jasti S.; Mohanam, Sanjeeva

    2012-01-01

    Background Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. Methods and Findings Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells. Conclusions Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells. PMID:22363512

  13. 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.

  14. Neuroblastoma GOTO cells are hypersensitive to disruption of lipid rafts.

    Science.gov (United States)

    Tomioka, Ryosaku; Minami, Natsumi; Kushida, Ai; Horibe, Shiho; Izumi, Ippei; Kato, Akira; Fukushima, Keiko; Ideo, Hiroko; Yamashita, Katsuko; Hirose, Shigehisa; Saito, Yuji

    2009-11-06

    GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Cell Proliferation and Cytotoxicity Assays.

    Science.gov (United States)

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  17. CDDO and ATRA Instigate Differentiation of IMR32 Human Neuroblastoma Cells

    National Research Council Canada - National Science Library

    Namrata Chaudhari; Priti Talwar; Christian Lefebvre D'hellencourt; Palaniyandi Ravanan

    2017-01-01

    ...(11)-dien-28-oic acid (CDDO) on human neuroblastoma IMR32 cells. Our results demonstrate that treatment with low concentration of CDDO and particularly in combination with all trans retinoic acid (ATRA...

  18. 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.

  19. trans-4,4’-Dihydroxystilbene (DHS) inhibits human neuroblastoma tumor growth and induces mitochondrial and lysosomal damages in neuroblastoma cell lines

    Science.gov (United States)

    Saha, Bhaskar; Patro, Birija Sankar; Koli, Mrunesh; Pai, Ganesh; Ray, Jharna; Bandyopadhyay, Sandip K.; Chattopadhyay, Subrata

    2017-01-01

    In view of the inadequacy of neuroblastoma treatment, five hydroxystilbenes and resveratrol (Resv) were screened for their cytotoxic property against human neuroblastoma cell lines. The mechanism of cytotoxic action of the most potent compound, trans-4,4’-dihydroxystilbene (DHS) was investigated in vitro using human neuroblastoma cell lines. DHS was also tested in a mouse xenograft model of human neuroblastoma tumor. The MTT, sub-G1, annexin V and clonogenic assays as well as microscopy established higher cytotoxicity of DHS than Resv to the IMR32 cell line. DHS (20 μM) induced mitochondrial membrane permeabilization (MMP) in the cells, as revealed from JC-1 staining, cytochrome c and ApaF1 release and caspases-9/3 activation. DHS also induced lysosomal membrane permeabilization (LMP) to release cathepsins B, L and D, and the cathepsins inhibitors partially reduced MMP/caspase-3 activation. The ROS, produced by DHS activated the p38 and JNK MAPKs to augment the BAX activity and BID-cleavage, and induce LMP and MMP in the cells. DHS (100 mg/kg) also inhibited human neuroblastoma tumor growth in SCID mice by 51%. Hence, DHS may be a potential chemotherapeutic option against neuroblastoma. The involvement of an independent LMP as well as a partially LMP-dependent MMP by DHS is attractive as it provides options to target both mitochondria and lysosome. PMID:29088756

  20. trans-4,4'-Dihydroxystilbene (DHS) inhibits human neuroblastoma tumor growth and induces mitochondrial and lysosomal damages in neuroblastoma cell lines.

    Science.gov (United States)

    Saha, Bhaskar; Patro, Birija Sankar; Koli, Mrunesh; Pai, Ganesh; Ray, Jharna; Bandyopadhyay, Sandip K; Chattopadhyay, Subrata

    2017-09-26

    In view of the inadequacy of neuroblastoma treatment, five hydroxystilbenes and resveratrol (Resv) were screened for their cytotoxic property against human neuroblastoma cell lines. The mechanism of cytotoxic action of the most potent compound, trans-4,4'-dihydroxystilbene (DHS) was investigated in vitro using human neuroblastoma cell lines. DHS was also tested in a mouse xenograft model of human neuroblastoma tumor. The MTT, sub-G1, annexin V and clonogenic assays as well as microscopy established higher cytotoxicity of DHS than Resv to the IMR32 cell line. DHS (20 μM) induced mitochondrial membrane permeabilization (MMP) in the cells, as revealed from JC-1 staining, cytochrome c and ApaF1 release and caspases-9/3 activation. DHS also induced lysosomal membrane permeabilization (LMP) to release cathepsins B, L and D, and the cathepsins inhibitors partially reduced MMP/caspase-3 activation. The ROS, produced by DHS activated the p38 and JNK MAPKs to augment the BAX activity and BID-cleavage, and induce LMP and MMP in the cells. DHS (100 mg/kg) also inhibited human neuroblastoma tumor growth in SCID mice by 51%. Hence, DHS may be a potential chemotherapeutic option against neuroblastoma. The involvement of an independent LMP as well as a partially LMP-dependent MMP by DHS is attractive as it provides options to target both mitochondria and lysosome.

  1. 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.

  2. Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells.

    Science.gov (United States)

    Herrmann, A; Rice, M; Lévy, R; Pizer, B L; Losty, P D; Moss, D; Sée, V

    2015-02-09

    Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies.

  3. 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.

  4. Targeting Suppressive Myeloid Cells Potentiates Checkpoint Inhibitors to Control Spontaneous Neuroblastoma.

    Science.gov (United States)

    Mao, Yumeng; Eissler, Nina; Blanc, Katarina Le; Johnsen, John Inge; Kogner, Per; Kiessling, Rolf

    2016-08-01

    Neuroblastoma is the most common extracranial solid cancer type in childhood, and high-risk patients have poor prognosis despite aggressive multimodal treatment. Neuroblastoma-driven inflammation contributes to the induction of suppressive myeloid cells that hamper efficient antitumor immune responses. Therefore, we sought to enhance antitumor immunity by removing immunosuppression mediated by myeloid cells. The prognostic values of myeloid cells are demonstrated by analyzing genomic datasets of neuroblastoma patients. The impact of tumor-derived factors on myelopoiesis and local induction of suppressive myeloid cells is dissected by in vitro culture models using freshly isolated human CD34(+) hematopoietic stem cells, primary human monocytes, and murine bone marrow cells. To test the therapeutic efficacy of BLZ945 as a monotherapy or in combination with checkpoint inhibitors, we used a transgenic murine model (TH-MYCN) that develops aggressive spontaneous neuroblastoma. We report that infiltrating CSF-1R(+) myeloid cells predict poor clinical outcome in patients with neuroblastoma. In vitro, neuroblastoma-derived factors interfere with early development of myeloid cells and enable suppressive functions on human monocytes through M-CSF/CSF-1R interaction. In a transgenic mouse model (TH-MYCN) resembling high-risk human neuroblastoma, antagonizing CSF-1R with a selective inhibitor (BLZ945) modulates the induction of human and murine suppressive myeloid cells and efficiently limit tumor progression. While checkpoint inhibitors are insufficient in controlling tumor growth, combining BLZ945 with PD-1/PD-L1 blocking antibodies results in superior tumor control. Our results demonstrate the essential role of CSF-1R signaling during the induction of suppressive myeloid cells and emphasize its clinical potential as an immunotherapy for human cancers. Clin Cancer Res; 22(15); 3849-59. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Histone deacetylase 8 in neuroblastoma tumorigenesis.

    Science.gov (United States)

    Oehme, Ina; Deubzer, Hedwig E; Wegener, Dennis; Pickert, Diana; Linke, Jan-Peter; Hero, Barbara; Kopp-Schneider, Annette; Westermann, Frank; Ulrich, Scott M; von Deimling, Andreas; Fischer, Matthias; Witt, Olaf

    2009-01-01

    The effects of pan-histone deacetylase (HDAC) inhibitors on cancer cells have shown that HDACs are involved in fundamental tumor biological processes such as cell cycle control, differentiation, and apoptosis. However, because of the unselective nature of these compounds, little is known about the contribution of individual HDAC family members to tumorigenesis and progression. The purpose of this study was to evaluate the role of individual HDACs in neuroblastoma tumorigenesis. We have investigated the mRNA expression of all HDAC1-11 family members in a large cohort of primary neuroblastoma samples covering the full spectrum of the disease. HDACs associated with disease stage and survival were subsequently functionally evaluated in cell culture models. Only HDAC8 expression was significantly correlated with advanced disease and metastasis and down-regulated in stage 4S neuroblastoma associated with spontaneous regression. High HDAC8 expression was associated with poor prognostic markers and poor overall and event-free survival. The knockdown of HDAC8 resulted in the inhibition of proliferation, reduced clonogenic growth, cell cycle arrest, and differentiation in cultured neuroblastoma cells. The treatment of neuroblastoma cell lines as well as short-term-culture neuroblastoma cells with an HDAC8-selective small-molecule inhibitor inhibited cell proliferation and clone formation, induced differentiation, and thus reproduced the HDAC8 knockdown phenotype. Global histone 4 acetylation was not affected by HDAC8 knockdown or by selective inhibitor treatment. Our data point toward an important role of HDAC8 in neuroblastoma pathogenesis and identify this HDAC family member as a specific drug target for the differentiation therapy of neuroblastoma.

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

    Science.gov (United States)

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

    2015-04-01

    Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  7. Glycolysis-respiration relationships in a neuroblastoma cell line.

    Science.gov (United States)

    Swerdlow, Russell H; E, Lezi; Aires, Daniel; Lu, Jianghua

    2013-04-01

    Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. 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.

  9. MMSET is highly expressed and associated with aggressiveness in neuroblastoma.

    Science.gov (United States)

    Hudlebusch, Heidi Rye; Skotte, Julie; Santoni-Rugiu, Eric; Zimling, Zarah Glad; Lees, Michael James; Simon, Ronald; Sauter, Guido; Rota, Rossella; De Ioris, Maria Antonietta; Quarto, Micaela; Johansen, Jens Vilstrup; Jørgensen, Mette; Rechnitzer, Catherine; Maroun, Lisa Leth; Schrøder, Henrik; Petersen, Bodil Laub; Helin, Kristian

    2011-06-15

    MMSET (WHSC1/NSD2) is a SET domain-containing histone lysine methyltransferase the expression of which is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have shown that MMSET mRNA levels are increased in other tumor types as well. We have carried out immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n = 164). The expression level of MMSET in neuroblastomas was significantly associated with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and postchemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we show that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation. Importantly, we show that MMSET is required for proliferation of neuroblastoma cells and brain-derived neural stem cells. Taken together, our results suggest that MMSET is implicated in neuroblastomagenesis possibly by supporting proliferation of progenitor cells and negatively regulating their differentiation. In this respect, MMSET might be a strong candidate therapeutic target in a subset of neuroblastomas with unfavorable prognosis.

  10. Unusual chromaffin cell differentiation of a neuroblastoma after chemotherapy and radiotherapy: report of an autopsy case with immunohistochemical evaluations.

    Science.gov (United States)

    Miyauchi, Jun; Kiyotani, Chikako; Shioda, Yoko; Kumagai, Masaaki; Honna, Toshiro; Matsuoka, Kentaro; Masaki, Hidekazu; Aiba, Motohiko; Hata, Jun-ichi; Tsunematsu, Yukiko

    2004-04-01

    Neuroblastomas are derived from neural crest cells that are capable of multilineage differentiation. Ganglionic neuronal differentiation of childhood neuroblastoma is seen with increasing age, leading to more differentiated tumors called ganglioneuroblastomas or ganglioneuromas. Despite the fact that neuroblastomas most often arise from the adrenal medulla, chromaffin-cell differentiation in neuroblastomas is not widely recognized. Tumor cells with a chromaffin-cell nature have only been detected using histochemical techniques in neuroblastoma cell lines or focal areas of certain in vivo tumors. We describe a neuroblastoma that exhibited an unusual differentiation toward chromaffin cells in a patient that had been treated with surgery, intensive chemotherapy, and radiotherapy. Although a biopsy specimen of the retroperitoneal primary tumor was extensively necrotic, possibly because of a previous chemotherapy regimen, surgically resected metastatic tumors of bilateral ovaries were viable and diagnosed as poorly differentiated neuroblastomas according to the International Neuroblastoma Pathology Classification system. However, metastatic tumors of bilateral lungs examined at the time of autopsy exhibited histologic features similar to those of a pheochromocytoma/paraganglioma, and immunohistochemical examinations demonstrated that these tumors were composed of extra-adrenal chromaffin cells. This case confirms that neuroblastomas in childhood can transform into pheochromocytoma/paraganglioma-like tumors under special conditions.

  11. Akt2 regulates metastatic potential in neuroblastoma.

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    Jingbo Qiao

    Full Text Available Activation of PI3K/AKT pathway correlates with poor prognosis in patients with neuroblastoma. Our previous studies have demonstrated that PI3K/AKT signaling is critical for the oncogenic transformations induced by gastrin-releasing peptide (GRP and its receptor, GRP-R, in neuroblastoma. Moreover, PI3K/AKT-dependent oncogenic transformations require N-myc, an extensively studied oncogene in neuroblastoma. Whether AKT directly regulates the expression of N-myc oncogene is yet to be determined. Here, we report a novel finding that of the three AKT isoforms, AKT2 specifically regulated N-myc expression in neuroblastoma cells. We also confirmed that GRP-R is upstream of AKT2 and in turn, regulated N-myc expression via AKT2 in neuroblastoma cells. Functional assays demonstrated that attenuation of AKT2 impaired cell proliferation and anchorage-independent cell growth, and decreased the secretion of angiogenic factor VEGF in vitro. Furthermore, silencing AKT2 inhibited migration and invasion of neuroblastoma cells in vitro. Xenografts established by injecting AKT2 silenced human neuroblastoma cells into murine spleen expressed decreased levels of AKT2 and resulted in fewer liver metastases compared to controls in vivo. Hence, our study highlights the potential molecular mechanism(s mediating the oncogenic role of GRP/GRP-R and demonstrates a novel role for AKT2 in neuroblastoma tumorigenesis, indicating that targeting the GRP/GRP-R/AKT2 axis may be important for developing novel therapeutics in the treatment of clinically aggressive neuroblastoma.

  12. Phenolic diterpenes derived from Hyptis incana induce apoptosis and G(2)/M arrest of neuroblastoma cells.

    Science.gov (United States)

    Tabata, Keiichi; Kim, Myongjun; Makino, Mitsuko; Satoh, Mitsuru; Satoh, Yoshio; Suzuki, Takashi

    2012-11-01

    Neuroblastoma is one of the most commonly encountered solid tumors in the pediatric age group, and the prognosis of patients with advanced neuroblastoma is very poor. In this study, the antitumor effects of five phenolic diterpenes derived from Hyptis incana (Lamiaceae), a Brazilian medicinal plant, were examined on neuroblastoma cells. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic nuclear shrinkage was monitored by Hoechst 33342 staining. The cell-cycle status was evaluated by flow cytometry and protein alterations were monitored by western blotting. Differentiated cells were photographed and counted in a randomized fashion. All of the examined compounds exhibited significant cytotoxicity towards the neuroblastoma cells. In particular, 7-ethoxyrosmanol had a high degree of efficacy. Nuclear condensation and degradation of procaspase-3 and -9 were observed after treatment of the cells with these compounds. Moreover, phenolic diterpenes induced cell-cycle arrest in the G(2)/M phase. Rosmanol and epirosmanol tended to induce differentiation. Phenolic diterpenes isolated from H. incana have multiple antitumor effects on neuroblastoma cells.

  13. Calcium signaling and cell proliferation.

    Science.gov (United States)

    Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

    2015-11-01

    Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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

  15. Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.

    Science.gov (United States)

    Bailey, Trisha L; Wang, Mian; Solocinski, Jason; Nathan, Britto P; Chakraborty, Nilay; Menze, Michael A

    2015-12-01

    A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to cell culture plates were incubated for 24 h at varying concentrations of trehalose, proline, sucrose and combinations of these compounds. Cells were cryopreserved for 24 h and cell viability post-freezing and thawing was quantified by trypan blue exclusion assay. On average, only 13.5% of adherent cells survived freezing in the presence of 10% Me2SO alone (control). Pre-incubation of cells with medium containing both trehalose and proline severely decreased cell proliferation, but increased cell recovery to about 53% of control. Furthermore, characterization using Raman microspectroscopy revealed that the addition of both trehalose and proline to 10% Me2SO substantially increased the size, and altered the nature, of ice crystals formed during freezing. Our results suggest that pre-incubation of Neuro-2a cells with trehalose and proline in combination provides cell protection along with alterations of ice structure in order to increase cell survival post-freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas.

    Science.gov (United States)

    Castino, Roberta; Pace, Deborah; Démoz, Marina; Gargiulo, Marco; Ariatta, Chiara; Raiteri, Elisabetta; Isidoro, Ciro

    2002-02-20

    Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen. Copyright 2001 Wiley-Liss, Inc.

  17. Regression of orthotopic neuroblastoma in mice by targeting the endothelial and tumor cell compartments

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    Stridsberg Mats

    2009-03-01

    Full Text Available Abstract Background High-risk neuroblastoma has an overall five-year survival of less than 40%, indicating a need for new treatment strategies such as angiogenesis inhibition. Recent studies have shown that chemotherapeutic drugs can inhibit angiogenesis if administered in a continuous schedule. The aim of this study was primarily to characterize tumor spread in an orthotopic, metastatic model for aggressive, MYCN-amplified neuroblastoma and secondarily to study the effects of daily administration of the chemotherapeutic agent CHS 828 on tumor angiogenesis, tumor growth, and spread. Methods MYCN-amplified human neuroblastoma cells (IMR-32, 2 × 106 were injected into the left adrenal gland in SCID mice through a flank incision. Nine weeks later, a new laparotomy was performed to confirm tumor establishment and to estimate tumor volume. Animals were randomized to either treatment with CHS 828 (20 mg/kg/day; p.o. or vehicle control. Differences between groups in tumor volume were analyzed by Mann-Whitney U test and in metastatic spread using Fisher's exact test. Differences with p Results The orthotopic model resembled clinical neuroblastoma in respect to tumor site, growth and spread. Treatment with CHS 828 resulted in tumor regression (p Conclusion The metastatic animal model in this study resembled clinical neuroblastoma and is therefore clinically relevant for examining new treatment strategies for this malignancy. Our results indicate that daily scheduling of CHS 828 may be beneficial in treating patients with high-risk neuroblastoma.

  18. Phospholipase Cβ1 regulates proliferation of neuronal cells.

    Science.gov (United States)

    Garwain, Osama; Valla, Kaitlyn; Scarlata, Suzanne

    2018-01-12

    Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cβ (PLCβ), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCβ1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCβ1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G1 phase, a cytosolic population of PLCβ1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27Kip1. Binding of PLCβ1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gαq by carbachol causes movement of PLCβ from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gαq moves PLCβ1 to the membrane, reverses G1 arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCβ1 that occurs at the onset of differentiation arrests cells in the G1 phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCβ1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cβ1 regulates proliferation of neuronal cells.

  19. 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

  20. 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.

  1. 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

    2017-09-10

    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.

  2. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    Science.gov (United States)

    2014-09-01

    prognostic marker in neuroblastoma, Cancer Res 71, 4314-4324. 6. Le, M. T., Xie, H., Zhou, B., Chia , P. H., Rizk, P., Um, M., Udolph, G., Yang, H...matter,   or   any   new   and   useful   improvement   thereof,   which,  taken  together,  include   practically  everything...is  an   important   therapeutic   strategy   for  neuroblastoma.  We  developed  a  direct   functional  high-­‐content

  3. Galectin-3 impairment of MYCN-dependent apoptosis-sensitive phenotype is antagonized by nutlin-3 in neuroblastoma cells.

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    Veronica Veschi

    Full Text Available MYCN amplification occurs in about 20-25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.

  4. Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma.

    Science.gov (United States)

    Gonzalez, Sergio; Naranjo, Araceli; Serrano, Lisa M; Chang, Wen-Chung; Wright, Christine L; Jensen, Michael C

    2004-06-01

    Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma. Copyright 2004 John Wiley & Sons, Ltd.

  5. 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].

  6. Retinoic acid evoked-differentiation of neuroblastoma cells predominates over growth factor stimulation: an automated image capture and quantitation approach to neuritogenesis.

    Science.gov (United States)

    Simpson, P B; Bacha, J I; Palfreyman, E L; Woollacott, A J; McKernan, R M; Kerby, J

    2001-11-15

    To facilitate the characterization of compounds that have positive growth factor mimetic effects on neuritogenesis, we have implemented a high-throughput functional assay which measures, in a multiparametric manner, the proliferation and differentiation characteristics of cells in a microtiter plate. Conditions were established using chronic incubation of SH-SY5Y human neuroblastoma cells with retinoic acid (RA) and/or nerve growth factor (NGF) in which discernible alterations in proliferation, growth, and differentiation of cells were induced. SH-SY5Y cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on Cellomics ArrayScanII was utilized to quantify the effects of these treatments on cell characteristics. NGF and retinoic acid were found to increase multiple parameters of SH-SY5Y differentiation, including an increased proportion of cells having neurites and increased extent of branching. However, marked differences in the effects of these compounds on SH-SY5Y growth and differentiation were also detected: whereas NGF increased cell number, RA treatment decreased cell number, and RA but not NGF caused significant elongation of neurites. This study quantifies and characterizes the effects of differentiating and proliferating agents on a human-derived neuroblastoma cell line. The high-content, rapid-throughput nature of this assay makes it ideal for functional identification and characterization of compounds regulating cell behavior.

  7. Oncogenic mutations of ALK kinase in neuroblastoma.

    Science.gov (United States)

    Chen, Yuyan; Takita, Junko; Choi, Young Lim; Kato, Motohiro; Ohira, Miki; Sanada, Masashi; Wang, Lili; Soda, Manabu; Kikuchi, Akira; Igarashi, Takashi; Nakagawara, Akira; Hayashi, Yasuhide; Mano, Hiroyuki; Ogawa, Seishi

    2008-10-16

    Neuroblastoma in advanced stages is one of the most intractable paediatric cancers, even with recent therapeutic advances. Neuroblastoma harbours a variety of genetic changes, including a high frequency of MYCN amplification, loss of heterozygosity at 1p36 and 11q, and gain of genetic material from 17q, all of which have been implicated in the pathogenesis of neuroblastoma. However, the scarcity of reliable molecular targets has hampered the development of effective therapeutic agents targeting neuroblastoma. Here we show that the anaplastic lymphoma kinase (ALK), originally identified as a fusion kinase in a subtype of non-Hodgkin's lymphoma (NPM-ALK) and more recently in adenocarcinoma of lung (EML4-ALK), is also a frequent target of genetic alteration in advanced neuroblastoma. According to our genome-wide scans of genetic lesions in 215 primary neuroblastoma samples using high-density single-nucleotide polymorphism genotyping microarrays, the ALK locus, centromeric to the MYCN locus, was identified as a recurrent target of copy number gain and gene amplification. Furthermore, DNA sequencing of ALK revealed eight novel missense mutations in 13 out of 215 (6.1%) fresh tumours and 8 out of 24 (33%) neuroblastoma-derived cell lines. All but one mutation in the primary samples (12 out of 13) were found in stages 3-4 of the disease and were harboured in the kinase domain. The mutated kinases were autophosphorylated and displayed increased kinase activity compared with the wild-type kinase. They were able to transform NIH3T3 fibroblasts as shown by their colony formation ability in soft agar and their capacity to form tumours in nude mice. Furthermore, we demonstrate that downregulation of ALK through RNA interference suppresses proliferation of neuroblastoma cells harbouring mutated ALK. We anticipate that our findings will provide new insights into the pathogenesis of advanced neuroblastoma and that ALK-specific kinase inhibitors might improve its clinical outcome.

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

    Science.gov (United States)

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

    2008-01-01

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

  9. I-131-Metaiodobenzylguanidine therapy with allogeneic cord blood stem cell transplantation for recurrent neuroblastoma

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    Sato Yuya

    2012-10-01

    Full Text Available Abstract Iodine-131-metaiodiobenzylguanidine (131I-MIBG therapy combined with allogeneic cord blood stem cell transplantation (SCT was used to treat a 4-year-old girl with recurrent neuroblastoma. The patient experienced relapse 2 years after receiving first-line therapies, which included chemotherapy, surgical resection, irradiation, and autologous peripheral SCT. Although 131I-MIBG treatment did not achieve complete remission, the size of the tumor was reduced after treatment. Based on our findings, we suggest that 131I-MIBG treatment with myeloablative allogeneic SCT should be considered as first-line therapy for high-risk neuroblastoma patients when possible.

  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. Exendin-4 induces cell adhesion and differentiation and counteracts the invasive potential of human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Paola Luciani

    Full Text Available Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R, thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i the evaluation of neurite-like protrusions in 3D cell cultures, ii the analysis of the expression of neuronal markers and iii electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Neuroblastoma Screening

    Science.gov (United States)

    ... Neuroblastoma Treatment for more information about neuroblastoma. Most cases of neuroblastoma are diagnosed before 1 year of ... to Use This Summary PDQ is a registered trademark. The content of PDQ documents can be used ...

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Targeting ATP7A to increase the sensitivity of neuroblastoma cells to retinoid therapy.

    Science.gov (United States)

    Cheung, B B; Marshall, G M

    2011-09-01

    Following the discovery that defective retinoid signaling directly contributes to tumorigenesis, and, that retinoids have an anti-cancer effect in vitro and in vivo, retinoids have become part of the routine care in children with neuroblastoma at the stage of minimal residual disease. However, many patients still relapse following retinoid therapy, demonstrating the need for more effective retinoids and better assays to predict retinoid sensitivity in cancer cells. Recent evidence suggests that the copper metabolism gene, ATP7A, is retinoid-regulated and an important component of the retinoic acid receptor β (RARβ) anticancer effect in neuroblastoma cells. To highlight and further develop the concept of using ATP7A as a target in retinoid therapy, and combination therapy with copper chelators in neuroblastoma, the current literature and abstracts related to the clinical application of retinoids, the function of ATP7A and the clinical application of copper chelators are summarized. We propose that strategies targeting the copper export protein, ATP7A, in combination therapy with retinoids and copper depletion therapy, may have great therapeutic potential in the clinical treatment of neuroblastoma and other malignancies.

  19. 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

  20. Identification of neuroblastoma stem cells by characterization of side population cells in the human neuroblastoma SK-N-SH cell line.

    Science.gov (United States)

    Qi, Shiqin; Zheng, Jicui; Zhu, Haitao; Yang, Lin; Xiao, Xianmin

    2010-12-01

    The purpose of the study was to sort side population (SP) cells from the neuroblastoma SK-N-SH cell line and to systematically investigate whether this population has stem cell characteristics. Side population and non-SP cells were separated from the SK-N-SH cell line by flow cytometry, and their morphologic characteristics were analyzed by light and electron microscopy. We also used Western blotting to analyze marker proteins, Cell Counting Kit-8 assay for proliferative ability, series differentiation studies for differentiation properties, and Matrigel invasion study and tumorigenicity assay for malignant potential. The SK-N-SH SP cells expressed high levels of stem cell markers, had high proliferative and malignant abilities, and had the capacity for rapid differentiation. The non-SP cells expressed differentiated cell marker proteins at high levels, had low proliferative and malignant abilities, and exhibited slow differentiation. The SK-N-SH SP cells have cancer stem cell-like properties. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. 3D culturing and differentiation of SH-SY5Y neuroblastoma cells on bacterial nanocellulose scaffolds.

    Science.gov (United States)

    Innala, Marcus; Riebe, Ilse; Kuzmenko, Volodymyr; Sundberg, Johan; Gatenholm, Paul; Hanse, Eric; Johannesson, Sara

    2014-10-01

    A new in vitro model, mimicking the complexity of nerve tissue, was developed based on a bacterial nanocellulose (BNC) scaffold that supports 3D culturing of neuronal cells. BNC is extracellularly excreted by Gluconacetobacter xylinus (G. xylinus) in the shape of long non-aggregated nanofibrils. The cellulose network created by G. xylinus has good mechanical properties, 99% water content, and the ability to be shaped into 3D structures by culturing in different molds. Surface modification with trimethyl ammonium beta-hydroxypropyl (TMAHP) to induce a positive surface charge, followed by collagen I coating, has been used to improve cell adhesion, growth, and differentiation on the scaffold. In the present study, we used SH-SY5Y neuroblastoma cells as a neuronal model. These cells attached and proliferated well on the BNC scaffold, as demonstrated by scanning electron microscopy (SEM) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Following neuronal differentiation, we demonstrated functional action potentials (APs) by electrophysiological recordings, indicating the presence of mature neurons on the scaffolds. In conclusion, we have demonstrated for the first time that neurons can attach, proliferate, and differentiate on BNC. This 3D model based on BNC scaffolds could possibly be used for developing in vitro disease models, when combined with human induced pluripotent stem (iPS) cells (derived from diseased patients) for detailed investigations of neurodegenerative disease mechanisms and in the search for new therapeutics.

  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

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gouget, B. E-mail: gouget@drecam.cea.fr; 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. Cell culture and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma.

    Science.gov (United States)

    Chand, Damini; Yamazaki, Yasuo; Ruuth, Kristina; Schönherr, Christina; Martinsson, Tommy; Kogner, Per; Attiyeh, Edward F; Maris, John; Morozova, Olena; Marra, Marco A; Ohira, Miki; Nakagawara, Akira; Sandström, Per-Erik; Palmer, Ruth H; Hallberg, Bengt

    2013-03-01

    Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

  6. Cell culture and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

    Directory of Open Access Journals (Sweden)

    Damini Chand

    2013-03-01

    Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK receptor tyrosine kinase (RTK, which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs. Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP, which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T. In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase and STAT3 (signal transducer and activator of transcription 3 in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i gain-of-function ligand-independent mutations such as ALKF1174l, (ii kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a and (iii ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066, albeit with differing levels of sensitivity.

  7. 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.

  8. Signaling pathways in PACAP regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Falktoft, Birgitte; Georg, Birgitte; Fahrenkrug, Jan

    2009-01-01

    Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene...... in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells....... expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and approximately 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists...

  9. Signaling pathways in PACAP regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

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

    2009-01-01

    Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene...... in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells. (C) 2009 Elsevier Ltd. All rights reserved Udgivelsesdato: 2009/10...... expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and similar to 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists...

  10. Increasing antiproliferative properties of endocannabinoids in N1E-115 neuroblastoma cells through inhibition of their metabolism.

    Directory of Open Access Journals (Sweden)

    Laurie Hamtiaux

    Full Text Available 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.

  11. Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma

    Science.gov (United States)

    Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J.; Singh, Ugra S.

    2014-01-01

    Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

  12. MicroRNA-497 increases apoptosis in MYCN amplified neuroblastoma cells by targeting the key cell cycle regulator WEE1.

    OpenAIRE

    Creevey, Laura; Ryan, Jacqueline; Harvey, Harry; Bray, Isabella M.; Meehan, Maria; Khan, Adnan R.; Stallings, Raymond L.

    2013-01-01

    PUBLISHED Neuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted t...

  13. Clinical Significance of Tumor-Associated Inflammatory Cells in Metastatic Neuroblastoma

    Science.gov (United States)

    Asgharzadeh, Shahab; Salo, Jill A.; Ji, Lingyun; Oberthuer, André; Fischer, Matthias; Berthold, Frank; Hadjidaniel, Michael; Liu, Cathy Wei-Yao; Metelitsa, Leonid S.; Pique-Regi, Roger; Wakamatsu, Peter; Villablanca, Judith G.; Kreissman, Susan G.; Matthay, Katherine K.; Shimada, Hiroyuki; London, Wendy B.; Sposto, Richard; Seeger, Robert C.

    2012-01-01

    Purpose Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature. Methods Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts. Results Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively. Conclusion These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets. PMID:22927533

  14. 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....

  15. Sodium ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake.

    Science.gov (United States)

    Carosio, Roberta; Zuccari, Guendalina; Orienti, Isabella; Mangraviti, Salvatore; Montaldo, Paolo G

    2007-08-30

    Neuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred. We could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells. Sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

  16. Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

    Directory of Open Access Journals (Sweden)

    Orienti Isabella

    2007-08-01

    Full Text Available Abstract Background Neuroblastoma (NB is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred. Results We could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC, further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells. Conclusion Sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

  17. The role of apoptosis in Listeria monocytogenes neural infection: listeriolysin O interaction with neuroblastoma Neuro-2a cells.

    Science.gov (United States)

    Parra, Maria C; Baquero, Fernando; Perez-Diaz, Jose C

    2008-01-01

    Listeria monocytogenes is the etiological agent of meningitis that affects individuals at high risk such as pregnant women, neonates, the elderly and immunocompromised individuals. Infection by this intracellular pathogen can be lethal if not diagnosed and treated. Mouse neuroblastoma Neuro-2a cells, a neuron-like cell line, were infected with L. monocytogenes. In this study apoptotic changes of neuroblastoma Neuro-2a cells infected with strains of Listeria producing different listeriolysin O levels are investigated by cytotoxicity assay, cellular viability assay, DAPI staining, intranucleosomal DNA fragmentation test, and monoclonal antibodies against ss-DNA. Results show that after internalization, the bacteria induced morphological, functional and genetic changes in the cells characteristic of apoptosis, which was dose-and time-dependent on listeriolysin O. Neuroblastoma Neuro-2a cells represent an interesting model cell line to further the understanding of Listeria pathogenesis within the central nervous system.

  18. Activating mutations in ALK provide a therapeutic target in neuroblastoma.

    Science.gov (United States)

    George, Rani E; Sanda, Takaomi; Hanna, Megan; Fröhling, Stefan; Luther, William; Zhang, Jianming; Ahn, Yebin; Zhou, Wenjun; London, Wendy B; McGrady, Patrick; Xue, Liquan; Zozulya, Sergey; Gregor, Vlad E; Webb, Thomas R; Gray, Nathanael S; Gilliland, D Gary; Diller, Lisa; Greulich, Heidi; Morris, Stephan W; Meyerson, Matthew; Look, A Thomas

    2008-10-16

    Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

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

    Science.gov (United States)

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

    2016-03-01

    Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Grzegorz Raszewski

    2015-12-01

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

  1. Control of cell proliferation by Myc

    DEFF Research Database (Denmark)

    Bouchard, C; Staller, P; Eilers, M

    1998-01-01

    Myc proteins are key regulators of mammalian cell proliferation. They are transcription factors that activate genes as part of a heterodimeric complex with the protein Max. This review summarizes recent progress in understanding how Myc stimulates cell proliferation and how this might contribute ...

  2. Delta-9-tetrahydrocannabinol depresses inward sodium current in mouse neuroblastoma cells.

    Science.gov (United States)

    Turkanis, S A; Partlow, L M; Karler, R

    1991-01-01

    Whole-cell voltage-clamp techniques were used in order to define the effects of delta-9-tetrahydrocannabinol (THC) on the voltage-gated sodium current in neuroblastoma cells. With regard to the inward sodium current, THC decreased the peak amplitude and increased both the time to peak and tau for recovery. The reversal potential was unchanged, suggesting that channel selectivity for sodium was not altered by the drug. With regard to the outward sodium current, THC had no effect on the peak amplitude, time to peak or tau for recovery. This functional alteration of the voltage-gated sodium channel may contribute to the depressant effects of the cannabinoid.

  3. Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell.

    Science.gov (United States)

    Mora, J; Cheung, N K; Juan, G; Illei, P; Cheung, I; Akram, M; Chi, S; Ladanyi, M; Cordon-Cardo, C; Gerald, W L

    2001-09-15

    The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

  4. 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.

  5. 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.

  6. Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

    Directory of Open Access Journals (Sweden)

    Gingras Denis

    2009-12-01

    Full Text Available Abstract Background Neuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14, an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression. Methods Tyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome, and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices. Results Although there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006 or in neuroblastoma samples from patients younger than one year (P = 0.0002. In vitro experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells. Conclusion Overall

  7. 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.

  8. Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

    Directory of Open Access Journals (Sweden)

    Jirapa Chetsawang

    2010-01-01

    Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

  9. Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

    Directory of Open Access Journals (Sweden)

    A L Furfaro

    Full Text Available The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM, fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1, the modulatory subunit of γ-glutamylcysteine ligase (GCLM and the transporter for cysteine (x-CT, enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 μM reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

  10. 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.

  11. Metyrapone, an inhibitor of cytochrome oxidases, does not affect viability in a neuroblastoma cell model of bilirubin toxicity

    Directory of Open Access Journals (Sweden)

    Maria N. Naguib Leerberg

    2014-01-01

    Conclusion: Herein we have shown that metyrapone does not increase cell death in neuroblastoma cells in culture exposed to bilirubin. Our results question the relationship between the oxidative mechanism evaluated by spectrophotometry and cell viability. Our findings add to the discussion on whether bilirubin oxidation represents a potentially important protective mechanism in neurons challenged by hyperbilirubinemia.

  12. Chemoresistance acquisition induces a global shift of expression of aniogenesis-associated genes and increased pro-angogenic activity in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Doerr Hans W

    2009-09-01

    Full Text Available Abstract Background Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology. Results Bioinformatics analysis of gene expression data revealed that expression of angiogenesis-associated genes significantly differs between chemosensitive and chemoresistant neuroblastoma cells. A subsequent systematic analysis of a panel of 14 chemosensitive and chemoresistant neuroblastoma cell lines in vitro and in animal experiments indicated a consistent shift to a more pro-angiogenic phenotype in chemoresistant neuroblastoma cells. The molecular mechanims underlying increased pro-angiogenic activity of neuroblastoma cells are individual and differ between the investigated chemoresistant cell lines. Treatment of animals carrying doxorubicin-resistant neuroblastoma xenografts with doxorubicin, a cytotoxic drug known to exert anti-angiogenic activity, resulted in decreased tumour vessel formation and growth indicating chemoresistance-associated enhanced pro-angiogenic activity to be relevant for tumour progression and to represent a potential therapeutic target. Conclusion A bioinformatics approach allowed to identify a relevant chemoresistance-associated shift in neuroblastoma cell biology. The chemoresistance-associated enhanced pro-angiogenic activity observed in neuroblastoma cells is relevant for tumour progression and represents a potential therapeutic target.

  13. 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...

  14. Idebenone induces apoptotic cell death in the human dopaminergic neuroblastoma SHSY-5Y cells.

    Science.gov (United States)

    Tai, Kwok-Keung; Pham, L; Truong, D D

    2011-11-01

    Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 μM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 μM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 μM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

  15. Automatic detection and genetic profiling of disseminated neuroblastoma cells.

    Science.gov (United States)

    Méhes, G; Luegmayr, A; Hattinger, C M; Lörch, T; Ambros, I M; Gadner, H; Ambros, P F

    2001-01-01

    Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

  16. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

    Energy Technology Data Exchange (ETDEWEB)

    Marzinke, Mark A. [Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544 (United States); Clagett-Dame, Margaret, E-mail: dame@biochem.wisc.edu [Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544 (United States); Pharmaceutical Science Division, University of Wisconsin-Madison, Madison, WI 53705-2222 (United States)

    2012-01-01

    The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21{sup Cip1}, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G{sub 1}/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer Calmin is a retinoic acid-responsive gene. Black-Right-Pointing-Pointer Calmin promotes cell cycle exit in N2A cells. Black-Right-Pointing-Pointer Calmin overexpression increases p21Cip1 and decreases cyclin D1. Black-Right-Pointing-Pointer Calmin is required for RA-induced growth inhibition and neurite outgrowth.

  17. Graphene oxide - gelatin nanohybrids as functional tools for enhanced Carboplatin activity in neuroblastoma cells.

    Science.gov (United States)

    Makharza, Sami; Vittorio, Orazio; Cirillo, Giuseppe; Oswald, Steffen; Hinde, Elizabeth; Kavallaris, Maria; Büchner, Bernd; Mertig, Michael; Hampel, Silke

    2015-06-01

    Preparation of Nanographene oxide (NGO) - Gelatin hybrids for efficient treatment of Neuroblastoma. Nanohybrids were prepared via non-covalent interactions. Spectroscopic tools have been used to discriminate the chemical states of NGO prior and after gelatin coating, with UV visible spectroscopy revealing the maximum binding capacity of gelatin to NGO. Raman and X-ray photoelectron spectroscopy (XPS) demonstrated NGO and Gelatin_NGO nanohybrids through a new chemical environments produced after noncovalent interaction. Microscopic analyses, atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to estimate the thickness of samples and the lateral width in the nanoscale, respectively. The cell viability assay validated Gelatin_NGO nanohybrids as a useful nanocarrier for Carboplatin (CP) release and delivery, without obvious signs of toxicity. The nano-sized NGO (200 nm and 300 nm) did not enable CP to kill the cancer cells efficiently, whilst the CP loaded Gel_NGO 100 nm resulted in a synergistic activity through increasing the local concentration of CP inside the cancer cells. The nanohybrids provoked high stability and dispersibility in physiological media, as well as enhanced the anticancer activity of the chemotherapy agent Carboplatin (CP) in human neuroblastoma cells.

  18. Immune Therapies for Neuroblastoma

    Science.gov (United States)

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

    2009-01-01

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

  19. Targeted inhibition of NMYC by peptide nucleic acid in N-myc amplified human neuroblastoma cells: cell-cycle inhibition with induction of neuronal cell differentiation and apoptosis.

    Science.gov (United States)

    Pession, Andrea; Tonelli, Roberto; Fronza, Raffaele; Sciamanna, Elena; Corradini, Roberto; Sforza, Stefano; Tedeschi, Tullia; Marchelli, Rosangela; Montanaro, Lorenzo; Camerin, Consuelo; Franzoni, Monica; Paolucci, Guido

    2004-02-01

    We developed an antisense peptide nucleic acid (PNA) targeted against a unique sequence in the terminus of the 5'-UTR of N-myc, designed for selective inhibition of NMYC in neuroblastoma cells. Fluorescent microscopy showed carrier-free delivery of the PNA to two human neuro-blastoma cell lines: GI-LI-N (N-myc-amplified) and GI-CA-N (N-myc-unamplified). Only in the former, PNA treatment determined 70% cell-viability reduction (at 48 h). In N-myc-amplified GI-LI-N cells, the PNA determined NMYC-translation inhibition (Western blotting), accumulation of cells in G1, induction of differentiation and apoptosis. Selectivity of the PNA was demonstrated by altering three point mutations. These findings should encourage development of a PNA-based tumor-specific agent for neuroblastoma (or other neoplasms) with N-myc overexpression.

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

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    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

  1. Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

    Science.gov (United States)

    Jamil, S; Cedervall, J; Hultman, I; Ali, R; Margaryan, N V; Rasmuson, A; Johnsen, J I; Sveinbjörnsson, B; Dalianis, T; Kanter, L; Orrego, A; Strizzi, L; Hendrix, M J C; Sandstedt, B; Kogner, P; Ahrlund-Richter, L

    2013-09-01

    Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem‑cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell‑induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non‑random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

  2. Immune Therapies for Neuroblastoma

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

    2009-01-01

    Neuroblastoma, a solid tumor arising from developing cells of the sympathetic nervous system, is the most common extracranial tumor in children. The prognosis for high-risk neuroblastoma remains poor with conventional treatment, and new approaches are therefore being explored to treat this disease. One such alternative therapy that holds promise is immune therapy. We review here the recent advances in 4 types of immune therapy – cytokine, vaccine, antibody, and cellular therapy – to treat neu...

  3. Scorpion (Odontobuthus doriae) venom induces apoptosis and inhibits DNA synthesis in human neuroblastoma cells.

    Science.gov (United States)

    Zargan, Jamil; Sajad, Mir; Umar, Sadiq; Naime, M; Ali, Shakir; Khan, Haider A

    2011-02-01

    Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 μg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 μg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 μg/ml. Two concentrations viz. 50 and 100 μ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 μg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 μg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties

  4. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

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    Susana Alvarez

    Full Text Available There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells promoter constructed with a dominant negative version of NFAT (dn-NFAT. Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl5,5-diphenyltetrazolium bromide and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

  5. 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.

  6. Modeling the electric potential across neuronal membranes: the effect of fixed charges on spinal ganglion neurons and neuroblastoma cells.

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    Thiago M Pinto

    Full Text Available We present a model for the electric potential profile across the membranes of neuronal cells. We considered the resting and action potential states, and analyzed the influence of fixed charges of the membrane on its electric potential, based on experimental values of membrane properties of the spinal ganglion neuron and the neuroblastoma cell. The spinal ganglion neuron represents a healthy neuron, and the neuroblastoma cell, which is tumorous, represents a pathological neuron. We numerically solved the non-linear Poisson-Boltzmann equation for the regions of the membrane model we have adopted, by considering the densities of charges dissolved in an electrolytic solution and fixed on both glycocalyx and cytoplasmic proteins. Our model predicts that there is a difference in the behavior of the electric potential profiles of the two types of cells, in response to changes in charge concentrations in the membrane. Our results also describe an insensitivity of the neuroblastoma cell membrane, as observed in some biological experiments. This electrical property may be responsible for the low pharmacological response of the neuroblastoma to certain chemotherapeutic treatments.

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

    Science.gov (United States)

    Petro, Kimberly A; Schengrund, Cara-Lynne

    2009-01-01

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

  8. Omega-3 Polyunsaturated Fatty Acids Trigger Cell Cycle Arrest and Induce Apoptosis in Human Neuroblastoma LA-N-1 Cells

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    Wai Wing So

    2015-08-01

    Full Text Available Omega-3 (n-3 fatty acids are dietary long-chain fatty acids with an array of health benefits. Previous research has demonstrated the growth-inhibitory effect of n-3 fatty acids on different cancer cell lines in vitro, yet their anti-tumor effects and underlying action mechanisms on human neuroblastoma LA-N-1 cells have not yet been reported. In this study, we showed that docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA exhibited time- and concentration-dependent anti-proliferative effect on the human neuroblastoma LA-N-1 cells, but had minimal cytotoxicity on the normal or non-tumorigenic cells, as measured by MTT reduction assay. Mechanistic studies indicated that DHA and EPA triggered G0/G1 cell cycle arrest in LA-N-1 cells, as detected by flow cytometry, which was accompanied by a decrease in the expression of CDK2 and cyclin E proteins. Moreover, DHA and EPA could also induce apoptosis in LA-N-1 cells as revealed by an increase in DNA fragmentation, phosphatidylserine externalization and mitochondrial membrane depolarization. Up-regulation of Bax, activated caspase-3 and caspase-9 proteins, and down-regulation of Bcl-XL protein, might account for the occurrence of apoptotic events. Collectively, our results suggest that the growth-inhibitory effect of DHA and EPA on LA-N-1 cells might be mediated, at least in part, via triggering of cell cycle arrest and apoptosis. Therefore, DHA and EPA are potential anti-cancer agents which might be used for the adjuvant therapy or combination therapy with the conventional anti-cancer drugs for the treatment of some forms of human neuroblastoma with minimal toxicity.

  9. Ethanol enhances tau accumulation in neuroblastoma cells that inducibly express tau

    Science.gov (United States)

    Gendron, Tania F.; McCartney, Sharon; Causevic, Ena; Ko, Li-wen; Yen, Shu-Hui

    2008-01-01

    Chronic alcohol consumption causes pathological changes in the brain and neuronal loss. Ethanol toxicity may partially result from the perturbation of microtubule associated proteins, like tau. Tau dysfunction is well known for its involvement in certain neurodegenerative diseases, such as Alzheimer's disease. In the present study, the effect of ethanol on tau was examined using differentiated human neuroblastoma cells that inducibly express the 4R0N isoform of tau via a tetracycline-off expression system. During tau induction, ethanol exposure (1.25-5 mg/ml) dose-dependently increased tau protein levels and reduced cell viability. The increase in cell death likely resulted from tau accumulation since increased levels of tau were sufficient to reduce cell viability and ethanol was toxic to cells expressing tau but not to non-induced controls. Tau accumulation did not result from greater tetracycline-off induction since ethanol neither increased tau mRNA expression nor the expression of the tetracycline-controlled transactivator. Additionally, ethanol increased endogenous tau protein levels in neuroblastoma cells lacking the tetracycline-off induction system for tau. Ethanol delayed tau clearance suggesting ethanol impedes its degradation. Though ethanol inhibited neither cathepsin B, cathepsin D, nor chymotrypsin-like activity, it did significantly reduce calpain 1 expression and activity. Calpain I knockdown by shRNA increased tau levels indicating that calpain participates in tau degradation in this model. Moreover, the activation of calpain, by the calcium ionophore A23187, partially reversed the accumulation of tau resulting from ethanol exposure. Impaired calpain-mediated degradation may thus contribute to the increased accumulation of tau caused by ethanol. PMID:18672021

  10. MicroRNA mediates DNA demethylation events triggered by retinoic acid during neuroblastoma cell differentiation.

    Science.gov (United States)

    Das, Sudipto; Foley, Niamh; Bryan, Kenneth; Watters, Karen M; Bray, Isabella; Murphy, Derek M; Buckley, Patrick G; Stallings, Raymond L

    2010-10-15

    Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.

  11. Quantitative analysis of PPT1 interactome in human neuroblastoma cells

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    Enzo Scifo

    2015-09-01

    Full Text Available Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1 or CLN1, cause Infantile NCL (INCL, MIM#256730. PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS. Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015 42–53.

  12. Synergistic interactions between PBDEs and PCBs in human neuroblastoma cells.

    Science.gov (United States)

    Pellacani, C; Tagliaferri, S; Caglieri, A; Goldoni, M; Giordano, G; Mutti, A; Costa, L G

    2014-04-01

    Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants. Exposure to these chemicals has been associated with developmental neurotoxicity, endocrine dysfunction, and reproductive disorders. Humans and wildlife are generally exposed to a mixture of these environmental pollutants, highlighting the need to evaluate the potential effects of combined exposures. In this study, we investigated the cytotoxic effects of the combined exposure to two PBDEs and two PCBs in a human neuronal cell line. 2,2',4,4'-Tetrabromodiphenyl ether, 2,2',4,4',5-pentabromodiphenyl ether, PCB-126 (3,3',4,4',5-pentachlorobiphenyl; a dioxin-like PCB), and PCB-153 (2,2',4,4',5,5'-hexachlorobiphenyl; a non-dioxin-like PCB) were chosen, because their concentrations are among the highest in human tissues and the environment. The results suggest that the nature of interactions is related to the PCB structure. Mixtures of PCB-153 and both PBDEs had a prevalently synergistic effect. In contrast, mixtures of each PBDE congener with PCB-126 showed additive effects at threshold concentrations, and synergistic effects at higher concentrations. These results emphasize the concept that the toxicity of xenobiotics may be affected by possible interactions, which may be of significance given the common coexposures to multiple contaminants. Copyright © 2012 Wiley Periodicals, Inc.

  13. A novel dysfunctional p53 mutation in the human neuroblastoma cell line TGW.

    Science.gov (United States)

    Sugiyama, Hisahiko; Arita, Michitsune; Min, Zhenghua; Zhong, Xioling; Iwasaki, Iwao; Hirano, Keihachiro; Shimatake, Hiroyuki; Hemmi, Hiromichi

    2003-12-01

    Mutations of p53 are rare in primary and advanced neuroblastomas. The p53 gene was studied in a TGW cell line established from a TNB1 xenograft, derived from metastasized neuroblastoma. The p53 protein level in TGW was elevated at baseline. Treatment with doxorubicin to induce genotoxic stress neither altered the p53 protein level nor induced p21 protein within 24 hours. DNA sequencing analysis revealed a novel triplet deletion mutation at codon 282 (R282del) of the p53 gene, a mutation also found in TNB1, indicating that the mutation occurred in the relapsed tumor. The mutant was incapable of transactivation and had no effect on the transactivational activity of the wild-type p53 gene product in reporter assays using a plasmid possessing a p53 responsive element of p21, bax or mdm2. These results suggest that the mutant p53R282del found in TGW is a non-functional mutant and has no dominant negative nature.

  14. Blue light inhibits proliferation of melanoma cells

    Science.gov (United States)

    Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2016-03-01

    Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

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

    Science.gov (United States)

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

    2008-04-01

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

  16. 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

  17. Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.

    Science.gov (United States)

    Huang, Jen-Ming; Sheard, Michael A; Ji, Lingyun; Sposto, Richard; Keshelava, Nino

    2010-12-01

    As p53 loss of function (LOF) confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt), and LOF TP53 (n = 4) or p14(ARF) deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (P flavopiridol combination in CHLA-90. The combination caused reduction in the expression of G(2)/M proteins (cyclin B1, Mad2, MPM2) in 2 cell lines with mt TP53 but not in those with wt TP53. Plk1 expression was reduced in all treated lines. Small interfering RNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. The combination of HDAC inhibitor and flavopiridol may be a unique approach to treating neuroblastomas with p53 LOF, one that evokes induction of mitotic failure. ©2010 AACR.

  18. Functional Sphere Profiling Reveals the Complexity of Neuroblastoma Tumor-Initiating Cell Model

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    Aurélie Coulon

    2011-10-01

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

  19. Acute mitochondrial and chronic toxicological effects of 1-methyl-4-phenylpyridinium in human neuroblastoma cells.

    Science.gov (United States)

    Stephans, Stacy E; Miller, Gary W; Levey, Allan I; Greenamyre, J Timothy

    2002-10-01

    At low micromolar concentrations, 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively kills nigrostriatal dopaminergic neurons by mechanisms believed to involve impairment of mitochondrial complex I. A human neuroblastoma cell line expressing the dopamine transporter (DAT) was utilized to examine the effects of MPP+ on acute physiologic responses and subsequent cell death. Acute responses were measured by microphysiometry and by monitoring mitochondrial membrane potential with [3H]tetraphenylphosphonium (TPP+) uptake. MPP+ (10 microM) increased extracellular proton excretion in DAT-expressing cells within 2-3 min, but had no effect in untransfected cells. The lipophilic complex I inhibitor, rotenone, increased proton excretion in both cell lines. In DAT-expressing cells, mitochondrial membrane potential was reduced within I h of 10 microM MPP+ exposure. Rotenone reduced mitochondrial membrane potential in both cell lines. MPP+ caused apoptotic death of DAT-transfected cells 2-3 days after drug application, but did not kill untransfected cells. Thus, MPP+ produces immediate mitochondrial impairment only in cells that express DAT, and these changes occur days before overt cellular toxicity. The magnitude, time course and nature of these changes were similar to those produced by rotenone, confirming the site of action of MPP+ as mitochondrial complex I. These immediate mitochondrial effects appear to be an accurate predictor of subsequent cell death.

  20. Anti-angiogenesis in neuroblastoma.

    Science.gov (United States)

    Ribatti, Domenico

    2013-06-01

    The nature of the angiogenic balance in neuroblastoma is complex, and a spectrum of angiogenesis stimulators and inhibitors have been detected in neuroblastoma tumours. The complex relationships between angiogenic cascade and anti-angiogenic agents in the tumour vascular phase have indicated that anti-angiogenesis can be considered as a strategy for the adjuvant therapy of neuroblastoma. The major goal is to establish if inhibition of angiogenesis is a realistic therapeutic strategy for inhibiting tumour cell dissemination and the formation of metastasis in neuroblastoma. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines.

    Science.gov (United States)

    Peng, Jiaojiao; Zhu, Shenghe; Hu, Lili; Ye, Pingping; Wang, Yifei; Tian, Qin; Mei, Mingzhu; Chen, Hao; Guo, Xiaofeng

    2016-10-02

    Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.

  2. [Humoral regulation of stem cell proliferation].

    Science.gov (United States)

    Musashi, M; Ogawa, M

    1991-05-01

    The central feature of hematopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The recruitment of the dormant stem cells into cell cycle may be promoted by such factors as interleukin (IL)-1, IL-6, granulocyte-colony stimulating factor (G-CSF), and newly discovered IL-11. The effects of IL-1 on stem cells may be indirect. Once the stem cells leave Go and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. Though several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a stochastic process. The proliferation of early hemopoietic progenitors requires the presence of IL-3 and/or IL-4, and the intermediate process appears to be supported by granulocyte/macrophage-CSF (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin (erythropoiesis), G-CSF (neutrophil production), and IL-5 (eosinophilopoiesis). Thus, hemopoietic proliferation appears to be regulated by a cascade of factors directed at different developmental stages.

  3. Immunohistochemical Assessment of Proliferating Cell Nuclear ...

    African Journals Online (AJOL)

    Background: Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S‑phase of the cell cycle. ... Two sections were taken from each one for H and E. Other sections were stained according to super sensitive polymer horseradish peroxidase method for identifying PCNA expression.

  4. Proteomic Analysis of the Effect of Extremely Low-Frequency Electromagnetic Fields (ELF-EMF) With Different Intensities in SH-SY5Y Neuroblastoma Cell Line.

    Science.gov (United States)

    Rezaie-Tavirani, Mostafa; Hasanzadeh, Hadi; Seyyedi, Samaneh; Zali, Hakimeh

    2017-01-01

    Introduction: During the last 3 decades, human is exposed to extremely low frequency electromagnetic fields (ELF-EMF) emitted by power lines and electronic devices. It is now well accepted that ELF-EMF are able to produce a variety of biological effects, although the molecular mechanism is unclear and controversial. Investigation of different intensities effects of 50 Hz ELF-EMF on cell morphology and protein expression is the aim of this study. Methods: SH-SY5Y human neuroblastoma cell line was exposed to 0.5 and 1 mT 50 Hz (ELF-EMF) for 3 hours. Proteomics techniques were used to determine the effects of these fields on protein expression. Bioinformatic and statistical analysis of proteomes were performed using Progensis SameSpots software. Results: Our results showed that exposure to ELF-EMF changes cell morphology and induces a dose-dependent decrease in the proliferation rate of the cells. The proteomic studies and bioinformatic analysis indicate that exposure to 50 Hz ELF-EMF leads to alteration of cell protein expression in both dose-dependent and intensity dependent manner, but the later is more pronounced. Conclusion: Our data suggests that increased intensity of ELF-EMF may be associated with more alteration in cell protein expression, as well as effect on cell morphology and proliferation.

  5. 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.

  6. Fatty acids and breast cancer cell proliferation.

    Science.gov (United States)

    Hardy, R W; Wickramasinghe, N S; Ke, S C; Wells, A

    1997-01-01

    We and others have shown that fatty acids are important regulators of breast cancer cell proliferation. In particular individual fatty acids specifically alter EGF-induced cell proliferation in very different ways. This regulation is mediated by an EGFR/G-protein signaling pathway. Understanding the molecular mechanisms of how this signaling pathway functions and how fatty acids regulate it will provide important information on the cellular and molecular basis for the association of dietary fat and cancer. Furthermore these in vitro studies may explain data previously obtained from in vivo animal studies and identify "good" as well as "bad" fatty acids with respect to the development of cancer.

  7. The impact of Zika virus infection on human neuroblastoma (SH-SY5Y) cell line.

    Science.gov (United States)

    Luplertlop, Natthanej; Suwanmanee, San; Muangkaew, Watcharamat; Ampawong, Sumate; Kitisin, Thitinan; Poovorawan, Yong

    2017-01-01

    An increase in Zika virus (ZIKV) epidemic during the last decade has become a major global concern as the virus affects both newborns and adult humans. Earlier studies have shown the impact of ZIKV infection in developing human foetus. However, effective in vitro model of target cells for studying the ZIKV infection in adult human neurons is not available. This study aimed to establish the use of human neuroblastoma cell line (SH-SY5Y) for studying an infection of ZIKV in vitro. ZIKV growth kinetics, viral toxicity, and SH-SY5Y cell vialibity were determined after ZIKV infection in SH-SY5Y cells in vitro. ZIKV-infected SH-SY5Y cells were morphologically analysed and compared with nonhuman primate Vero cells. Furthermore, the susceptibility of SH-SY5Y cells to ZIKV infection was also determined. The results showed that ZIKV efficiently infects SH-SY5Y cell lines in vitro. Gradual changes of several cellular homeostasis parameters including cell viability, cytotoxicity, and cell morphology were observed in ZIKVinfected SH-SY5Y cells when compared to mock-treated or non-human primate cells. Interestingly, ZIKV particles were detected in the nucleoplasmic compartment of the infected SH-SY5Y cells. The results suggest that ZIKV particle can be detected in the nucleoplasmic compartment of the infected SH-SY5Y cells beside the known viral replicating cytoplasmic area. Hence, SH-SY5Y cells can be used as an in vitro adult human neuronal cell-based model, for further elucidating the ZIKV biology, and highlight other possible significance of Zika virus distribution through nuclear localization, which may correlate to the neuropathological defects in ZIKV-infected adult humans.

  8. Histone deacetylase inhibitors repress chondrosarcoma cell proliferation.

    Science.gov (United States)

    Zhu, Jiaxue; Gu, Jianhua; Ma, Jie; Xu, Zhixing; Tao, Hairong

    2015-01-01

    Due to the high resistance to conventional therapy, there is still no convincingly effective treatment for chondrosarcoma. As a promising new treatment strategy, histone deacetylase inhibitors (HDACIs) have been reported to induce cell arrest, apoptosis and differentiation in some kinds of malignancies, but how HDACi exert their effects on chondrosarcoma is not well understood yet. We investigated the effects of HDACIs trichostatin A (TSA) and sodium valproate (VPA) on chondrosarcoma cells in vitro and in vivo. The cell proliferation and cell cycle were examined in two chondrosarcoma cell lines, SW1353 and JJ012, by MTS and flow cytometry assays, respectively. The in vivo effects of HDACIs were investigated by assessing the chondrosarcoma growth in a mouse xenograft model. Our results showed that TSA and VPA significantly repressed the proliferation of chondrosarcoma cells in a concentration-dependent manner. Flow cytometry indicated that TSA arrested the cell cycle in G2/M phase and VPA arrested the cell cycle in G1 phase. The tumor growth was markedly suppressed in mice treated with TSA and VPA. HDACIs significantly repress the proliferation of chondrosarcoma cells in vitro and in vivo. Our findings imply that HDACIs may provide a novel therapeutic target for the treatment of chondrosarcoma.

  9. A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

    OpenAIRE

    Fenteany, G; Standaert, R F; Reichard, G A; Corey, E J; Schreiber, S L

    1994-01-01

    Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacyst...

  10. 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.

  11. 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

  12. 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....

  13. A protein involved in central nervous system myelination: localization in the extracellular matrix and induction in neuroblastoma cells.

    Science.gov (United States)

    Notterpek, L M; Rome, L H

    1994-01-01

    G21.3, a monoclonal antibody previously shown to block central nervous system (CNS) myelination, is now demonstrated to recognize an extracellular matrix (ECM) component. The antigen is present on the surface of neurons but not oligodendrocytes and is highly abundant in the white matter of the adult rat brain; however, it is not found in isolated myelin. Double immunostaining studies indicate a neuronal and ependymal cell source of the G21.3 antigen and a developmental expression pattern distinct from known markers of CNS myelination. The antigen is found at low levels in non-neuronal tissue and is mainly localized to basement membranes. G21.3 immunoreactive proteins are upregulated by retinoic acid-induced differentiation of SK-N-SHF neuroblastoma cells. These results suggest that the G21.3 antigen is an axolemma-associated ECM component with roles in postnatal CNS development and cell-matrix interactions during morphological differentiation of neuroblastoma cells.

  14. Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity.

    Science.gov (United States)

    Kim, In Su; Koppula, Sushruta; Park, Pyo-Jam; Kim, Ee Hwa; Kim, Chan Gil; Choi, Wahn Soo; Lee, Kwang Ho; Choi, Dong-Kug

    2009-12-10

    Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications. To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP(+)), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms. The effects of CM against MPP(+)-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells. CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis. These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.

  15. Angiogenesis in neuroblastoma.

    Science.gov (United States)

    Ribatti, Domenico; Marimpietri, Danilo; Pastorino, Fabio; Brignole, Chiara; Nico, Beatrice; Vacca, Angelo; Ponzoni, Mirco

    2004-12-01

    Angiogenesis is a biological process by which new capillaries are formed from preexisting vessels. It occurs in physiological and pathological conditions, such as tumors, where a specific turning point is the transition from the avascular to the vascular phase. Tumor angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells able to stimulate the growth of the host's blood vessels. In neuroblastoma, the most common extracranial solid tumor of infancy and childhood, angiogenesis also appears to play an important role in determining tumor phenotype. The nature of the angiogenic balance in neuroblastoma is complex, and a spectrum of angiogenesis stimulators, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), and inhibitors, such as tissue inhibitors of matrix metalloproteinases (MMPs), have been detected in neuroblastoma tumors. Moreover, an increased production of MMP-2 and -9 has been also observed in advanced stages of tumor, favoring degradation of extracellular matrix and enhancing tumor dissemination. High tumor vascularity is correlated with widely disseminated disease, MYCN amplification, unfavorable histology, and poor outcome. In contrast, low tumor vascularity is associated with prognostically favorable features, such as a localized disease and favorable histology. It is becoming increasingly evident that agents that interfere with blood vessel formation also block tumor progression. Preclinical studies suggest that antiangiogenic strategies may be effective in the treatment of neuroblastoma. A major goal is the determination of whether inhibition of angiogenesis is a realistic way of inhibiting tumor cell dissemination and formation of metastasis in neuroblastoma.

  16. Noscapine induced apoptosis via downregulation of survivin in human neuroblastoma cells having wild type or null p53.

    Directory of Open Access Journals (Sweden)

    Shiwang Li

    Full Text Available Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. Noscapine, a nontoxic natural compound, can trigger apoptosis in many cancer types. We now show that p53 is dispensable for Noscapine-induced cell death in neuroblastoma cell lines, proapoptotic response to this promising chemopreventive agent is mediated by suppression of survivin protein expression. The Noscapine treatment increased levels of total and Ser(15-phosphorylated p53 protein in SK-SY5Y cells, but the proapoptotic response to this agent was maintained even after knockdown of the p53 protein level. Exposure of SK-SY5Y and LA1-5S cells to Noscapine resulted in a marked decrease in protein and mRNA level of survivin as early as 12 hours after treatment. Ectopic expression of survivin conferred statistically significant protection against Noscapine-mediated cytoplasmic histone-associated apoptotic DNA fragmentation. Also, the Noscapine-induced apoptosis was modestly but statistically significantly augmented by RNA interference of survivin in both cell lines. Furthermore, Noscapine-induced apoptotic cell death was associated with activation of caspase-3 and cleavage of PARP. In conclusion, the present study provides novel insight into the molecular circuitry of Noscapine-induced apoptosis to indicate suppression of survivin expression as a critical mediator of this process.

  17. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation.

    Science.gov (United States)

    Akter, Jesmin; Takatori, Atsushi; Islam, Md Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

    2014-10-10

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Novel anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates modulate the expression of p53-MYCN associated micro RNAs in neuroblastoma cells and cause cell cycle arrest and apoptosis.

    Science.gov (United States)

    Ramaiah, M Janaki; Pushpavalli, Sreerangam N C V L; Lavanya, A; Bhadra, Kaustav; Haritha, V; Patel, Nibedita; Tamboli, Jaki R; Kamal, Ahmed; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-10-15

    It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. N-Myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Juan C Corredor

    2016-01-01

    Full Text Available N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB. Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVδM51 and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVδM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses.

  20. p21Waf1/Cip1 is a common target induced by short-chain fatty acid HDAC inhibitors (valproic acid, tributyrin and sodium butyrate) in neuroblastoma cells.

    Science.gov (United States)

    Rocchi, Paola; Tonelli, Roberto; Camerin, Consuelo; Purgato, Stefania; Fronza, Raffaele; Bianucci, Fabrizio; Guerra, Francesco; Pession, Andrea; Ferreri, Anna Maria

    2005-06-01

    Histone acetyltransferase and histone deacetylase (HDAC) determine the acetylation status of histones, and thereby control the regulation of gene expression. HDAC inhibitors have been found to inhibit the growth of a variety of tumor cells in vitro and in vivo. We demonstrated previously that the short-chain fatty acid compound butyrate and its derivative tributyrin (both HDAC inhibitors) arrest cell growth and induce differentiation in human neuroblastoma (NB) cells. In the current study we investigated the effect of the HDAC inhibitor valproic acid (VPA) on proliferation and differentiation in human NB cells (SJ-N-KP, AF8). Treatment with VPA resulted in a strong inhibition of cell proliferation and induction of cell differentiation, as revealed by neurite outgrowth and increase of acetylcholinesterase specific activity. Moreover, we addressed the question of whether the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) are involved in the mechanism of action of members of the short-chain fatty acids class (VPA, sodium butyrate and tributyrin) of HDAC inhibitors, in human NB cells. We demonstrated that p21(Cip1) is a common target of induction of transcription and protein expression for all the three compounds, while only VPA induced a concomitant increase of p27(Kip1) gene expression. These results suggest that p21(Cip1) could be involved in the inhibition of proliferation and induction of differentiation in human NB cells induced by treatment with VPA or tributyrin or sodium butyrate. Moreover, p21(Cip1) could be applied in the molecular monitoring of drug action in the possible therapeutic application of these short-chain fatty acid members of HDAC inhibitors for human NB treatment.

  1. FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells.

    Science.gov (United States)

    Han, Weidong; Wu, Zhiqiang; Zhao, Yali; Meng, Yuanguang; Si, Yiling; Yang, Jie; Fu, Xiaobing; Yu, Li

    2009-07-01

    Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix-loop-helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1-4) via an N-terminal loop-helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix-loop-helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2-E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.

  2. NSAIDs and Cell Proliferation in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Raj Ettarh

    2010-06-01

    Full Text Available Colon cancer is common worldwide and accounts for significant morbidity and mortality in patients. Fortunately, epidemiological studies have demonstrated that continuous therapy with NSAIDs offers real promise of chemoprevention and adjunct therapy for colon cancer patients. Tumour growth is the result of complex regulation that determines the balance between cell proliferation and cell death. How NSAIDs affect this balance is important for understanding and improving treatment strategies and drug effectiveness. NSAIDs inhibit proliferation and impair the growth of colon cancer cell lines when tested in culture in vitro and many NSAIDs also prevent tumorigenesis and reduce tumour growth in animal models and in patients, but the relationship to inhibition of tumour cell proliferation is less convincing, principally due to gaps in the available data. High concentrations of NSAIDs are required in vitro to achieve cancer cell inhibition and growth retardation at varying time-points following treatment. However, the results from studies with colon cancer cell xenografts are promising and, together with better comparative data on anti-proliferative NSAID concentrations and doses (for in vitro and in vivo administration, could provide more information to improve our understanding of the relationships between these agents, dose and dosing regimen, and cellular environment.

  3. induced apoptosis of neuroblastoma

    African Journals Online (AJOL)

    Affiliated Hospital to Changchun Traditional Chinese Medicine University, 4Department of Pediatrics, The First Hospital of Jilin. University, Changchun ... treatment of cancer using a neuroblastoma (NB) cell model. Method: Cell viability assay ... long-term survival rate in the late-stage patients is less than 40 % even with ...

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

    Directory of Open Access Journals (Sweden)

    Zhi-gao Sun

    2016-01-01

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

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

    Science.gov (United States)

    Sun, Zhi-Gao; Chen, Li-Ping; Wang, Fa-Wei; Xu, Cheng-Yong; Geng, Miao

    2016-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Effects of Agelas oroides and Petrosia ficiformis crude extracts on human neuroblastoma cell survival.

    Science.gov (United States)

    Ferretti, Cristina; Marengo, Barbara; De Ciucis, Chiara; Nitti, Mariapaola; Pronzato, Maria Adelaide; Marinari, Umberto Maria; Pronzato, Roberto; Manconi, Renata; Domenicotti, Cinzia

    2007-01-01

    Among marine sessile organisms, sponges (Porifera) are the major producers of bioactive secondary metabolites that defend them against predators and competitors and are used to interfere with the pathogenesis of many human diseases. Some of these biological active metabolites are able to influence cell survival and death, modifying the activity of several enzymes involved in these cellular processes. These natural compounds show a potential anticancer activity but the mechanism of this action is largely unknown. In this study, we investigated the effects of two Mediterranean sponges, Agelas oroides and Petrosia ficiformis on the viability of human neuroblastoma cells. Upon treatment with the methanolic extract of Petrosia ficiformis, a marked cytotoxic effect was observed at any concentration or time of exposure. In contrast, a time- and dose-dependent effect was monitored for Agelas oroides that induced the development of apoptotic features and ROS production in LAN5 cells. These events were suppressed by calpeptin or zVAD and by vitamin C suggesting that the cell death caused by Agelas oroides was calpain- and caspase-dependent and of oxidative nature. Comet assay showed that this methanolic extract was not able to produce a genotoxic effect. Future studies will be applied to investigate the effect of isolated bioactive compounds from crude extract of this sponge which are potentially useful for cancer therapeutics.

  8. Shotgun proteomics and network analysis of neuroblastoma cell lines treated with curcumin.

    Science.gov (United States)

    D'Aguanno, Simona; D'Agnano, Igea; De Canio, Michele; Rossi, Claudia; Bernardini, Sergio; Federici, Giorgio; Urbani, Andrea

    2012-04-01

    Curcumin is a natural compound with recognized anti-inflammatory properties, but its anticancer activity is still object of study. We provided an unsupervised molecular investigation of the main proteome rearrangements involved in the cellular response to curcumin in a human neuroblastoma cell line sensitive to cisplatin and its resistant counterpart by a comparative proteomic approach. Shotgun analysis demonstrated that 66 proteins were differentially expressed in response to 24 h treatment with 40 μM curcumin in sensitive cells, whereas 32 proteins were significantly modulated in treated resistant cells. Functional analysis revealed that proteins involved in cellular assembly and organization, biosynthesis and glycolysis were down-regulated by curcumin treatment. Proteome changes were associated to cell cycle arrest in the G2/M phase and accumulation of polyubiquitinated proteins, also confirmed by flow cytometry and immunoblotting analysis, but not to a significant increment of reactive oxygen species production. Since the polyubiquitination of proteins influences a wide range of cellular pathways, the inhibition of the ubiquitin-proteasome system may be the main way through which curcumin performs its multi-target activity.

  9. 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.

  10. Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes.

    Science.gov (United States)

    Sugimoto, N; Ozutsumi, K; Matsuda, M; Higashida, H; Miki, N

    1983-12-01

    We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.

  11. 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.

  12. 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...

  13. 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

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

    Science.gov (United States)

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

    2014-04-01

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

  15. 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.

  16. 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.

  17. Jak Inhibitors Modulate Production of Replication Competent Zika Virus in Human Hofbauer, Trophoblasts, and Neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Christina Gavegnano

    2017-05-01

    Full Text Available Zika Virus (ZIKV is a Flavivirus that has been implicated in brain deformations, birth defects, and microcephaly of unborn fetuses and associated with Guillain-Barre syndrome.  Mechanisms responsible for transmission of ZIKV across the placenta to the fetus are incompletely understood.  Herein, we define key events modulating infection in clinically relevant cells, including primary placental macrophages (human hofbauer cells; HC, trophoblasts, and neuroblastoma cells. Consistent with previous findings, HC and trophoblasts are permissive to ZIKV infection. Decrease of interferon signaling by Jak 1/2 inhibition (via ruxolitinib significantly increased ZIKV replicationin HC, trophoblasts, and neuroblasts. Enhanced ZIKV production in ruxolitinib treated HC was associated with increased expression of HLA-DR and DC-SIGN. Nucleoside analogs blocked ruxolitinib-mediated production of extracellular virus. Although low-level ZIKV infection occurred in untreated HC and trophoblasts, the produced virus was incapable of infecting naïve Vero cells.  These deficient virions from untreated HC present “thin-coats” suggesting immature virion structure. Blocking Jak 1/2 signaling (with ruxolitinib restored replication competence as virions produced under these conditions confer CPE in naïve Vero cells.  These data demonstrate that Jak-STAT signaling directly impacts the ability of primary placental cells to produce replication competent virus and is a key gatekeeper in production of mature virions in clinically relevant cells including HC and trophoblasts. Design of targeted agents to prevent ZIKV replication in the placenta should consider Jak 1/2 signaling and the impact of its block on ZIKV infection and subsequent transmission to the fetus.

  18. Proliferation conditions for human satellite cells

    DEFF Research Database (Denmark)

    Gaster, M; Beck-Nielsen, H; Schrøder, H D

    2001-01-01

    Primary satellite cell cultures have become an important tool as a model system for skeletal muscles. A common problem in human satellite cell culturing is fibroblast overgrowth. We combined N-CAM (Leu19) immunocytochemical staining of satellite cells (Sc) with stereological methods to estimate...... the fraction of Sc in culture. Evaluation of different culture conditions allowed us to find proliferation conditions preferentially for Sc: a) Sc should be cultured on surfaces coated with ECM-gel. b) Primary cell culture should be inoculated in DMEM supplemented with 10% fetal calf serum to increase cell...... adherence. c) Change of media to DMEM supplemented with 2% Ultroser-G and 2% FCS after 24 h.d) Before subcultivation, cells should be preplated for 30 min. The fractional content of Sc in passage four when applying this method of cultivation was 0.82 +/- 0.07 (mean +/- SE, N = 10). Our method enabled us...

  19. 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.

  20. Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

    OpenAIRE

    Fu, T; Okano, Y; Zhang, W.; OZEKI, T.; Mitsui, Y; Nozawa, Y.

    1990-01-01

    Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or E...

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

    Directory of Open Access Journals (Sweden)

    Margarita Gutova

    2017-03-01

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

  2. Neuroprotective Effects of Castanea sativa Mill. Bark Extract in Human Neuroblastoma Cells Subjected to Oxidative Stress.

    Science.gov (United States)

    Brizi, Claudia; Santulli, Chiara; Micucci, Matteo; Budriesi, Roberta; Chiarini, Alberto; Aldinucci, Carlo; Frosini, Maria

    2016-02-01

    One of the major features of neurodegenerative disease is the selective vulnerability of different neuronal populations that are affected in a progressive and often stereotyped manner. Despite the susceptible neuronal population varies between diseases, oxidative stress is implicated as the major pathogenic process in all of them. Natural Extract of Castanea sativa Mill. bark (ENC), recently characterized in its phenolic composition, acts as antioxidant and cardioprotective agent. Its neuroprotettive properties, however, have never been investigated. The aim of this study was to assess neuroprotection of ENC in in vitro models of oxidative-stress-mediate injury. Human neuroblastoma SH-SY5Y cells treated with glutamate (50 mM for 24 h) or hydrogen peroxide (25 μM for 1 h followed by 24 with medium) were used. The results showed that the addition of ENC (1-50 μg/ml) to cell medium before the neuronal damage provided neuroprotection in both experimental models used, while its addition after the injury was ineffective. In conclusion, the present results suggest that ENC could be a valuable support as dietary supplement, combining beneficial preventive neuroprotettive effects with a high antioxidant activity. © 2015 Wiley Periodicals, Inc.

  3. Effect of ELF-EMF Exposure on Human Neuroblastoma Cell Line: a Proteomics Analysis.

    Science.gov (United States)

    Hasanzadeh, Hadi; Rezaie-Tavirani, Mostafa; Seyyedi, Samaneh Sadat; Zali, Hakimeh; Heydari Keshel, Saeid; Jadidi, Majid; Abedelahi, Ali

    2014-01-01

    Extremely low frequency electromagnetic fields (ELF-EMF) have been common in daily life all over the world. They have produced by power lines and electrical appliances, but higher levels of them have raised a lot of concerns about their carcinogenesis. Both epidemiological and laboratory studies have suggested that EMFs might increase cancer incidence, including acute childhood leukemia, brain and breast cancer. In the present study, SH-SY5Y human neuroblastoma cell line has exposed to 2mT, 50 Hz magnetic field for 3 h. Next, effect of this exposure on protein expression including over-expression or under-expression has assessed by proteomics. Bioinformatics and statistical analysis using progenesis same spot software on the obtained 2D electrophoresis has shown that expression of 189 proteins in exposed group has changed relative to control. Besides, PCA analysis has verified results of clustering, and has shown that protein data has clustered according to experimental conditions. The results of this study have shown that ELF-EMF changes cell morphology via altering protein expression, but more profound studies have needed to determine the kind of proteins altered.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Paolo Fardin

    2010-01-01

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

  6. Identification of multiple hypoxia signatures in neuroblastoma cell lines by l1-l2 regularization and data reduction.

    Science.gov (United States)

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

    2010-01-01

    Hypoxia is a condition of low oxygen tension occurring in the tumor and negatively correlated with the progression of the disease. We studied the gene expression profiles of nine neuroblastoma cell lines grown under hypoxic conditions to define gene signatures that characterize hypoxic neuroblastoma. The l(1)-l(2) regularization applied to the entire transcriptome identified a single signature of 11 probesets discriminating the hypoxic state. We demonstrate that new hypoxia signatures, with similar discriminatory power, can be generated by a prior knowledge-based filtering in which a much smaller number of probesets, characterizing hypoxia-related biochemical pathways, are analyzed. l(1)-l(2) regularization identified novel and robust hypoxia signatures within apoptosis, glycolysis, and oxidative phosphorylation Gene Ontology classes. We conclude that the filtering approach overcomes the noisy nature of the microarray data and allows generating robust signatures suitable for biomarker discovery and patients risk assessment in a fraction of computer time.

  7. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma.

    Science.gov (United States)

    Janoueix-Lerosey, Isabelle; Lequin, Delphine; Brugières, Laurence; Ribeiro, Agnès; de Pontual, Loïc; Combaret, Valérie; Raynal, Virginie; Puisieux, Alain; Schleiermacher, Gudrun; Pierron, Gaëlle; Valteau-Couanet, Dominique; Frebourg, Thierry; Michon, Jean; Lyonnet, Stanislas; Amiel, Jeanne; Delattre, Olivier

    2008-10-16

    Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

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

    Directory of Open Access Journals (Sweden)

    Yung-Ting Kuo

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

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

    Directory of Open Access Journals (Sweden)

    Pan Yeung

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

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Integrative genomics identifies LMO1 as a neuroblastoma oncogene.

    Science.gov (United States)

    Wang, Kai; Diskin, Sharon J; Zhang, Haitao; Attiyeh, Edward F; Winter, Cynthia; Hou, Cuiping; Schnepp, Robert W; Diamond, Maura; Bosse, Kristopher; Mayes, Patrick A; Glessner, Joseph; Kim, Cecilia; Frackelton, Edward; Garris, Maria; Wang, Qun; Glaberson, Wendy; Chiavacci, Rosetta; Nguyen, Le; Jagannathan, Jayanti; Saeki, Norihisa; Sasaki, Hiroki; Grant, Struan F A; Iolascon, Achille; Mosse, Yael P; Cole, Kristina A; Li, Hongzhe; Devoto, Marcella; McGrady, Patrick W; London, Wendy B; Capasso, Mario; Rahman, Nazneen; Hakonarson, Hakon; Maris, John M

    2011-01-13

    Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.

  12. Deoxyhypusine synthase (DHPS) inhibitor GC7 induces p21/Rb-mediated inhibition of tumor cell growth and DHPS expression correlates with poor prognosis in neuroblastoma patients.

    Science.gov (United States)

    Bandino, Andrea; Geerts, Dirk; Koster, Jan; Bachmann, André S

    2014-12-01

    Neuroblastoma (NB) is an aggressive pediatric malignancy that typically occurs in infants and children under the age of 5 years. High-stage tumors relapse frequently even after aggressive multimodal treatment, resulting in therapy resistance and eventually in patient death. Clearly, new biologically-targeted drugs are needed that more efficiently suppress tumor growth and prevent relapse. We and others previously showed that polyamines such as spermidine play an essential role in NB tumorigenesis and that DFMO, an inhibitor of the central polyamine synthesis gene ODC, is effective in vitro and in vivo, prompting its evaluation in NB clinical trials. However, the specific molecular actions of polyamines remain poorly defined. Spermidine and deoxyhypusine synthase (DHPS) are essential components in the hypusination-driven post-translational activation of eukaryotic initiation factor 5A (eIF5A). We assessed the role of DHPS in NB and the impact of its inhibition by N(1)-guanyl-1,7-diaminoheptane (GC7) on tumor cell growth using cell proliferation assays, Western blot, immunofluorescence microscopy, and Affymetrix micro-array mRNA expression analyses in NB tumor samples. We found that GC7 inhibits NB cell proliferation in a dose-dependent manner, through induction of the cell cycle inhibitor p21 and reduction of total and phosphorylated Rb proteins. Strikingly, high DHPS mRNA expression correlated significantly with unfavorable clinical parameters, including poor patient survival, in a cohort of 88 NB tumors (all P DHPS are key contributing factors in NB tumor proliferation through regulation of the p21/Rb signaling axis.

  13. Beyond cell proliferation in avian facial morphogenesis.

    Science.gov (United States)

    Linde-Medina, Marta; Hallgrímsson, Benedikt; Marcucio, Ralph

    2016-03-01

    The upper jaw in vertebrates forms from several prominences that arise around the stomodeum or primitive mouth. These prominences undergo coordinated growth and morphogenesis to fuse and form the face. Undirected, regionalized cell proliferation is thought to be the driving force behind the morphogenesis of the facial prominences. However, recent findings suggest that directed cell behaviors in the mesenchyme (e.g., directed cell division, directed cell movement, convergent extension) might be required for successful face formation. Here we discuss the evidence for this view and how directed behaviors may interact with the basement membrane to regulate morphogenesis of the facial region. We believe that future research in these largely unexplored areas could significantly impact our understanding of facial morphogenesis. © 2016 Wiley Periodicals, Inc.

  14. Antitumor activity of nifurtimox observed in a patient with neuroblastoma.

    Science.gov (United States)

    Saulnier Sholler, Giselle L; Kalkunte, Satyan; Greenlaw, Carla; McCarten, Kathleen; Forman, Edwin

    2006-10-01

    Chemotherapy-resistant neuroblastoma is a difficult disease to treat with poor survival. We treated a patient with neuroblastoma who had progressed on conventional chemotherapy. This 5-year-old girl with chemotherapy-resistant neuroblastoma developed Chagas disease at the start of salvage chemotherapy for which she was also started on nifurtimox. The neuroblastoma response to these treatments resulted in clinical remission. In vitro, treatment of a neuroblastoma cell line with nifurtimox resulted in decreased cell viability whereas no effect was seen on an endothelial cell line. Nifurtimox shows promise as a potential new treatment for neuroblastoma and warrants further testing.

  15. Eosinophil peroxidase signals via epidermal growth factor-2 to induce cell proliferation.

    LENUS (Irish Health Repository)

    Walsh, Marie-Therese

    2011-11-01

    Eosinophils exert many of their inflammatory effects in allergic disorders through the degranulation and release of intracellular mediators, including a set of cationic granule proteins that include eosinophil peroxidase. Studies suggest that eosinophils are involved in remodeling. In previous studies, we showed that eosinophil granule proteins activate mitogen-activated protein kinase signaling. In this study, we investigated the receptor mediating eosinophil peroxidase-induced signaling and downstream effects. Human cholinergic neuroblastoma IMR32 and murine melanoma B16.F10 cultures, real-time polymerase chain reaction, immunoprecipitations, and Western blotting were used in the study. We showed that eosinophil peroxidase caused a sustained increase in both the expression of epidermal growth factor-2 (HER2) and its phosphorylation at tyrosine 1248, with the consequent activation of extracellular-regulated kinase 1\\/2. This, in turn, promoted a focal adhesion kinase-dependent egress of the cyclin-dependent kinase inhibitor p27(kip) from the nucleus to the cytoplasm. Eosinophil peroxidase induced a HER2-dependent up-regulation of cell proliferation, indicated by an up-regulation of the nuclear proliferation marker Ki67. This study identifies HER2 as a novel mediator of eosinophil peroxidase signaling. The results show that eosinophil peroxidase, at noncytotoxic levels, can drive cell-cycle progression and proliferation, and contribute to tissue remodeling and cell turnover in airway disease. Because eosinophils are a feature of many cancers, these findings also suggest a role for eosinophils in tumorigenesis.

  16. Biocytin and biotin uptake into NB2a neuroblastoma and C6 astrocytoma cells.

    Science.gov (United States)

    Baur, Barbara; Suormala, Terttu; Baumgartner, E Regula

    2002-01-25

    Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence of high extracellular Na+ (Km 0.4 microM, Vmax 20 pmol/min/mg), K+ (Km 1.7 microM, Vmax 32 pmol/min/mg), or choline+ (Km 0.1 microM, Vmax 5 pmol/min/mg). Two transport systems (Km1 17 microM, Vmax1 53 pmol/min/mg; Km2 314 microM, Vmax2 360 pmol/min/mg) were identified for biotin with only system 1 being Na+-dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (kD 0.6 microl/min/mg) and a single saturable mechanism (Km 70 microM, Vmax 119 pmol/min/mg) at high extracellular Na+. High extracellular K+ enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.

  17. Cu-Zn superoxide dismutase activates muscarinic acetylcholine M1 receptor pathway in neuroblastoma cells.

    Science.gov (United States)

    Damiano, Simona; Petrozziello, Tiziana; Ucci, Valentina; Amente, Stefano; Santillo, Mariarosaria; Mondola, Paolo

    2013-01-01

    Muscarinic receptors (mAChRs) control several neuronal functions and are widely expressed in the central nervous system (CNS): M1 subtype represents the predominant mAChR in the CNS. Previously, we showed that antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) is secreted by many cellular lines and specifically interacts with cell surface membrane of human neuroblastoma SK-N-BE cells thus activating phospholipase C (PLC) transduction pathway and increasing intracellular calcium concentration ([Ca(2+)](i)). In addition, we demonstrated that a small amount of SOD1 is contained in large core dense vesicles and that it is secreted in response to depolarization induced by elevated extracellular K(+) concentration. In the present study, we investigated the involvement of muscarinic M1 receptors in SOD1-induced activation of PLC transduction pathway. We showed that, in SK-N-BE cells, SOD1 was able to activate muscarinic M1 receptor producing a phosphorylation of ERK 1/2 and Akt in dose- and time-dependent manner. Interestingly, in the presence of the M1 antagonist pirenzepine, ERK 1/2 and Akt phosphorylation induced by SOD1 was remarkably prevented. This effect was mimicked by knocking-down M1 receptor using two sequences of RNA silencing (siRNA). At functional level, siRNAs against M1 receptor were able to prevent the increase in [Ca(2+)](i) induced by SOD1. The same inhibitory effect on [Ca(2+)](i) changes was produced by the M1 antagonist pirenzepine. Collectively, the results of this study demonstrated that SOD1 could activate a transductional pathway through the involvement of M1 muscarinic receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. 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.

  19. PKC Activation Counteracts ADAM10 Deficit in HuD-Silenced Neuroblastoma Cells.

    Science.gov (United States)

    Marchesi, Nicoletta; Amadio, Marialaura; Colombrita, Claudia; Govoni, Stefano; Ratti, Antonia; Pascale, Alessia

    2016-09-06

    Neuronal ELAV/Hu (nELAV) are RNA-binding proteins that mainly regulate gene expression by increasing the stability and/or translation rate of target mRNAs bearing ARE (adenine and uracil-rich elements) sequences. Among nELAV target transcripts there is ADAM10, an α-secretase involved in the non-amyloidogenic processing of the amyloid-β protein precursor (AβPP) which leads to the production of the neuroprotective sAβPPα peptide. The aim of this study was to evaluate if nELAV depletion affects ADAM10 expression in human SH-SY5Y neuroblastoma cells. We also studied the effects of Bryostatin-1, a molecule able to activate nELAV protein cascade. The specific HuD/nELAV gene silencing decreased both nELAV and ADAM10 protein contents; similar results were obtained by Aβ40 treatment in wild-type SH-SY5Y cells. In HuD-silenced cells, the exposure to Bryostatin-1 counteracted both nELAV and ADAM10 proteins downregulation, by restoring nELAV/ADAM10 basal levels. We also found that sAβPPα release, which seemed not to be compromised by Aβ40 challenge or HuD-silencing, was favored by Bryostatin-1. Overall, these findings strongly suggest that a deficiency in nELAV content negatively affects ADAM10 expression and may play a role in neurodegenerative diseases, which may benefit by molecules activating ELAV cascade.

  20. Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells.

    Science.gov (United States)

    Pezzini, Francesco; Bettinetti, Laura; Di Leva, Francesca; Bianchi, Marzia; Zoratti, Elisa; Carrozzo, Rosalba; Santorelli, Filippo M; Delledonne, Massimo; Lalowski, Maciej; Simonati, Alessandro

    2017-05-01

    Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Briggs, D.J.

    1989-01-01

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

  2. Korean Red Ginseng inhibits apoptosis in neuroblastoma cells via estrogen receptor β-mediated phosphatidylinositol-3 kinase/Akt signaling

    OpenAIRE

    Nguyen, Cuong Thach; Luong, Truc Thanh; Kim, Gyu-Lee; Pyo, Suhkneung; Rhee, Dong-Kwon

    2014-01-01

    Background: Ginseng has been shown to exert antistress effects both in vitro and in vivo. However, the effects of ginseng on stress in brain cells are not well understood. This study investigated how Korean Red Ginseng (KRG) controls hydrogen peroxide-induced apoptosis via regulation of phosphatidylinositol-3 kinase (PI3K)/Akt and estrogen receptor (ER)-β signaling. Methods: Human neuroblastoma SK-N-SH cells were pretreated with KRG and subsequently exposed to H2O2. The ability of KRG to i...

  3. 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.

  4. Oxidative stress induced pulmonary endothelial cell proliferation is ...

    African Journals Online (AJOL)

    Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

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

    Science.gov (United States)

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

    2017-03-01

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

  6. Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

    Directory of Open Access Journals (Sweden)

    Ana P. Berbegall

    2014-06-01

    Full Text Available Neuroblastoma (NB is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68, and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients.

  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. TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells

    Science.gov (United States)

    Sutton, Selina K.; Koach, Jessica; Tan, Owen; Liu, Bing; Carter, Daniel R.; Wilmott, James S.; Yosufi, Benafsha; Haydu, Lauren E.; Mann, Graham J.; Thompson, John F.; Long, Georgina V.; Liu, Tao; McArthur, Grant; Zhang, Xu Dong; Scolyer, Richard A.; Cheung, Belamy B.; Marshall, Glenn M.

    2014-01-01

    High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. PMID:25333256

  9. 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.

  10. Cell proliferation alterations in Chlorella cells under stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: cid@udc.es [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)

    2009-09-14

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  11. A secreted factor represses cell proliferation in Dictyostelium

    OpenAIRE

    Brock, Debra A.; Gomer, Richard H.

    2005-01-01

    Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that i...

  12. Immunomodulatory effects of turmeric: Proliferation of spleen cells in mice.

    Science.gov (United States)

    Mustafa, Rafid; Blumenthal, Elliott

    2017-01-01

    Experiments were conducted to examine the effects of turmeric on spleen cell proliferation. Cell suspensions of spleen cells from young and aged mice were treated with or without conconavalin A (Con-A) as a proliferation stimulant, and with and without turmeric (20 mg/mL) in different concentrations. Spleen cells from young mice that received turmeric showed significant increase in spleen cell proliferation (P turmeric showed no significant increase in T lymphocytes. The data indicates that turmeric increases the ability of spleen cells in young mice to proliferate, in vitro.

  13. Effects of grape seed proanthocyanidin on 5-hydroxytryptamine(3) receptors in NCB-20 neuroblastoma cells.

    Science.gov (United States)

    Li, Qing-Zhong; Cho, Hyeong-Seok; Jeun, Seung-Hyun; Kim, Ki Jung; Choi, Se Joon; Sung, Ki-Wug

    2011-01-01

    Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)(3) receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3-100 µg/ml) and 3 µM 5-HT (near EC(50)) produced a slight inhibition of 5-HT-induced inward peak current (I(5-HT)) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I(5-HT) in an irreversible, concentration- and time-dependent manner (IC(50)=6.5±0.4 µg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 µM 5-HT, near-maximal concentration (IC(50)=22.1±0.4 µg/ml, Hill coefficient=2.4±0.1). High concentrations (≧30 µg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 µM 5-HT. Further studies showed that pretreatment of 20 µg/ml proanthocyanidin caused not only a rightward shift of the dose-response curve for 5-HT (EC(50) shift from 2.7±0.4 to 6.2±0.5 µM), but also a decreased E(max) (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT(3) receptors did not show a significant difference within the testing holding potential ranges (-50-+30 mV). These results suggest that proanthocyanidin inhibits 5-HT(3) receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.

  14. Equine Hoof Canker: Cell Proliferation and Morphology.

    Science.gov (United States)

    Apprich, V; Licka, T; Zipfl, N; Tichy, A; Gabriel, C

    2017-07-01

    Hoof canker is described as progressive pododermatitis of the equine hoof with absent epidermal cornification and extensive proliferation of the dermal papillary body; however, in-depth research on the type of proliferative activity has not yet been reported. The aim of the present study was to determine cell-specific proliferation patterns together with morphological analysis of hoof canker tissue. Tissues removed during surgery from 19 horses presented for treatment of canker were compared with similar postmortem tissues of healthy hooves of 10 horses. Morphological alterations visible in light microscopy were assessed semiquantitatively and graded for severity. Proliferative activity was evaluated by means of anti-PCNA (proliferative cell nuclear antigen) and anti-Ki67 immunohistochemistry. Histologically, canker tissue showed 5 major morphological alterations-the presence of lacunae, vacuoles, giant cells, hemorrhage, and inflammation-not seen in control tissue. Also, there was a notable koilocytotic appearance of keratinocytes in canker tissue. Immunohistochemistry revealed increased levels of PCNA protein expression in keratinocytes and fibroblasts of canker tissue compared with control tissue. In control tissue, keratinocytes showed higher levels of Ki67 compared with canker tissue, while the dermal fibroblasts of both groups showed similar levels of Ki67, indicating similar proliferative activity of less than 3% of total dermal fibroblasts. These results demonstrate that, in contrast to previous reports, there is no evidence for increased proliferative activity of the dermal papillary body associated with hoof canker. Increased levels of PCNA protein expression and morphological alterations indicate that dysregulation of keratinocyte differentiation constitutes a key event in equine hoof canker development.

  15. Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells.

    Science.gov (United States)

    Takamatsu, Yuki; Uchida, Leo; Morita, Kouichi

    2015-08-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases, which result in a high mortality ratio and neurological sequelae after recovery. Understanding the mechanism of neuropathogenicity in these viral infections is important for the development of specific antiviral therapy. Here, we focused on human-derived neuronal and glial cells to understand the cellular responses against WNV and JEV infection. It was demonstrated that early IFN-β induction regulated virus replication in glioblastoma tbl98G cells, whereas delayed IFN-β induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results, which showed different IFN responses between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms for neuropathology in these viral infections.

  16. Direct effects of riluzole on 5-hydroxytryptamine (5-HT)3 receptor-activated ion currents in NCB-20 neuroblastoma cells.

    Science.gov (United States)

    Kim, Ki Jung; Cho, Hyeong Seok; Choi, Se Joon; Jeun, Seung Hyun; Kim, Seong Yun; Sung, Ki-Wug

    2008-05-01

    The pharmacological action of riluzole, a drug that has been approved as a neuroprotective agent for the treatment of amyotrophic lateral sclerosis, has not yet been established. We examined the effects of riluzole on 5-hydroxytryptamine (5-HT)3) receptors in NCB-20 neuroblastoma cells using the whole-cell voltage clamp technique combined with a fast drug application method. Co-application of riluzole (1 - 300 microM, 5 s) produced a dose-dependent reduction in peak amplitudes and in the rise slope of the currents induced by 2 microM 5-HT. In addition, 5-HT3-mediated currents evoked by dopamine, a partial 5-HT3-receptor agonist, were inhibited by riluzole co-application. These inhibitory effects were clearly shown at low concentrations of 5-HT. The decay time constants of the receptor desensitization and deactivation were also significantly attenuated by riluzole. G-protein inhibitors (pertussis toxin and guanosine 5'-[beta-thio] diphosphate) did not completely block these inhibitory actions of riluzole. These results indicate that riluzole inhibits 5-HT3-induced ion currents directly by slowing channel activation in NCB-20 neuroblastoma cells.

  17. Reactive oxygen species regulate the levels of dual oxidase (Duox1-2 in human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Simona Damiano

    Full Text Available Dual Oxidases (DUOX 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13 and in rat brain and they are induced by platelet derived growth factor (PDGF. The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox (NOX2, or of the other membrane subunit of NADPH oxidase, p22(phox, blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.

  18. Skin Cell Proliferation Stimulated by Microneedles

    Science.gov (United States)

    Liebl, Horst; Kloth, Luther C.

    2012-01-01

    A classical wound may be defined as a disruption of tissue integrity. Wounds, caused by trauma from accidents or surgery, that close via secondary intention rely on the biological phases of healing, i.e., hemostasis, inflammation, proliferation, and remodeling (HIPR). Depending on the wound type and severity, the inflammation phase begins immediately after injury and may last for an average of 7–14 days. Concurrent with the inflammation phase or slightly delayed, cell proliferation is stimulated followed by the activation of the remodeling (maturation) phase. The latter phase can last as long as 1 year or more, and the final healed state is represented by a scar tissue, a cross-linked collagen formation that usually aligns collagen fibers in a single direction. One may assume that skin microneedling that involves the use of dozens or as many as 200 needles that limit penetration to 1.5 mm over 1 cm2 of skin would cause trauma and bleeding followed by the classical HIPR. However, this is not the case or at least the HIPR phases are significantly curtailed and healing never ends in a scar formation. Conversely dermabrasion used in aesthetic medicine for improving skin quality is based on “ablation” (destruction or wounding of superficial skin layers), which requires several weeks for healing that involves formation of new skin layers. Such procedures provoke an acute inflammatory response. We believe that a less intense inflammatory response occurs following microneedle perforation of the skin. However, the mechanism of action of microneedling appears to be different. Here we review the potential mechanisms by which microneedling of the skin facilitates skin repair without scarring after the treatment of superficial burns, acne, hyperpigmentation, and the non-advancing periwound skin surrounding the chronic ulcerations of the integument. PMID:24527373

  19. Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study

    Directory of Open Access Journals (Sweden)

    Hermanova Marketa

    2010-05-01

    Full Text Available Abstract Background We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2 and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA and inhibitors of lipoxygenases (LOX and cyclooxygenases (COX. This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Methods Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Results Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2 or SH-SY5Y after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX in combination with ATRA in both cell lines. Conclusions Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

  20. Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

    OpenAIRE

    Armstrong, J. L.; Ruiz, M.; Boddy, A V; Redfern, C P F; Pearson, A D J; Veal, G J

    2005-01-01

    Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid bi...

  1. Satellite cell proliferation in adult skeletal muscle

    Science.gov (United States)

    Booth, Frank W. (Inventor); Thomason, Donald B. (Inventor); Morrison, Paul R. (Inventor); Stancel, George M. (Inventor)

    1995-01-01

    Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

  2. Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lingling [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Zhao, Yingmin [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin [Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Gu, Jian [Department of Hematology, Yangzhou University School of Clinical Medicine, Yangzhou 225001 (China); Yu, Duonan, E-mail: duonan@yahoo.com [Department of Pediatrics, Jingjiang People' s Hospital, Yangzhou University, Jingjiang 214500 (China); Noncoding RNA Center, Yangzhou University, Yangzhou 225001 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Disease, Yangzhou 225001 (China); Institute of Comparative Medicine, Yangzhou University, Yangzhou 225001 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou 225001 (China)

    2016-06-10

    Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.

  3. 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.

  4. Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models.

    Directory of Open Access Journals (Sweden)

    Nishant Mohan

    Full Text Available Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA plasmid transfection and genistein (GST treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88 and upregulation of autophagy inhibiting marker molecules (p62 and mTOR in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

  5. Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells.

    Science.gov (United States)

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

    2013-11-01

    Polysulfide is a bound sulfur species derived from endogenous H2S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Cell proliferation and apoptosis in gastric cancer and intestinal metaplasia

    OpenAIRE

    Nora Manoukian Forones; Ana Paula Souza Carvalho; Oswaldo Giannotti-Filho; Laércio Gomes Lourenço; Celina Tizuko Fujiyama Oshima

    2005-01-01

    BACKGROUND: Higher proliferation is commonly observed in cancer cells. Apoptosis can be a useful measure of a tumor cell kinetic. Alteration of the balance between proliferation and apoptosis is associated with cancer. AIM: To study proliferation and apoptosis on gastric cancer and in intestinal metaplasia. METHODOLOGY: Twenty-two samples from gastric adenocarcinomas and 22 biopsies from intestinal metaplasia were studied. The apoptotic bodies in hematoxylin-eosin slides and the expression of...

  7. Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

    Science.gov (United States)

    Fu, T; Okano, Y; Zhang, W; Ozeki, T; Mitsui, Y; Nozawa, Y

    1990-11-15

    Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.

  8. Neural differentiation of the human neuroblastoma cell line IMR32 induces production of a thyrotropin-releasing hormone-like peptide

    NARCIS (Netherlands)

    J.M.M. Rondeel (Jan); W. Klootwijk (Willem); E. Linkels; W.J. de Greef (W.); T.J. Visser (Theo)

    1994-01-01

    textabstractThe human neuroblastoma cell line IMR32 produces and secretes substantial amounts of TRH-immunoreactivity (TRH-IR) as measured with radioimmunoassay (RIA) using the nonspecific antiserum 4319. It was found that synthesis of TRH-IR is dependent on neural differentiation: under serum-free

  9. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation.

    Directory of Open Access Journals (Sweden)

    José Manuel Tirado-Vélez

    Full Text Available Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40-70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

  10. Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hongsheng [Department of Histology and Embryology, Guangdong Medical College, Dongguan 523808, Guangdong (China); Wu, Fenping [The 7th People’s Hospital of Chengdu, Chengdu 610041, Sichuan (China); Wang, Yan [The Second School of Clinical Medicine, Guangdong Medical College, Dongguan 523808, Guangdong (China); Yan, Chong [School of Pharmacy, Guangdong Medical College, Dongguan 523808, Guangdong (China); Su, Wenmei, E-mail: wenmeisutg@126.com [Oncology of Affiliated Hospital Guangdong Medical College, Zhanjiang 524000, Guangdong (China)

    2014-08-08

    Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found that Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.

  11. 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

    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...... 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...

  12. 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.

  13. Withania somnifera water extract as a potential candidate for differentiation based therapy of human neuroblastomas.

    Science.gov (United States)

    Kataria, Hardeep; Wadhwa, Renu; Kaul, Sunil C; Kaur, Gurcharan

    2013-01-01

    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.

  14. 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

  15. 2,2',4,4'-Tetrabromodiphenyl ether promotes human neuroblastoma SH-SY5Y cells migration via the GPER/PI3K/Akt signal pathway.

    Science.gov (United States)

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

    2016-02-01

    Neuroblastoma is the predominant tumor of early childhood. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) has the highest concentration among all polybrominated diphenyl ether (PBDE) congeners in human body, particularly for children. Considering that accumulating evidences showed developmental neurotoxicity of PBDE, there is an urgent need to investigate the effects of BDE-47 on the development of neuroblastoma. This study revealed that BDE-47 had limited effects on the cytotoxicity while significantly increased the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells. This was further confirmed by the results that BDE-47 treatment significantly downregulated the expression of E-cadherin and zona occludin-1 and upregulated the expression of matrix metalloproteinase-9 (MMP-9). Silencing of MMP-9 by specific small interfering RNA significantly abolished the BDE-47-induced migration and invasion of SH-SY5Y cells. Further, the signals G protein-coupled estrogen receptor 1 (GPER)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) mediated the BDE-47-induced upregulation of MMP-9 and in vitro migration of SH-SY5Y cells since G15 (GPER inhibitor) and LY 294002 (PI3K/Akt inhibitor) significantly abolished the effects of BDE-47. Our results revealed that BDE-47 significantly triggered the metastasis of human neuroblastoma SH-SY5Y cells via upregulation of MMP-9 by the GPER/PI3K/Akt signal pathway. This study revealed for the first time that BDE-47 can promote the migration of SH-SY5Y cells. It also provided a better understanding about the metastasis of human neuroblastoma induced by environmental endocrine disruptors. © The Author(s) 2015.

  16. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  17. 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

  18. 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.

  19. Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells.

    Science.gov (United States)

    Choi, Jin-Sung; Choi, Bok Hee; Ahn, Hye Sook; Kim, Myung-Jun; Rhie, Duck-Joo; Yoon, Shin Hee; Min, Do Sik; Jo, Yang-Hyeok; Kim, Myung-Suk; Sung, Ki-Wug; Hahn, Sang June

    2003-12-01

    The effect of fluoxetine (Prozac) on 5-hydroxytryptamine(3) (5-HT(3))-mediated currents in NCB-20 neuroblastoma cells was examined using the whole-cell patch-clamp technique. Fluoxetine produced a significant reduction of peak amplitude without altering the activation time course of 5-HT(3)-mediated currents. These effects were concentration-dependent, with an IC(50) value of 4.15 microM. No voltage dependence was evident in fluoxetine's block of 5-HT(3)-mediated currents over the entire voltage range tested. The extent of block by pre-application of fluoxetine was significantly greater than that by co-application. Fluoxetine also increased the apparent rate of current desensitization to 5-HT application. Using a first-order kinetics analysis, the open-channel blocking rate constants were 0.06 microM(-1)s(-1) (k(+1), association rate constant) and 0.05 s(-1) (k(-1), dissociation rate constant), with an apparent K(d) (=k(-1)/k(+1)) of 0.83 microM. This value is close to an IC(50) of 1.11 microM obtained from the reduction in tau, the time constant of desensitization. Intracellular application of fluoxetine for long durations had no effect on the amplitude or kinetics of 5-HT(3)-mediated currents. Similarly, norfluoxetine, the major metabolite of fluoxetine, reduced the peak current, and enhanced the rate of current desensitization in a concentration-dependent manner with an IC(50) of 2.66 microM, indicating that norfluoxetine is more potent than fluoxetine in blocking 5-HT(3)-mediated currents. These results indicate that, at clinically relevant concentrations, fluoxetine and its metabolite, norfluoxetine, block 5-HT(3)-mediated currents in NCB-20 neuroblastoma cells.

  20. Significance of pleural effusion in neuroblastoma.

    Science.gov (United States)

    Gupta, Himesh; Conrad, John; Khoury, Joseph D; McGregor, Lisa M; Krasin, Matthew J; Dome, Jeffrey S; Santana, Victor M; Davidoff, Andrew M

    2007-12-01

    Pleural effusion is uncommon at diagnosis of neuroblastoma in children. Because the presence of malignant cells in pleural fluid may significantly change the management and outcome of patients with neuroblastoma, we retrospectively analyzed a cohort of neuroblastoma patients who presented with pleural effusion at the time of diagnosis to determine the incidence, presentation, stage, treatment, and outcome of these patients. We reviewed the presenting features of 295 patients with the diagnosis of neuroblastoma who received treatment at St. Jude Children's Research Hospital between 1991 and 2005. Patients were chosen for further analysis if pleural effusion had been identified on chest radiographs or computed tomography (CT) scans at diagnosis Thirty-one out of 295(10.5%) patients with neuroblastoma had pleural effusion identified at time of presentation. International neuroblastoma staging system (INSS) risk stratification was high risk in 29 cases and intermediate risk and low risk in 1 case each. The primary site of disease was abdomen in 26 patients; mediastinum in 5. We conducted cytologic analysis of pleural fluid of nine patients; the specimen of seven contained malignant cells. Eighteen of 31 patients died of progressive or recurrent disease. In patients with neuroblastoma, pleural effusion is usually associated with unfavorable biologic features and high-risk disease. Pleural fluid should be examined cytologically and at a time when the results would change the risk stratification. There was no statistically significant difference in the survival rate of the patients with high-risk neuroblastoma with or without malignant pleural effusion. 2007 Wiley-Liss, Inc

  1. Therapeutic touch stimulates the proliferation of human cells in culture.

    Science.gov (United States)

    Gronowicz, Gloria A; Jhaveri, Ankur; Clarke, Libbe W; Aronow, Michael S; Smith, Theresa H

    2008-04-01

    Our objective was to assess the effect of Therapeutic Touch (TT) on the proliferation of normal human cells in culture compared to sham and no treatment. Several proliferation techniques were used to confirm the results, and the effect of multiple 10-minute TT treatments was studied. Fibroblasts, tendon cells (tenocytes), and bone cells (osteoblasts) were treated with TT, sham, or untreated for 2 weeks, and then assessed for [(3)H]-thymidine incorporation into the DNA, and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The number of PCNA-stained cells was also quantified. For 1 and 2 weeks, varying numbers of 10-minute TT treatments were administered to each cell type to determine whether there was a dose-dependent effect. TT administered twice a week for 2 weeks significantly stimulated proliferation of fibroblasts, tenocytes, and osteoblasts in culture (p = 0.04, 0.01, and 0.01, respectively) compared to untreated control. These data were confirmed by PCNA immunocytochemistry. In the same experiments, sham healer treatment was not significantly different from the untreated cultures in any group, and was significantly less than TT treatment in fibroblast and tenocyte cultures. In 1-week studies involving the administration of multiple 10-minute TT treatments, four and five applications significantly increased [(3)H]-thymidine incorporation in fibroblasts and tenocytes, respectively, but not in osteoblasts. With different doses of TT for 2 weeks, two 10-minute TT treatments per week significantly stimulated proliferation in all cell types. Osteoblasts also responded to four treatments per week with a significant increase in proliferation. Additional TT treatments (five per week for 2 weeks) were not effective in eliciting increased proliferation compared to control in any cell type. A specific pattern of TT treatment produced a significant increase in proliferation of fibro-blasts, osteoblasts, and tenocytes in culture. Therefore, TT may

  2. Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.

    Science.gov (United States)

    Tonelli, Roberto; Purgato, Stefania; Camerin, Consuelo; Fronza, Raffaele; Bologna, Fabrizio; Alboresi, Simone; Franzoni, Monica; Corradini, Roberto; Sforza, Stefano; Faccini, Andrea; Shohet, Jason M; Marchelli, Rosangela; Pession, Andrea

    2005-05-01

    We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

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

    Science.gov (United States)

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

    2009-09-01

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

  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. Metformin inhibits the proliferation of benign prostatic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Zongwei Wang

    Full Text Available Benign prostatic hyperplasia (BPH is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1 promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R and regulating cell cycle.BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA.Metformin (0.5-10mM, 6-48h significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the expression of IGF-1R

  7. Dying endothelial cells stimulate proliferation of malignant glioma cells via a caspase 3-mediated pathway

    OpenAIRE

    Mao, Ping; Smith, Luke; XIE, WANFU; Wang, Maode

    2013-01-01

    Emerging evidence has indicated that apoptotic cells have a compensatory effect on the proliferation of neighboring cells. However, the potential role of dying vascular endothelial cells (ECs) in glioma tumor proliferation remains unclear. In the present study, three glioma cell lines were cocultured with dying ECs under various conditions to evaluate the effect of dying ECs on tumor proliferation using alamarBlue and trypan blue assays to assess cell proliferation and viability, respectively...

  8. A Neural Network Based Workstation for Automated Cell Proliferation Analysis

    Science.gov (United States)

    2001-10-25

    proliferation analysis, of cytological microscope images. The software of the system assists the expert biotechnologist during cell proliferation and...work was supported by the Programa de Apoyo a Proyectos de Desarrollo e Investigacíon en Informática REDII 2000. We thank Blanca Itzel Taboada for

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

    Directory of Open Access Journals (Sweden)

    Su Pei-Yi

    2012-08-01

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

  10. Ketamine suppresses the proliferation of rat C6 glioma cells.

    Science.gov (United States)

    Niwa, Hidetomo; Furukawa, Ken-Ichi; Seya, Kazuhiko; Hirota, Kazuyoshi

    2017-10-01

    The present study investigated the effects of N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine, on the growth of gliomas. To analyze the effects of ketamine treatment, rat C6 glioma cells arising from astrocytes, and RNB cells representing non-malignant astrocytes, were examined. In ketamine-treated C6 cells, the gene expression changes associated with cell proliferation following ketamine treatment were evaluated using a cDNA microarray. A cell proliferation assay was performed to analyze the dose-dependent proliferation of C6 glioma and RNB cells following culture (72 h) with ketamine treatment (0-100 µM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed following cell incubation with/without ketamine, to confirm if the ketamine-induced cell death of C6 glioma and RNB cells were due to apoptosis. In addition, cell proliferation and TUNEL assays were performed following cell incubations with a selective NMDAR antagonist, D-2-amino-5-phosphonovaleric acid (D-AP5). Analysis of the cDNA microarray indicated that the growth of C6 glioma cells were suppressed by the effects of ketamine. Furthermore, results of the proliferation assay confirmed that ketamine treatment inhibited C6 cell proliferation, most notably at a dose of 30 µM (n=7, 66.4%; Pcells, with a significant effect on the rate of death observed at all tested concentrations (3, 10, 30 and 100 µM). Results of the aforementioned proliferation and TUNEL assay experiments were reproduced when ketamine was replaced with a selective NMDAR antagonist, D-AP5. However, the NMDARantagonist-induced effects were not observed in RNB cell cultures. Although it would be premature to apply the results from the present study to human cases, these results indicated that ketamine is an anesthetic candidate providing potential benefit for glioma resection.

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Evaluation of the Cell Proliferation Process of Ovarian Follicles in Hypothyroid Rats by Proliferation Cell Nuclear Antigen Immunohistochemical Technique

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    M. Moghaddam Dorafshani

    2012-10-01

    Full Text Available Introduction & Objective: The normal females reproductive function , needs hypothalamus-hypophysis-ovarian extensive hormonal messages. Primary hypothyroidism is characterized by reduced production and secretion of thyroid hormones. During follicular growth PCNA (Proliferating Cell Nuclear Antigen and cycklin D complex play an important role in regulating cell proliferation .This study aimed to determine the cell proliferation index and how this process changes induced by thyroid hormone decreased in rat ovarian follicles.Materials & Methods: In this experimental study, 20 Wistar female rats were divided into experimental and control groups. Experimental group was chemically thyroidectomized by administering propylthiouracil (PTU (500 mg per liter of drinking water. The control group received normal drinking water. After three weeks rats were killed and their ovaries dissected and fixed for the histological preparation. Cell proliferation was determined by PCNA and stereological methods were used for counting cells.Results: Cell proliferation index showed a significant decrease in the frequency of follicular growth from prenatal to graafian follicles in hypothyroidism groups(P0.05 . PCNA expression determined that Primary follicle growth begins earlier. Positive PCNA cells were not observed in primordial follicles of the groups.Conclusion: According to the results of our study, this hypothesis is raised that granulosa cells in growing follicles may be increased by follicle adjacent cells in ovarian stroma . Hormonal changes following the reduction of thyroid hormones may greatly affect the cell proliferation index and lead to faster follicle degeneration.(Sci J Hamadan Univ Med Sci 2012; 19 (3:5-15

  13. Genetics Home Reference: neuroblastoma

    Science.gov (United States)

    ... activating mutations of the ALK kinase receptor in neuroblastoma. Nature. 2008 Oct 16;455(7215):967-70. doi: ... JM. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 2008 Oct 16;455(7215):930-5. doi: ...

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

    Science.gov (United States)

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

    2017-01-01

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

  15. Chemical Methods to Induce Beta-Cell Proliferation

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    Amedeo Vetere

    2012-01-01

    Full Text Available Pancreatic beta-cell regeneration, for example, by inducing proliferation, remains an important goal in developing effective treatments for diabetes. However, beta cells have mainly been considered quiescent. This “static” view has recently been challenged by observations of relevant physiological conditions in which metabolic stress is compensated by an increase in beta-cell mass. Understanding the molecular mechanisms underlining these process could open the possibility of developing novel small molecules to increase beta-cell mass. Several cellular cell-cycle and signaling proteins provide attractive targets for high throughput screening, and recent advances in cell culture have enabled phenotypic screening for small molecule-induced beta-cell proliferation. We present here an overview of the current trends involving small-molecule approaches to induce beta-cell regeneration by proliferation.

  16. 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).

  17. Neuroblastoma: molecular pathogenesis and therapy.

    Science.gov (United States)

    Louis, Chrystal U; Shohet, Jason M

    2015-01-01

    Neuroblastoma is a developmental tumor of young children arising from the embryonic sympathoadrenal lineage of the neural crest. Neuroblastoma is the primary cause of death from pediatric cancer for children between the ages of one and five years and accounts for ∼13% of all pediatric cancer mortality. Its clinical impact and unique biology have made this aggressive malignancy the focus of a large concerted translational research effort. New insights into tumor biology are driving the development of new classification schemas. Novel targeted therapeutic approaches include small-molecule inhibitors as well as epigenetic, noncoding-RNA, and cell-based immunologic therapies. In this review, recent insights regarding the pathogenesis and biology of neuroblastoma are placed in context with the current understanding of tumor biology and tumor/host interactions. Systematic classification of patients coupled with therapeutic advances point to a future of improved clinical outcomes for this biologically distinct and highly aggressive pediatric malignancy.

  18. Lysosome vacuolation disrupts the completion of autophagy during norephedrine exposure in SH-SY5Y human neuroblastoma cells.

    Science.gov (United States)

    Funakoshi, Takeshi; Aki, Toshihiko; Unuma, Kana; Uemura, Koichi

    2013-01-15

    In our current study, we examined the mechanism underlying neuronal cell injuries caused by norephedrine in SH-SY5Y human neuroblastoma cells. Norephedrine was found to induce cytoplasmic vacuolation and a resultant loss of cell viability. In the cells treated with norephedrine also, an autophagic marker LC3 was converted to its LC3-II activated form, suggesting the induction of autophagy. In cells transfected with RFP-LC3 and GFP-LAMP1, a punctate patterning of LC3 expression and colocalization of LAMP1 with the formed vacuoles were observed, highlighting the lysosomal nature of the vacuoles and their association with autophagosomes. An autophagic flux assay using tfLC3 (mRFP-GFP-LC3) indicated the formation of autophagosomes and autolysosomes by norephedrine stimulation at an early timepoint (∼3 h). However, at a later timepoint (∼6 h), both the dilation of autolysosomes/lysosomes and the neutralization of the vacuolar pH were also observed. These results thus indicate that norephedrine induces autophagy at an early timepoint and cell death with lysosomal dysfunction and autophagy disruption at a later timepoint. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

    Directory of Open Access Journals (Sweden)

    Yumiko Mitome-Mishima

    Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

  20. Catecholamines Inhibit Gastric Epithelial [RGM-1] Cell Proliferation ...

    African Journals Online (AJOL)

    adrenergic blocker) significantly (but not totally) reversed the inhibitory action of ADR on cell proliferation. Furthermore, procaterol (selective beta-2 agonist) but not dobutamine (selective beta-1 agonist) had effects similar to those produced by ...

  1. A Confocal and Electron Microscopic Comparison of Interferon β–Induced Changes in Vesicular Stomatitis Virus Infection of Neuroblastoma and Nonneuronal Cells

    Science.gov (United States)

    D'Agostino, Paul M.

    2010-01-01

    Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNβ results in a 104-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNβ, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNβ-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18–24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNβ-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells, we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of

  2. Hypoxia promotes adipose-derived stem cell proliferation via VEGF

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    Phuc Van Pham

    2016-01-01

    Full Text Available Adipose-derived stem cells (ADSCs are a promising mesenchymal stem cell source with therapeutic applications. Recent studies have shown that ADSCs could be expanded in vitro without phenotype changes. This study aimed to evaluate the effect of hypoxia on ADSC proliferation in vitro and to determine the role of vascular endothelial growth factor (VEGF in ADSC proliferation. ADSCs were selectively cultured from the stromal vascular fraction obtained from adipose tissue in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. ADSCs were cultured under two conditions: hypoxia (5% O2 and normal oxygen (21% O2. The effects of the oxygen concentration on cell proliferation were examined by cell cycle and doubling time. The expression of VEGF was evaluated by the ELISA assay. The role of VEGF in ADSC proliferation was studied by neutralizing VEGF with anti-VEGF monoclonal antibodies. We found that the ADSC proliferation rate was significantly higher under hypoxia compared with normoxia. In hypoxia, ADSCs also triggered VEGF expression. However, neutralizing VEGF with anti-VEGF monoclonal antibodies significantly reduced the proliferation rate. These results suggest that hypoxia stimulated ADSC proliferation in association with VEGF production. [Biomed Res Ther 2016; 3(1.000: 476-482

  3. [COMPARISON OF CYTOPROTECTIVE EFFECTS OF HEMANTANE AND AMANTADINE UNDER CONDITIONS OF 6-HYDROXYDOPAMINE NEUROTOXIN ACTION ON CULTURED HUMAN NEUROBLASTOMA CELLS].

    Science.gov (United States)

    Logvinov, I O; Antipova, T A; Nepoklonov, A V; Valdman, E A

    2016-01-01

    Potential neuroprotective activity of the novel antiparkinsonian drug hemantane (hydrochloride N-2-(adamantyl)-hexamethylenimine) in comparison to amantadine has been studied in various regimes of administration on human neuroblastoma SH-SY5Y cell line injury induced by 6-hydroxydopamine (6-OHDA), which is used as in vitro model of dopaminergic neurons for Parkinson's disease. Two regimes of hemantane and amantadine administration in a range of final concentrations 10⁻⁶-10⁻⁸ M were used either prior to or immediately after 6-OHDA introduction. MTT colorimetric assay was used to assess the viability of test cells. Significant decrease in viability of SH-SY5Y cells treated with 6-OHDA was observed. The addition of hemantane to cell medium produced cytoprotective effects in both regimes of administration--before and after 6-OHDA--at concentrations 10⁻⁷ M and 10⁻⁶-10⁻⁸ M, respectively. Amantadine in con- centrations 10⁻⁷-10⁻⁸ M was effective to increase cell survival only when administered after 6-OHDA. These results show that hemantane has a greater neu-roprotective potential in comparison to amantadine.

  4. Inhibition of brain tumor cell proliferation by alternating electric fields

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr [School of Biosystem and Biomedical Science, Korea University, Seoul 136-703 (Korea, Republic of); Sung, Jiwon; Jeong, Seonghoon; Yoon, Myonggeun, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr [Department of Bio-convergence Engineering, Korea University, Seoul 136-703 (Korea, Republic of); Koh, Eui Kwan [Seoul Center, Korea Basic Science Institute, Seoul 136-713 (Korea, Republic of)

    2014-11-17

    This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

  5. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    Science.gov (United States)

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  6. 7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

    African Journals Online (AJOL)

    melanin contents compared with control cells. Evaluation of tyrosinase activity. After culture and incubation with PEC or solvent for 72 h, cells were collected, washed twice with. PBS, and subjected to trypsinization. The cells were then treated with trypan blue (0.4 %). Cell lysates were prepared by treating the cells for 30.

  7. Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins.

    Directory of Open Access Journals (Sweden)

    Fabio Morandi

    Full Text Available Metastases in the bone marrow (BM are grim prognostic factors in patients with neuroblastoma (NB. In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin, CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.

  8. Regulation of germline stem cell proliferation downstream of nutrient sensing

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    Roy Richard

    2006-12-01

    Full Text Available Abstract Stem cells have recently attracted significant attention largely due to their potential therapeutic properties, but also because of their role in tumorigenesis and their resemblance, in many aspects, to cancerous cells. Understanding how stem cells are regulated, namely with respect to the control of their proliferation and differentiation within a functional organism, is thus primordial to safely profit from their therapeutic benefits. Here, we review recent advances in the understanding of germline stem cell proliferation control by factors that respond to the nutritional status and/or insulin signaling, through studies performed in C. elegans and Drosophila. Together, these data uncover some shared fundamental features that underlie the central control of cellular proliferation within a target stem cell population in an organism. These features may indeed be conserved in higher organisms and may apply to various other stem cell populations.

  9. A new parameter of growth inhibition for cell proliferation assays.

    Science.gov (United States)

    Fiorentino, Francesco P; Bagella, Luigi; Marchesi, Irene

    2017-10-12

    Cell proliferation assays are performed by four decades to test the anti-proliferative activity of natural products and synthetic compounds in cell cultures. In cancer research, they are widely employed to evaluate drug efficacy in in vitro tumor models, such as established cell lines, primary cultures, and recently developed three-dimensional tumor organoids. In this manuscript, we demonstrated that current employed parameters used by researchers to quantify in vitro growth inhibition, IC50 and GI50 , lead to a misinterpretation of results based on the exponential, and not linear, proliferation of the cells in culture. Therefore, we introduce a new parameter for the analysis of growth inhibition in cell proliferation assays, termed relative population doubling capacity, that can be employed to properly quantify the anti-proliferative activity of tested compounds and to compare drug efficacy between distinct cell models. © 2017 Wiley Periodicals, Inc.

  10. Neonatal pancreatic pericytes support β-cell proliferation

    Directory of Open Access Journals (Sweden)

    Alona Epshtein

    2017-10-01

    Conclusions: This study introduces pancreatic pericytes as regulators of neonatal β-cell proliferation. In addition to advancing current understanding of the physiological β-cell replication process, these findings could facilitate the development of protocols aimed at expending these cells as a potential cure for diabetes.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

    2010-01-01

    Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer’s disease (AD). Considerable attention has 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 (GSH) 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 damages, 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. PMID:18822368

  13. Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins.

    Science.gov (United States)

    Wu, Peng-Fei; Xie, Na; Zhang, Juan-Juan; Guan, Xin-Lei; Zhou, Jun; Long, Li-Hong; Li, Yuan-Long; Xiong, Qiu-Ju; Zeng, Jian-Hua; Wang, Fang; Chen, Jian-Guo

    2013-06-01

    Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson's disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

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    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  15. Proliferation of differentiated glial cells in the brain stem

    Directory of Open Access Journals (Sweden)

    Barradas P.C.

    1998-01-01

    Full Text Available Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions.

  16. 7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

    African Journals Online (AJOL)

    PEC) on melanoma cell lines. Methods: Cell viability was analyzed by trypan blue exclusion assays and the cell cycle by flow cytometry using ModFit LT software. Specifically, cells were stained with propidium iodide (0.5 mg/mL) supplemented ...

  17. Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells.

    Science.gov (United States)

    Fisher, Jonathan P H; Yan, Mengyong; Heuijerjans, Jennifer; Carter, Lisa; Abolhassani, Ayda; Frosch, Jennifer; Wallace, Rebecca; Flutter, Barry; Capsomidis, Anna; Hubank, Mike; Klein, Nigel; Callard, Robin; Gustafsson, Kenth; Anderson, John

    2014-11-15

    The majority of circulating human γδT lymphocytes are of the Vγ9Vδ2 lineage, and have T-cell receptor (TCR) specificity for nonpeptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focused on stimulation using ligands of the Vγ9Vδ2 receptor, whereas relatively little is known about variant blood γδT subsets and their potential role in cancer immunotherapy. To expand the full repertoire of γδT without bias toward specific TCRs, we made use of artificial antigen-presenting cells loaded with an anti γδTCR antibody that promoted unbiased expansion of the γδT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vδ2 TCR chains (Vδ2(+)), Vδ1 chains (Vδ1(+)), and TCR of other δ chain subtypes (Vδ1(neg)Vδ2(neg)). Both freshly isolated and expanded cells showed heterogeneity of differentiation markers, with a less differentiated phenotype in the Vδ1 and Vδ1(neg)Vδ2(neg) populations. Expanded cells were largely of an effector memory phenotype, although there were higher numbers of less differentiated cells in the Vδ1(+) and Vδ1(neg)Vδ2(neg) populations. Using neuroblastoma tumor cells and the anti-GD2 therapeutic mAb ch14.18 as a model system, all three populations showed clinically relevant cytotoxicity. Although killing by expanded Vδ2 cells was predominantly antibody dependent and proportionate to upregulated CD16, Vδ1 cells killed by antibody-independent mechanisms. In conclusion, we have demonstrated that polyclonal-expanded populations of γδT cells are capable of both antibody-dependent and -independent effector functions in neuroblastoma. ©2014 American Association for Cancer Research.

  18. Cell cycles and proliferation patterns in Haematococcus pluvialis

    Science.gov (United States)

    Zhang, Chunhui; Liu, Jianguo; Zhang, Litao

    2017-09-01

    Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation; far less attention has been paid to cell cycles and proliferation patterns. The purpose of this study was to clarify cell cycles and proliferation patterns in H. pluvialis microscopically using a camera and video recorder system. The complicated life history of H. pluvialis can be divided into two stages: the motile stage and the non-motile stage. All the cells can be classified into forms as follows: motile cell, nonmotile cell, zoospore and aplanospore. The main cell proliferation, both in the motile phase and non-motile phase in H. pluvialis, is by asexual reproduction. Under normal growth conditions, a motile cell usually produces two, sometimes four, and exceptionally eight zoospores. Under unfavorable conditions, the motile cell loses its flagella and transforms into a non-motile cell, and the non-motile cell usually produces 2, 4 or 8 aplanospores, and occasionally 20-32 aplanospores, which further develop into non-motile cells. Under suitable conditions, the non-motile cell is also able to release zoospores. The larger non-motile cells produce more than 16 zoospores, and the smaller ones produce 4 or 8 zoospores. Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase. There is, as yet, no convincing direct evidence for sexual reproduction.

  19. Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem.

    Science.gov (United States)

    Street, Ian H; Aman, Sitwat; Zubo, Yan; Ramzan, Aleena; Wang, Xiaomin; Shakeel, Samina N; Kieber, Joseph J; Schaller, G Eric

    2015-09-01

    The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell proliferation and cell elongation. Multiple phytohormones interact to control root growth, including ethylene, which is primarily known for its role in controlling root cell elongation. We find that ethylene also negatively regulates cell proliferation at the root meristem of Arabidopsis (Arabidopsis thaliana). Genetic analysis indicates that the inhibition of cell proliferation involves two pathways operating downstream of the ethylene receptors. The major pathway is the canonical ethylene signal transduction pathway that incorporates CONSTITUTIVE TRIPLE RESPONSE1, ETHYLENE INSENSITIVE2, and the ETHYLENE INSENSITIVE3 family of transcription factors. The secondary pathway is a phosphorelay based on genetic analysis of receptor histidine kinase activity and mutants involving the type B response regulators. Analysis of ethylene-dependent gene expression and genetic analysis supports SHORT HYPOCOTYL2, a repressor of auxin signaling, as one mediator of the ethylene response and furthermore, indicates that SHORT HYPOCOTYL2 is a point of convergence for both ethylene and cytokinin in negatively regulating cell proliferation. Additional analysis indicates that ethylene signaling contributes but is not required for cytokinin to inhibit activity of the root meristem. These results identify key elements, along with points of cross talk with cytokinin and auxin, by which ethylene negatively regulates cell proliferation at the root apical meristem. © 2015 American Society of Plant Biologists. All Rights Reserved.

  20. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi.

    Science.gov (United States)

    Parker, Aimee; Maclaren, Oliver J; Fletcher, Alexander G; Muraro, Daniele; Kreuzaler, Peter A; Byrne, Helen M; Maini, Philip K; Watson, Alastair J M; Pin, Carmen

    2017-02-01

    The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.-Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi. © The Author(s).

  1. Assessment of cell proliferation with resazurin-based fluorescent dye.

    Science.gov (United States)

    Czekanska, Ewa M

    2011-01-01

    The Alamar Blue assay is based on enzymatic reduction of indicator dye by viable cells and serves as an effective tool for assessing cell proliferation and as a screening technique. It can be applied in studies concentrating on animal, plant, yeast, and bacteria cells. Among the various methods for cell viability and cytotoxicity, it utilises all features of ideal and reliable test; it is one-step, sensitive, safe, non-toxic for cells, and cost-effective.

  2. Cell proliferation regulated by CO/sub 2/

    Energy Technology Data Exchange (ETDEWEB)

    Laszlo, E.; Novak, B.

    1986-01-01

    Microbial and mammalian cells don't proliferate when the CO/sub 2/ is removed from culture liquid. This means that CO/sub 2/ fixation is required for cell proliferation. CO/sub 2/ fixation is needed for the tricarboxylic acid cycle (TCAC), for purine and pyrimidine synthesis, and for lipid synthesis. Adding the purine and pyrimidine precursors and fatty acids into cell cultures, they examined the role of CO/sub 2/ in TCAC. When the CO/sub 2/ concentration increased inside the cell, increases were observed . By oversaturation of the respiration chain the alternative oxydases produce hydroxyl radicals, which cause DNA damage. The role of CO/sub 2/ in normal and abnormal cell proliferation is discussed.

  3. Cholesterol induces proliferation of chicken primordial germ cells.

    Science.gov (United States)

    Chen, Dongyang; Chen, Meijuan; Lu, Zhenping; Yang, Mengmeng; Xie, Long; Zhang, Wenxin; Xu, Huiyan; Lu, Kehuan; Lu, Yangqing

    2016-08-01

    Primordial germ cells (PGCs) are the precursors of sperm and eggs and may serve as suitable cells for use in research in developmental biology and transgenic animals. However, the long-term propagation of PGCs in vitro has so far been plagued by the loss of their germ cell characteristics. This is largely because of the scarcity of knowledge concerning cell division and proliferation in these cells and the poor optimization of the culture medium. The sonic hedgehog (SHH) signaling pathway is involved in proliferation of many types of cells, but little is known about its role in chicken PGCs. The results of the current study indicate that the proliferation of chicken PGCs increases significantly when cholesterol, a molecule that facilitates the trafficking of HH ligands, is supplemented in the culture medium. This effect was attenuated when an SHH antagonist, cyclopamine was added, suggesting the involvement of SHH signaling in this process. The characterization of PGCs treated with cholesterol has shown that these cells express germ-cell-related markers and retain their capability to colonize the embryonic gonad after re-introduction to vasculature of stage-15 HH embryos, indicating that proliferation of PGCs induced by cholesterol does not alter the germ cell characteristics of these cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres

    Directory of Open Access Journals (Sweden)

    Rebecca A. Dagg

    2017-06-01

    Full Text Available Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM, either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.

  5. Small molecule mediated proliferation of primary retinal pigment epithelial cells.

    Science.gov (United States)

    Swoboda, Jonathan G; Elliott, Jimmy; Deshmukh, Vishal; de Lichtervelde, Lorenzo; Shen, Weijun; Tremblay, Matthew S; Peters, Eric C; Cho, Charles Y; Lu, Bin; Girman, Sergej; Wang, Shaomei; Schultz, Peter G

    2013-07-19

    Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

  6. 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.

  7. Dietary bovine lactoferrin increases intestinal cell proliferation in neonatal piglets.

    Science.gov (United States)

    Reznikov, Elizabeth A; Comstock, Sarah S; Yi, Cuiyi; Contractor, Nikhat; Donovan, Sharon M

    2014-09-01

    Lactoferrin is a bioactive milk protein that stimulates cell proliferation in vitro; however, limited in vivo evidence exists to allow lactoferrin to be incorporated into infant formula. Herein, the effect of dietary bovine lactoferrin (bLF) on neonatal intestinal growth and maturation was investigated guided by the hypothesis that bLF would increase cellular proliferation leading to functional differences in neonatal piglets. Colostrum-deprived piglets were fed formula containing 0.4 [control (Ctrl)], 1.0 (LF1), or 3.6 (LF3) g bLF/L for the first 7 or 14 d of life. To provide passive immunity, sow serum was provided orally during the first 36 h of life. Intestinal cell proliferation, histomorphology, mucosal DNA concentration, enzyme activity, gene expression, and fecal bLF content were measured. Intestinal enzyme activity, DNA concentration, and villus length were unaffected by bLF. However, crypt proliferation was 60% greater in LF1- and LF3-fed piglets than in Ctrl piglets, and crypt depth and area were 20% greater in LF3-fed piglets than in Ctrl piglets. Crypt cells from LF3-fed piglets had 3-fold higher β-catenin mRNA expression than did crypt cells from Ctrl piglets. Last, feces of piglets fed bLF contained intact bLF, suggesting that some bLF was resistant to digestion and could potentially affect intestinal proliferation through direct interaction with intestinal epithelial cells. This study is the first to our knowledge to show that dietary bLF stimulates crypt cell proliferation in vivo. The increased β-catenin expression indicates that Wnt signaling may in part mediate the stimulatory effect of bLF on intestinal cell proliferation. © 2014 American Society for Nutrition.

  8. Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells.

    Science.gov (United States)

    Meng, Meng; Chen, Shuyang; Lao, Taotao; Liang, Dongming; Sang, Nianli

    2010-10-01

    Glutaminolysis and Warburg effect are the two most noticeable metabolic features of tumor cells whereas their biological significance in cell proliferation remains elusive. A widely accepted current hypothesis is that tumor cells use glutamine as a preferred carbon source for energy and reducing power, which has been used to explain both glutaminolysis and the Warburg effect. Here we provide evidence to show that supplying nitrogen, not the carbon skeleton, underlies the major biological importance of glutaminolysis for proliferating cells. We show alternative nitrogen supplying mechanisms rescue cell proliferation in glutamine-free media. Particularly, we show that ammonia is sufficient to maintain a long-term survival and proliferation of Hep3B in glutamine-free media. We also observed that nitrogen source restriction repressed carbon metabolic pathways including glucose utilization. Based on these new observations and metabolic pathways well established in published literature, we propose an alternative model that cellular demand for glutamate as a key molecule in nitrogen anabolism is the driving force of glutaminolysis in proliferating cells. Our model suggests that the Warburg effect may be a metabolic consequence secondary to the nitrogen anabolism.

  9. 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

  10. A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

    Science.gov (United States)

    Fenteany, G; Standaert, R F; Reichard, G A; Corey, E J; Schreiber, S L

    1994-04-12

    Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacystin altered only in the N-acetylcysteine moiety are active, while structural or stereochemical modifications of the gamma-lactam ring or the hydroxyisobutyl group lead to partial or complete loss of activity. The inactive compounds do not antagonize the effects of lactacystin in either neurite outgrowth or cell cycle progression assays. The response to lactacystin involves induction of a predominantly bipolar morphology that is maximal 16-32 h after treatment and is distinct from the response to several other treatments that result in morphological differentiation. Neurite outgrowth in response to lactacystin appears to be dependent upon microtubule assembly, actin polymerization, and de novo protein synthesis. The observed structure-activity relationships suggest that lactacystin and its related beta-lactone may act via acylation of one or more relevant target molecule(s) in the cell.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Improved selectivity of mIBG uptake into neuroblastoma cells in vitro and in vivo by inhibition of organic cation transporter 3 uptake using clinically approved corticosteroids.

    Science.gov (United States)

    Bayer, Melanie; Schmitt, Julia; Dittmann, Helmut; Handgretinger, Rupert; Bruchelt, Gernot; Sauter, Alexander W

    2016-09-01

    Radiolabeled meta-iodobenzylguanidine (mIBG) is used for imaging and therapy of neuroblastoma as well as pheochromocytoma. However, non-tumorous tissues also incorporate mIBG mainly by organic cation transporters (OCTs). In this study, we tested different clinically approved corticosteroids as potential inhibitors of the OCT3-mediated uptake in vitro and in vivo, to achieve a more selective mIBG tumor uptake. The in vitro incorporation of [(3)H]norepinephrine ([(3)H]NE), [(3)H]dopamine ([(3)H]DA) and [(123)I]mIBG in neuroblastoma cells (SK-N-SH, Kelly, IMR-32) and in HEK-293 cells transfected with human OCT3 was measured with and without supplemental corticosteroids (hydrocortisone, prednisolone, dexamethasone, corticosterone). The in vivo biodistribution of [(123)I]mIBG in absence and presence of corticosteroids was studied in non-tumor bearing NOD scid gamma mice. Retrospectively, we selected patients with and without corticosteroid treatment prior to [(123)I]mIBG scintigraphy. A concentration-dependent inhibitory effect of different corticosteroids on the [(3)H]NE and [(3)H]DA uptake via OCT3 was illustrated in vitro. The highest OCT3 inhibition was observed for corticosterone, but clinically used corticosteroids, showed also promising inhibitory effects. In contrast, the uptake in neuroblastoma cells was reduced only moderately. Hydrocortisone or prednisolone had only minor effects on [(123)I]mIBG uptake of both neuroblastoma cells, but reduced uptake in OCT3 expressing cells significantly. In mice tissues, [(123)I]mIBG uptake was inhibited by corticosteroids especially in the small intestine and kidney. Finally, in one patient with hydrocortisone treatment performed prior to [(123)I]mIBG scan, heart and liver uptake was reduced compared to untreated patients. The OCT3 is widely spread in many organs and responsible for non-targeted uptake of radiolabeled mIBG. In our study, clinically approved corticosteroids inhibited mIBG uptake in OCT3 expressing cells

  13. Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells

    Science.gov (United States)

    Park, Jeongyeon; Kwon, Jong Hwa; Kim, Nam

    2018-01-01

    Abstract Alzheimer’s disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cognitive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aβ), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aβ plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aβ processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aβ processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aβ peptide, was not significantly different between RF-EMF–exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observations suggest that RF-EMF exposure may not have a significant physiological effect on Aβ processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aβ processing with long-term and continuous exposure. PMID:29040655

  14. Monovalent ions control proliferation of Ehrlich Lettre ascites cells

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Preisler, Sarah; Pedersen, Stine Helene Falsig

    2010-01-01

    of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase...... effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine...

  15. High-dose chemotherapy and autologous haematopoietic stem cell rescue for children with high-risk neuroblastoma.

    Science.gov (United States)

    Yalçin, Bilgehan; Kremer, Leontien C M; van Dalen, Elvira C

    2015-10-05

    Despite the development of new treatment options, the prognosis of high-risk neuroblastoma patients is still poor; more than half of patients experience disease recurrence. High-dose chemotherapy and haematopoietic stem cell rescue (i.e. myeloablative therapy) might improve survival. This review is the second update of a previously published Cochrane review. Primary objectiveTo compare the efficacy, that is event-free and overall survival, of high-dose chemotherapy and autologous bone marrow or stem cell rescue with conventional therapy in children with high-risk neuroblastoma. Secondary objectivesTo determine adverse effects (e.g. veno-occlusive disease of the liver) and late effects (e.g. endocrine disorders or secondary malignancies) related to the procedure and possible effects of these procedures on quality of life. We searched the electronic databases The Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2014, issue 11), MEDLINE/PubMed (1966 to December 2014) and EMBASE/Ovid (1980 to December 2014). In addition, we searched reference lists of relevant articles and the conference proceedings of the International Society for Paediatric Oncology (SIOP) (from 2002 to 2014), American Society for Pediatric Hematology and Oncology (ASPHO) (from 2002 to 2014), Advances in Neuroblastoma Research (ANR) (from 2002 to 2014) and American Society for Clinical Oncology (ASCO) (from 2008 to 2014). We searched for ongoing trials by scanning the ISRCTN register (www.isrct.com) and the National Institute of Health Register (www.clinicaltrials.gov). Both registers were screened in April 2015. Randomised controlled trials (RCTs) comparing the efficacy of myeloablative therapy with conventional therapy in high-risk neuroblastoma patients. Two authors independently performed study selection, data extraction and risk of bias assessment. If appropriate, we pooled studies. The risk ratio (RR) and 95% confidence interval (CI) was calculated for dichotomous

  16. Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

    OpenAIRE

    Phillips Jonathan E; Bakthavatsalam Deenadayalan; Choe Jonathan M; Gomer Richard H

    2009-01-01

    Abstract Background Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the...

  17. Beta cell proliferation and growth factors

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis; Svensson, C; Møldrup, Annette

    1999-01-01

    Formation of new beta cells can take place by two pathways: replication of already differentiated beta cells or neogenesis from putative islet stem cells. Under physiological conditions both processes are most pronounced during the fetal and neonatal development of the pancreas. In adulthood little...... increase in the beta cell number seems to occur. In pregnancy, however, a marked hyperplasia of the beta cells is observed both in rodents and man. Increased mitotic activity has been seen both in vivo and in vitro in islets exposed to placental lactogen (PL), prolactin (PRL) and growth hormone (GH...... cloned a novel GH/PRL stimulated rat islet gene product, Pref-1 (preadipocyte factor-1). This protein contains six EGF-like motifs and may play a role both in embryonic pancreas differentiation and in beta cell growth and function. In summary, the increasing knowledge about the mechanisms involved...

  18. Nature of tumor control by permanently and transiently modified GD2 chimeric antigen receptor T cells in xenograft models of neuroblastoma.

    Science.gov (United States)

    Singh, Nathan; Liu, Xiaojun; Hulitt, Jessica; Jiang, Shuguang; June, Carl H; Grupp, Stephan A; Barrett, David M; Zhao, Yangbing

    2014-11-01

    Chimeric antigen receptor (CAR) therapy has begun to demonstrate success as a novel treatment modality for hematologic malignancies. The success observed thus far has been with T cells permanently engineered to express chimeric receptors. T cells engineered using RNA electroporation represent an alternative with the potential for similar efficacy and greater safety when initially targeting novel antigens. Neuroblastoma is a common pediatric solid tumor with the potential to be targeted using immunotherapy. We performed xenograft studies in NSG mice in which we assessed the efficacy of both permanently modified and transiently modified CAR T cells directed against the neuroblastoma antigen GD2 in both local and disseminated disease models. Disease response was monitored by tumor volume measurement and histologic examination, as well as in vivo bioluminescence. RNA-modified GD2 CAR T cells mediated rapid tumor destruction when delivered locally. A single infusion of lentivirally modified GD2 CAR T cells resulted in long-term control of disseminated disease. Multiple infusions of RNA GD2 CAR T cells slowed the progression of disseminated disease and improved survival, but did not result in long-term disease control. Histologic examination revealed that the transiently modified cells were unable to significantly penetrate the tumor environment when delivered systemically, despite multiple infusions of CAR T cells. Thus, we demonstrate that RNA-modified GD2 CAR T cells can mediate effective antitumor responses in vivo, and permanently modified cells are able to control disseminated neuroblastoma in xenograft mice. Lack of long-term disease control by RNA-engineered cells resulted from an inability to penetrate the tumor microenvironment. ©2014 American Association for Cancer Research.

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

    Science.gov (United States)

    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.

  20. Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells.

    Science.gov (United States)

    Lovinger, D M; Sung, K W; Zhou, Q

    2000-02-14

    Alcohol potentiation of 5-HT3 receptors was examined in NCB-20 neuroblastoma cells using whole-cell patch-clamp electrophysiological techniques. Activation of the receptor with the weak partial agonist dopamine (DA) was used to examine alcohol effects under conditions of full agonist occupancy, but low probability of channel opening. Dopamine activation of the receptor increased in a concentration-dependent manner (EC50=0.28 mM), and on average maximal responses to DA were 8.0+/-0.8% of the maximal response to 5-HT. Ethanol (EtOH) and trichloroethanol (TCEt) potentiated DA-activated ion current mediated by 5-HT3 receptors. Potentiation of responses to a maximally effective dopamine concentration averaged 52.0+/-8.0% for EtOH and 567+/-43% for TCEt, which was comparable to the potentiation observed when receptors were activated by a low concentration of 5-HT. The alcohols increased both the potency and efficacy with which dopamine activated the receptor. The observation that alcohols increase the maximal efficacy of dopamine activation of the receptor indicates that one action of alcohols on the 5-HT3 receptor is to increase the probability of channel opening independent of any effect on agonist affinity.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Differential effects of delta-9-tetrahydrocannabinol and its 11-hydroxy metabolite on sodium current in neuroblastoma cells.

    Science.gov (United States)

    Turkanis, S A; Karler, R; Partlow, L M

    1991-09-27

    Whole-cell voltage-clamp techniques were used to study the comparative effects of delta-9-tetrahydrocannabinol (THC) and its principal metabolite, 11-hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC), on the voltage-gated sodium current in neuroblastoma cells. The parent compound markedly depressed the inward sodium current with minimal reduction of the outward current, demonstrating that the effects of the drug were related to the membrane potential. In addition, THC reduced the reversal potential, indicating that the drug modified the ion selectivity of the channel. 11-OH-THC similarly depressed inward sodium current; however, in marked contrast to the effects of the parent compound, the drug equally depressed the outward voltage-gated sodium current, indicating that its effects were not related to the membrane potential. Furthermore, 11-OH-THC differed from THC in that it did not alter the reversal potential. The results demonstrate that THC and its 11-OH metabolite both reduce inward sodium current, but their effects on the outward current and ion selectivity are distinctly different. The sum of the actions of these two cannabinoids on the voltage-gated sodium channel provides a plausible cellular basis for THC's depression of action potentials in vivo and for some of its central depressant effects.

  3. Cell proliferation in cubozoan jellyfish Tripedalia cystophora and Alatina moseri.

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    Daniela Gurska

    Full Text Available Cubozoans (box jellyfish undergo remarkable body reorganization throughout their life cycle when, first, they metamorphose from swimming larvae to sessile polyps, and second, through the metamorphosis from sessile polyps to free swimming medusae. In the latter they develop complex structures like the central nervous system (CNS and visual organs. In the present study several aspects of cell proliferation at different stages of the life cycle of the box jellyfish Tripedalia cystophora and Alatina moseri have been examined through in vivo labeling of cells in the synthetic phase (S phase of the cell cycle. Proliferation zones were found in metamorphosing polyps, as well as in juvenile medusae, where both the rhopalia and pedalia have enhanced rates of proliferation. The results also indicate a rather fast cell turnover in the rhopalia including the rhopalial nervous system (RNS. Moreover, T. cystophora showed diurnal pattern of cell proliferation in certain body parts of the medusa, with higher proliferation rates at nighttime. This is true for two areas in close connection with the CNS: the stalk base and the rhopalia.

  4. Cell proliferation in cubozoan jellyfish Tripedalia cystophora and Alatina moseri.

    Science.gov (United States)

    Gurska, Daniela; Garm, Anders

    2014-01-01

    Cubozoans (box jellyfish) undergo remarkable body reorganization throughout their life cycle when, first, they metamorphose from swimming larvae to sessile polyps, and second, through the metamorphosis from sessile polyps to free swimming medusae. In the latter they develop complex structures like the central nervous system (CNS) and visual organs. In the present study several aspects of cell proliferation at different stages of the life cycle of the box jellyfish Tripedalia cystophora and Alatina moseri have been examined through in vivo labeling of cells in the synthetic phase (S phase) of the cell cycle. Proliferation zones were found in metamorphosing polyps, as well as in juvenile medusae, where both the rhopalia and pedalia have enhanced rates of proliferation. The results also indicate a rather fast cell turnover in the rhopalia including the rhopalial nervous system (RNS). Moreover, T. cystophora showed diurnal pattern of cell proliferation in certain body parts of the medusa, with higher proliferation rates at nighttime. This is true for two areas in close connection with the CNS: the stalk base and the rhopalia.

  5. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Original Research Article. Chloroquinone Inhibits Cell Proliferation and Induces. Apoptosis in Nasopharyngeal Carcinoma Cell Lines. Xin-Qing Yang, Hao Zheng, Qing Ye, Rui-Yu Li* and Yong Chen. Department of Otolaryngology Head and Neck Surgery, Fujian Provincial Clinical College, Fujian Medical University, China.

  6. Stimulation of cell proliferation by calcium and a calcimimetic compound

    NARCIS (Netherlands)

    Mailland, M; Waelchli, R; Ruat, M; Boddeke, HGWM; Seuwen, K

    Some mesenchymal cells respond to stimulation by specific cations with increased cell proliferation. In the present study we have investigated whether the parathyroid/kidney/brain calcium-sensing receptor (PCaR) can mediate such mitogenic responses. We have expressed the recombinant rat PCaR in

  7. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Purpose: To demonstrate the role of chloroquinone (CQ) in inducing apoptosis in HONE-1 and HNE-1, nasopharyngeal carcinoma (NPC) cell lines. Methods: Water-soluble tetrazolium salt (WST)-1 assay was used for the determination of cell proliferation while an inverted microscope was employed for the analysis of ...

  8. 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.

  9. 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.

  10. Cell Proliferation and Oncogenesis | Ally | South African ...

    African Journals Online (AJOL)

    The Progression of a normal cell to become a cancer cell is a Stepwise process involving multiple genetic and phenotypic alterations. Multiple obstacles must be overcome, which is thought to require several gene mutations. South African Gastroenterology Journal Vol. 6 (3) 2008: pp. 15-20 ...

  11. Emodin downregulates cell proliferation markers during DMBA ...

    African Journals Online (AJOL)

    Background: Cell-cycle disruption is the major characteristic features of neoplastic transformation and the status of cell-cycle regulators can thus be utilized to assess the prognostic significance in patients with cancer. The PCNA, cyclin D1, CDK4, CDK6 and survivin expression in the buccal mucosa was utilized to evaluate ...

  12. Controling stem cell proliferation - CKIs at work

    NARCIS (Netherlands)

    Bruggeman, SWM; van Lohuizen, M

    2006-01-01

    The cyclin-dependent kinase inhibitors or CKIs are well recognized as intrinsic regulators of the cell cycle. Here, we discuss recent data implicating their activity in restraining adult stem cell self-renewal, and the role that proteins regulating CKI expression play in this process.

  13. The regulation of angiogenesis in neuroblastoma.

    Science.gov (United States)

    Chlenski, Alexandre; Liu, Shuqing; Cohn, Susan L

    2003-07-18

    Angiogenesis is required for the growth and metastasis of malignant tumors, and high vascular density has been correlated with aggressive tumor growth in many types of cancer. This process is regulated by the local balance of stimulatory and inhibitory molecules produced by tumor cells, stromal cells, and the organ-specific environment. In neuroblastoma, a pediatric malignancy that is characterized by a broad spectrum of clinical behavior, angiogenesis also appears to play an important role in determining tumor phenotype. The nature of the angiogenic balance in neuroblastoma is complex, and a spectrum of angiogenesis stimulators and inhibitors has been detected in neuroblastoma tumors. This review summarizes our current understanding of the regulation of angiogenesis in neuroblastoma.

  14. Reciprocal control of cell proliferation and migration

    Directory of Open Access Journals (Sweden)

    De Donatis Alina

    2010-09-01

    Full Text Available Abstract In adult tissue the quiescent state of a single cell is maintained by the steady state conditions of its own microenvironment for what concern both cell-cell as well as cell-ECM interaction and soluble factors concentration. Physiological or pathological conditions can alter this quiescent state through an imbalance of both soluble and insoluble factors that can trigger a cellular phenotypic response. The kind of cellular response depends by many factors but one of the most important is the concentration of soluble cytokines sensed by the target cell. In addition, due to the intrinsic plasticity of many cellular types, every single cell is able, in response to the same stimulus, to rapidly switch phenotype supporting minimal changes of microenviromental cytokines concentration. Wound healing is a typical condition in which epithelial, endothelial as well as mesenchymal cells are firstly subjected to activation of their motility in order to repopulate the damaged region and then they show a strong proliferative response in order to successfully complete the wound repair process. This schema constitute the leitmotif of many other physiological or pathological conditions such as development vasculogenesis/angiogenesis as well as cancer outgrowth and metastasis. Our review focuses on the molecular mechanisms that control the starting and, eventually, the switching of cellular phenotypic outcome in response to changes in the symmetry of the extracellular environment.

  15. Arachidonoyl-Phospholipid Remodeling in Proliferating Murine T Cells

    Directory of Open Access Journals (Sweden)

    Ando Soichiro

    2004-01-01

    Full Text Available Abstract Backgound Previous studies have shown that the functional capacity of T cells may be modulated by the composition of fatty acids within, and the release of fatty acids from membrane phospholipids, particulary containing arachidonic acid (AA. The remodeling of AA within membrane phospholipids of resting and proliferating CD4+ and CD8+ T cells is examined in this study. Results Splenic T cells were cultured in the presence or absence of anti-CD3 mAb for 48 h then labeled with [3H]AA for 20 min. In unstimulated cells, labeled AA was preferentially incorporated into the phosphoglycerides, phosphatidylcholine (PC followed by phosphatidylinositol (PI and phosphatidylethanolamine (PE. During a subsequent chase in unlabeled medium unstimulated CD4+ and CD8+ T cells demonstrated a significant and highly selective transfer of free, labeled AA into the PC pool. In contrast, proliferating CD4+ and CD8+ T cells distributed labeled [3H]AA predominantly into PI followed by PC and PE. Following a chase in AA-free medium, a decline in the content of [3H]AA-PC was observed in association with a comparable increase in [3H]AA-PE. Subsequent studies revealed that the cold AA content of all PE species was increased in proliferating T cells compared with that in non-cycling cells, but that enrichment in AA was observed only in the ether lipid fractions. Finally, proliferating T cells preincubated with [3H]AA exhibited a significant loss of labeled arachidonate in the PC fraction and an equivalent gain in labeled AA in 1-alk-1'-enyl-2-arachidonoyl-PE during a chase in unlabeled medium. Conclusion This apparent unidirectional transfer of AA from PC to ether-containing PE suggests the existence of a CoA-independent transacylase system in T cells and supports the hypothesis that arachidonoyl phospholipid remodeling may play a role in the regulation of cellular proliferation.

  16. Electrospun fiber membranes enable proliferation of genetically modified cells.

    Science.gov (United States)

    Borjigin, Mandula; Eskridge, Chris; Niamat, Rohina; Strouse, Bryan; Bialk, Pawel; Kmiec, Eric B

    2013-01-01

    Polycaprolactone (PCL) and its blended composites (chitosan, gelatin, and lecithin) are well-established biomaterials that can enrich cell growth and enable tissue engineering. However, their application in the recovery and proliferation of genetically modified cells has not been studied. In the study reported here, we fabricated PCL-biomaterial blended fiber membranes, characterized them using physicochemical techniques, and used them as templates for the growth of genetically modified HCT116-19 colon cancer cells. Our data show that the blended polymers are highly miscible and form homogenous electrospun fiber membranes of uniform texture. The aligned PCL nanofibers support robust cell growth, yielding a 2.5-fold higher proliferation rate than cells plated on standard plastic plate surfaces. PCL-lecithin fiber membranes yielded a 2.7-fold higher rate of proliferation, while PCL-chitosan supported a more modest growth rate (1.5-fold higher). Surprisingly, PCL-gelatin did not enhance cell proliferation when compared to the rate of cell growth on plastic surfaces.

  17. Electrospun fiber membranes enable proliferation of genetically modified cells

    Science.gov (United States)

    Borjigin, Mandula; Eskridge, Chris; Niamat, Rohina; Strouse, Bryan; Bialk, Pawel; Kmiec, Eric B

    2013-01-01

    Polycaprolactone (PCL) and its blended composites (chitosan, gelatin, and lecithin) are well-established biomaterials that can enrich cell growth and enable tissue engineering. However, their application in the recovery and proliferation of genetically modified cells has not been studied. In the study reported here, we fabricated PCL-biomaterial blended fiber membranes, characterized them using physicochemical techniques, and used them as templates for the growth of genetically modified HCT116-19 colon cancer cells. Our data show that the blended polymers are highly miscible and form homogenous electrospun fiber membranes of uniform texture. The aligned PCL nanofibers support robust cell growth, yielding a 2.5-fold higher proliferation rate than cells plated on standard plastic plate surfaces. PCL-lecithin fiber membranes yielded a 2.7-fold higher rate of proliferation, while PCL-chitosan supported a more modest growth rate (1.5-fold higher). Surprisingly, PCL-gelatin did not enhance cell proliferation when compared to the rate of cell growth on plastic surfaces. PMID:23467983

  18. MicroRNAs-10a and -10b contribute to retinoic acid-induced differentiation of neuroblastoma cells and target the alternative splicing regulatory factor SFRS1 (SF2/ASF).

    Science.gov (United States)

    Meseguer, Salvador; Mudduluru, Giridhar; Escamilla, Juan Manuel; Allgayer, Heike; Barettino, Domingo

    2011-02-11

    MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.

  19. Actual proliferating index in oral squamous cell carcinoma and leukoplakia.

    Science.gov (United States)

    Chandak, Abhay R; Gadbail, Amol Ramchandra; Chaudhary, Minal S; Chandak, Shweta A; Wadhwani, Ritesh

    2011-08-01

      To examine the possible association between epithelial proliferation and disease progression in the oral mucosa using the actual proliferation index.   The actual proliferation index was measured by the Ki-67 labeling index and argyrophilic nucleolar organizer region count per nucleus. Immunohistochemistry was carried out for Ki-67 by using the molecular immunology borstel-1 clone in 20 leukoplakias, 20 oral squamous cell carcinomas, and 10 normal oral mucosae.   The argyrophilic nucleolar organizer region count per nucleus, Ki-67 labeling index, and actual proliferation index were significantly higher in oral squamous cell carcinoma, followed by leukoplakia and normal oral mucosa. Leukoplakia with dysplasia showed a significantly higher Ki-67 labeling index and actual proliferation index, compared to leukoplakia without dysphasia. There was a significant correlation of Bryne's histological malignancy grading with the argyrophilic nucleolar organizer region count and the Ki-67 labeling index. There was a significant positive correlation between the argyrophilic nucleolar organizer region count and the Ki-67 labeling index among all groups.   Leukoplakia or suspected epithelial dysplasia should be stained for argyrophilic nucleolar organizer regions and Ki-67. The actual proliferation index is not only useful as a prognostic factor, but could also be a promising treatment determining modality for patients with premalignant and malignant lesions. © 2011 Blackwell Publishing Asia Pty Ltd.

  20. Cerebellar neuroblastoma in an infant.

    Science.gov (United States)

    Nishio, S; Inamura, T; Morioka, T; Ishihara, S; Hirano, K; Murakami, N; Fukui, M

    2000-03-01

    A cerebellar neoplasm in an 8-month-old boy is reported. While this tumour was composed of small cells and had regions resembling desmoplastic medulloblastoma, it showed ultrastructural neuronal characteristics including bundles of microtubules in the cell processes, numerous synaptic vesicles, and occasional abortive or complete synapses. These characteristic features warranted the diagnosis of a neuroblastoma of the cerebellum. The nature of this rare intraparenchymal tumour in infants is also briefly discussed.

  1. Chemotherapy-Induced Apoptosis in a Transgenic Model of Neuroblastoma Proceeds Through p53 Induction

    Directory of Open Access Journals (Sweden)

    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.

  2. Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

    Science.gov (United States)

    Clarke, Scott T; Calderon, Veronica; Bradford, Jolene A

    2017-10-02

    The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  3. Development of bioengineering system for stem cell proliferation

    Science.gov (United States)

    Park, H. S.; Shah, R.; Shah, C.

    2016-08-01

    From last decades, intensive research in the field of stem cells proliferation had been promoted due to the unique property of stem cells to self-renew themselves into multiples and has potential to replicate into an organ or tissues and so it's highly demanding though challenging. Bioreactor, a mechanical device, works as a womb for stem cell proliferation by providing nutritious environment for the proper growth of stem cells. Various factors affecting stem cells growth are the bioreactor mechanism, feeding of continuous nutrients, healthy environment, etc., but it always remains a challenge for controlling biological parameters. The present paper unveils the design of mechanical device commonly known as bioreactor in tissues engineering and biotech field, use for proliferation of stem cells and imparts the proper growing condition for stem cells. This high functional bioreactor provides automation mixing of cell culture and stem cells. This design operates in conjunction with mechanism of reciprocating motion. Compare to commercial bioreactors, this proposed design is more convenient, easy to operate and less maintenance is required as bioreactor culture bag is made of polyethylene which is single use purpose. Development of this bioengineering system will be beneficial for better growth and expansion of stem cell

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells.

    Science.gov (United States)

    Zameer, Andleeb; Kasturirangan, Srinath; Emadi, Sharareh; Nimmagadda, Sridevi V; Sierks, Michael R

    2008-12-26

    The Amyloid-beta (Abeta) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Abeta are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Abeta using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Abeta, we identified an scFv that bound oligomeric Abeta specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Abeta aggregation and toxicity, and reduces the toxicity of preformed oligomeric Abeta towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Abeta scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Abeta in AD brains.

  6. Prostate progenitor cells proliferate in response to castration

    Directory of Open Access Journals (Sweden)

    Xudong Shi

    2014-07-01

    Full Text Available Androgen-deprivation is a mainstay of therapy for advanced prostate cancer but tumor regression is usually incomplete and temporary because of androgen-independent cells in the tumor. It has been speculated that these tumor cells resemble the stem/progenitor cells of the normal prostate. The purpose of this study was to examine the response of slow-cycling progenitor cells in the adult mouse prostate to castration. Proliferating cells in the E16 urogenital sinus were pulse labeled by BrdU administration or by doxycycline-controlled labeling of the histone-H2B GFP mouse. A small population of labeled epithelial cells in the adult prostate localized at the junction of the prostatic ducts and urethra. Fluorescence-activated cell sorting (FACS showed that GFP label-retaining cells were enriched for cells co-expressing stem cell markers Sca-1, CD133, CD44 and CD117 (4- marker cells; 60-fold enrichment. FACS showed, additionally, that 4-marker cells were androgen receptor positive. Castration induced proliferation and dispersal of E16 labeled cells into more distal ductal segments. When naïve adult mice were administered BrdU daily for 2 weeks after castration, 16% of 4-marker cells exhibited BrdU label in contrast to only 6% of all epithelial cells (P < 0.01. In sham-castrated controls less than 4% of 4-marker cells were BrdU labeled (P < 0.01. The unexpected and admittedly counter-intuitive finding that castration induced progenitor cell proliferation suggests that androgen deprivation therapy in men with advanced prostate cancer could not only exert pleiotrophic effects on tumor sub-populations but may induce inadvertent expansion of tumor stem cells.

  7. Transient processes in cell proliferation kinetics

    CERN Document Server

    Yakovlev, Andrej Yu

    1989-01-01

    A mathematician who has taken the romantic decision to devote himself to biology will doubtlessly look upon cell kinetics as the most simple and natural field of application for his knowledge and skills. Indeed, the thesaurus he is to master is not so complicated as, say, in molecular biology, the structural elements of the system, i. e. ceils, have been segregated by Nature itself, simple considerations of balance may be used for deducing basic equations, and numerous analogies in other areas of science also superficial add to one"s confidence. Generally speaking, this number of impression is correct, as evidenced by the very great theoretical studies on population kinetics, unmatched in other branches of mathematical biology. This, however, does not mean that mathematical theory of cell systems has traversed in its development a pathway free of difficulties or errors. The seeming ease of formalizing the phenomena of cell kinetics not infrequently led to the appearance of mathematical models lacking in adequ...

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Aerobic Glycolysis: Meeting the Metabolic Requirements of Cell Proliferation

    OpenAIRE

    Vander Heiden, Matthew G.; Lunt, Sophia Yunkyungkwon

    2011-01-01

    Warburg's observation that cancer cells exhibit a high rate of glycolysis even in the presence of oxygen (aerobic glycolysis) sparked debate over the role of glycolysis in normal and cancer cells. Although it has been established that defects in mitochondrial respiration are not the cause of cancer or aerobic glycolysis, the advantages of enhanced glycolysis in cancer remain controversial. Many cells ranging from microbes to lymphocytes use aerobic glycolysis during rapid proliferation, which...

  10. 7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

    African Journals Online (AJOL)

    formation of melanin in melanoma cells is induced via cAMP [18]. The activation and translocation of protein kinase A, and, consequently, CREB activation and gene transcription, are due to cAMP [19]. One of the genes expressed during this process is Mitf, which plays important roles in the regulation of melanocyte viability,.

  11. Glutathione, cell proliferation and differentiation | Ashtiani | African ...

    African Journals Online (AJOL)

    All organisms require an equivalent source for living. Reduced glutathione is the most abundant thiol containing protein in mammalian cells and organs. Glutathione was discovered by Hopkins in 1924 who published his findings in JBC. It is a three peptide containing glutamic acid, cystein and glycin and is found in reduced ...

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

    Directory of Open Access Journals (Sweden)

    Chao Wang

    2014-11-01

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

  13. 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.

  14. Amniotic Fluid Cells Proliferation in Normal and Down Syndrome Subjects

    Directory of Open Access Journals (Sweden)

    Honcea Adina

    2016-02-01

    Full Text Available Down Syndrome/Trisomy 21 is the most common chromosomal anomaly, and it represents the most common congenital cause of infants’ intellectual disability. Subjects with this syndrome are affected by degenerative processes caused by accelerated aging or unknown ethyologies. In recent years, accumulating evidence revealed increased potential of amniotic fluid-derived stem cells to be used in regenerative therapy. Our aim was to assess differences in immunophenotype, cell morphology and proliferation of amniotic fluid cells from normal and Down Syndrome pregnancies using a quantitative cytometry approach. Results revealed the emergence of a population of small sized cells in Down Syndrome derived amniotic fluid cells that are readily visible upon microscopic inspection. Hence, the fluorescence–based quantitative image cytometry determinations showed a tendency of decrease in both cell and nuclei size in trisomy, with no significant modification in nuclei circularity, as measured following actin cytoskeleton and nuclei labeling. The propensity of Ki67 positive cells was found to be increased in Down Syndrome derived cells (48.92% as compared to normal specimens (28.68%. However, cells in S and G2/M cell cycle phases decreased from 32.91% to 4.49% in diseased cells. Further studies are devoted to understanding the molecular basis of the observed differences in the proliferation ability of Down Syndrome amniotic cells, in order to evaluate the potential therapeutic effect of amniotic fluid stem cells for tissue regeneration in subjects with trisomy and to find correlations between amniotic cells phenotype and patient prognosis.

  15. Simvastatin Modulates Mesenchymal Stromal Cell Proliferation and Gene Expression

    Science.gov (United States)

    Zanette, Dalila Lucíola; Lorenzi, Julio Cesar Cetrulo; Panepucci, Rodrigo Alexandre; Palma, Patricia Vianna Bonini; dos Santos, Daiane Fernanda; Prata, Karen Lima; Silva, Wilson Araújo

    2015-01-01

    Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential. PMID:25874574

  16. 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.

  17. Paradoxical role of 3-methyladenine in pyocyanin-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    McFarland, Amelia J; Grant, Gary D; Perkins, Anthony V; Flegg, Cameron; Davey, Andrew K; Allsopp, Tristan J; Renshaw, Gillian; Kavanagh, Justin; McDermott, Catherine M; Anoopkumar-Dukie, Shailendra

    2013-01-01

    The role of autophagy in pyocyanin (PCN)-induced toxicity in the central nervous system (CNS) remains unclear, with only evidence from our group identifying it as a mechanism underlying toxicity in 1321N1 astrocytoma cells. Therefore, the aim of this study was to further examine the role of autophagy in PCN-induced toxicity in the CNS. To achieve this, we exposed 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells to PCN (0-100 μmol/L) and tested the contribution of autophagy by measuring the impact of the autophagy inhibitor 3-methyladenine (3-MA) using a series of biochemical and molecular markers. Pretreatment of 1321N1 astrocytoma cells with 3-MA (5 mmol/L) decreased the PCN-induced acidic vesicular organelle and autophagosome formation as measured using acridine orange and green fluorescent protein-LC3 -LC3 fluorescence, respectively. Furthermore, 3-MA (5 mmol/L) significantly protected 1321N1 astrocytoma cells against PCN-induced toxicity. In contrast pretreatment with 3-MA (5 mmol/L) increased PCN-induced toxicity in SH-SY5Y neuroblastoma cells. Given the influence of autophagy in inflammatory responses, we investigated whether the observed effects in this study involved inflammatory mediators. The PCN (100 μmol/L) significantly increased the production of interleukin-8 (IL-8), prostaglandin E2 (PGE₂), and leukotriene B4 (LTB₄) in both cell lines. Consistent with its paradoxical role in modulating PCN-induced toxicity, 3-MA (5 mmol/L) significantly reduced the PCN-induced production of IL-8, PGE₂, and LTB₄ in 1321N1 astrocytoma cells but augmented their production in SH-SY5Y neuroblastoma cells. In conclusion, we show here for the first time the paradoxical role of autophagy in mediating PCN-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells and provide novel evidence that these actions may be mediated by effects on IL-8, PGE₂, and LTB₄ production.

  18. Identification of Predictive Gene Markers for Multipotent Stromal Cell Proliferation.

    Science.gov (United States)

    Bellayr, Ian H; Marklein, Ross A; Lo Surdo, Jessica L; Bauer, Steven R; Puri, Raj K

    2016-06-01

    Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages, such as adipocytes, chondrocytes, and osteocytes. They can be isolated from numerous tissue sources, including bone marrow, adipose tissue, skeletal muscle, and others. Because of their differentiation potential and secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression. Cellular expansion is necessary to obtain sufficient numbers for use; however, MSCs exhibit a reduced capacity for proliferation and differentiation after several rounds of passaging. In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict proliferative quality. Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays. A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| >0.70) for eight MSC lines at multiple passages. These 24 identified genes were then confirmed using an additional set of MSCs from eight new donors using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The proliferative potential of the second set of MSCs was measured for each donor/passage for confluency fraction, fraction of EdU+ cells, and population doubling time. The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only seven of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, and SPC25) demonstrated significant correlation with MSC proliferation regardless of passage. Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the inclusion of non-MSC cell lines; therefore, this set of seven genes may be more strongly associated with MSC proliferative quality. Our results pave the way to determine the quality of an MSC population for a

  19. Molecular signatures of proliferation and quiescence in hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Teresa A Venezia

    2004-10-01

    Full Text Available Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.

  20. Radiolabeled metaiodobenzylguanidine for the treatment of neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    DuBois, Steven G. [Department of Pediatrics, UCSF School of Medicine, Box 0106, San Francisco, CA 94143-0106 (United States); Matthay, Katherine K. [Department of Pediatrics, UCSF School of Medicine, Box 0106, San Francisco, CA 94143-0106 (United States)], E-mail: matthayk@peds.ucsf.edu

    2008-08-15

    Introduction: Neuroblastoma is the most common pediatric extracranial solid cancer. This tumor is characterized by metaiodobenzylguanidine (MIBG) avidity in 90% of cases, prompting the use of radiolabeled MIBG for targeted radiotherapy in these tumors. Methods: The available English language literature was reviewed for original research investigating in vitro, in vivo and clinical applications of radiolabeled MIBG for neuroblastoma. Results: MIBG is actively transported into neuroblastoma cells by the norepinephrine transporter. Preclinical studies demonstrate substantial activity of radiolabeled MIBG in neuroblastoma models, with {sup 131}I-MIBG showing enhanced activity in larger tumors compared to {sup 125}I-MIBG. Clinical studies of {sup 131}I-MIBG in patients with relapsed or refractory neuroblastoma have identified myelosuppression as the main dose-limiting toxicity, necessitating stem cell reinfusion at higher doses. Most studies report a response rate of 30-40% with {sup 131}I-MIBG in this population. More recent studies have focused on the use of {sup 131}I-MIBG in combination with chemotherapy or myeloablative regimens. Conclusions: {sup 131}I-MIBG is an active agent for the treatment of patients with neuroblastoma. Future studies will need to define the optimal role of this targeted radiopharmaceutical in the therapy of this disease.

  1. Suppression of vascular smooth muscle cells' proliferation and ...

    African Journals Online (AJOL)

    This study aimed to determine the effects of valsartan on the proliferation and migration of isolated rat vascular smooth muscle cells (VSMCs) and the expression of phospho-p42/44 mitogen-activated protein kinase (MAPK) promoted by angiotensin II (Ang II). VSMCs from the rat thoracic aorta were cultured by ...

  2. Estimation of cell proliferation dynamics using CFSE data.

    Science.gov (United States)

    Banks, H T; Sutton, Karyn L; Thompson, W Clayton; Bocharov, Gennady; Roose, Dirk; Schenkel, Tim; Meyerhans, Andreas

    2011-01-01

    Advances in fluorescent labeling of cells as measured by flow cytometry have allowed for quantitative studies of proliferating populations of cells. The investigations (Luzyanina et al. in J. Math. Biol. 54:57-89, 2007; J. Math. Biol., 2009; Theor. Biol. Med. Model. 4:1-26, 2007) contain a mathematical model with fluorescence intensity as a structure variable to describe the evolution in time of proliferating cells labeled by carboxyfluorescein succinimidyl ester (CFSE). Here, this model and several extensions/modifications are discussed. Suggestions for improvements are presented and analyzed with respect to statistical significance for better agreement between model solutions and experimental data. These investigations suggest that the new decay/label loss and time dependent effective proliferation and death rates do indeed provide improved fits of the model to data. Statistical models for the observed variability/noise in the data are discussed with implications for uncertainty quantification. The resulting new cell dynamics model should prove useful in proliferation assay tracking and modeling, with numerous applications in the biomedical sciences.

  3. Epac inhibits migration and proliferation of human prostate carcinoma cells

    NARCIS (Netherlands)

    Grandoch, M.; Rose, A.; ter Braak, M.; Jendrossek, V.; Ruebben, H.; Fischer, J. W.; Schmidt, M.; Weber, A. A.

    2009-01-01

    BACKGROUND: It was recently found that cAMP mediates protein kinase A-independent effects through Epac proteins. The aim of this study was to investigate the role of Epac in migration and proliferation of prostate carcinoma cells. METHODS: The effect of Epac activation was determined by [(3)H

  4. Proliferation of Genetically Modified Human Cells on Electrospun Nanofiber Scaffolds

    Directory of Open Access Journals (Sweden)

    Mandula Borjigin

    2012-01-01

    Full Text Available Gene editing is a process by which single base mutations can be corrected, in the context of the chromosome, using single-stranded oligodeoxynucleotides (ssODNs. The survival and proliferation of the corrected cells bearing modified genes, however, are impeded by a phenomenon known as reduced proliferation phenotype (RPP; this is a barrier to practical implementation. To overcome the RPP problem, we utilized nanofiber scaffolds as templates on which modified cells were allowed to recover, grow, and expand after gene editing. Here, we present evidence that some HCT116-19, bearing an integrated, mutated enhanced green fluorescent protein (eGFP gene and corrected by gene editing, proliferate on polylysine or fibronectin-coated polycaprolactone (PCL nanofiber scaffolds. In contrast, no cells from the same reaction protocol plated on both regular dish surfaces and polylysine (or fibronectin-coated dish surfaces proliferate. Therefore, growing genetically modified (edited cells on electrospun nanofiber scaffolds promotes the reversal of the RPP and increases the potential of gene editing as an ex vivo gene therapy application.

  5. Estimation of Cell Proliferation Dynamics Using CFSE Data

    Science.gov (United States)

    Banks, H.T.; Sutton, Karyn L.; Thompson, W. Clayton; Bocharov, Gennady; Roose, Dirk; Schenkel, Tim; Meyerhans, Andreas

    2010-01-01

    Advances in fluorescent labeling of cells as measured by flow cytometry have allowed for quantitative studies of proliferating populations of cells. The investigations (Luzyanina et al. in J. Math. Biol. 54:57–89, 2007; J. Math. Biol., 2009; Theor. Biol. Med. Model. 4:1–26, 2007) contain a mathematical model with fluorescence intensity as a structure variable to describe the evolution in time of proliferating cells labeled by carboxyfluorescein succinimidyl ester (CFSE). Here, this model and several extensions/modifications are discussed. Suggestions for improvements are presented and analyzed with respect to statistical significance for better agreement between model solutions and experimental data. These investigations suggest that the new decay/label loss and time dependent effective proliferation and death rates do indeed provide improved fits of the model to data. Statistical models for the observed variability/noise in the data are discussed with implications for uncertainty quantification. The resulting new cell dynamics model should prove useful in proliferation assay tracking and modeling, with numerous applications in the biomedical sciences. PMID:20195910

  6. Neuroblastoma Treatment

    Science.gov (United States)

    ... Types Bladder Cancer Breast Cancer Colorectal Cancer Kidney (Renal Cell) Cancer Leukemia Liver Cancer Lung Cancer Lymphoma Pancreatic Cancer Prostate Cancer Skin Cancer Thyroid Cancer Uterine Cancer All ...

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

    Science.gov (United States)

    Wallace, Dorothy I; Dunham, Ann; Chen, Paula X; Chen, Michelle; Huynh, Milan; Rheingold, Evan; Prosper, Olivia

    2016-01-01

    Researchers have observed that response of tumor cells to treatment varies depending on whether the cells are grown in monolayer, as in vitro spheroids or in vivo. This study uses data from the literature on monolayer treatment of SK-N-SH neuroblastoma cells with 15-deoxy-PGJ2 and couples it with data on growth rates for untreated SK-N-SH neuroblastoma cells grown as multicellular spheroids. A linear model is constructed for untreated and treated monolayer data sets, which is tuned to growth, death, and cell cycle data for the monolayer case for both control and treatment with 15-deoxy-PGJ2. The monolayer model is extended to a five-dimensional nonlinear model of in vitro tumor spheroid growth and treatment that includes compartments of the cell cycle (G1, S, G2/M) as well as quiescent (Q) and necrotic (N) cells. Monolayer treatment data for 15-deoxy-PGJ2 is used to derive a prediction of spheroid response under similar treatments. For short periods of treatment, spheroid response is less pronounced than monolayer response. The simulations suggest that the difference in response to treatment of monolayer versus spheroid cultures observed in laboratory studies is a natural consequence of tumor spheroid physiology rather than any special resistance to treatment.

  8. 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.

  9. Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

    Science.gov (United States)

    Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

    2017-01-01

    A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G1 phase arrest. These results suggest that PANDA promotes G1-S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. Effect of Ultrasonic Vibration on Proliferation and Differentiation of Cells

    Directory of Open Access Journals (Sweden)

    Haruka Hino

    2016-12-01

    Full Text Available The effect of mechanical stimulation of vibration on proliferation and differentiation of cells has been studied in vitro. To apply the vibration on the cells, a piezoelectric element was attached on the outside surface of the bottom of the culture plate of six wells. The piezoelectric element was vibrated by sinusoidally alternating voltage at 1.0 MHz generated by a function generator. Five kinds of cells were used in the experiment: C2C12 (mouse myoblast cell, L929 (fibroblast connective tissue of mouse, Hepa1-6 (mouse hepatoma cell, HUVEC (human umbilical vein endothelial cell, and Neuro-2a (mouse neural crest-derived cell line. After the incubation for 24 hours, cells were exposed to the ultrasonic vibration intermittently for three days: for thirty minutes per day. At the end of the experiment, the number of cells was counted by colorimetric method with a microplate photometer. In the case of Neuro-2a, the total length of the neurite was calculated at the microscopic image. The experimental study shows following results. Cells are exfoliated by the strong vibration. Proliferation and differentiation of cells are accelerated with mild vibration. The optimum intensity of vibration depends on the kind of cells.

  11. More than the genes, the tumor microenvironment in neuroblastoma

    Science.gov (United States)

    Borriello, Lucia; Seeger, Robert C.; Asgharzadeh, Shahab; DeClerck, Yves A.

    2017-01-01

    Neuroblastoma is the second most common solid tumor in children. Since the seminal discovery of the role of amplification of the MYCN oncogene in the pathogenesis of neuroblastoma in the 1980s, much focus has been on the contribution of genetic alterations in the progression of this cancer. However it is now clear that not only genetic events play a role but that the tumor microenvironment (TME) substantially contributes to the biology of neuroblastoma. In this article, we present a comprehensive review of the literature on the contribution of the TME to the ten hallmarks of cancer in neuroblastoma and discuss the mechanisms of communication between neuroblastoma cells and the TME that underlie the influence of the TME on neuroblastoma progression. We end our review by discussing how the knowledge acquired over the last two decades in this field is now leading to new clinical trials targeting the TME. PMID:26597947

  12. Cancer-associated fibroblasts promote proliferation of endometrial cancer cells.

    Directory of Open Access Journals (Sweden)

    Kavita S Subramaniam

    Full Text Available Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin and hormonal (estrogen and progesterone receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175% when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51% (P<0.0001. These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001, suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR, also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP-1, interleukin (IL-6, IL-8, RANTES and vascular

  13. Inhibition of tracheal smooth muscle cell proliferation by phosphodiesterase inhibitors

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    Kazuko Masu

    1999-01-01

    Full Text Available Agents that increase intracellular cyclic 3',5'-adenosine monophosphate (cAMP, such as forskolin, prostaglandin (PGE2, salbutamol and 8-bromo-cAMP, have been shownto inhibit the proliferation of airway smooth-muscle (ASM cells in vitro. However, it has not yet been determined whether selective inhibitors of phosphodiesterase (PDE isoenzymes III and IV that catalyze cAMPto 5'-adenosine monophosphate have the ability to inhibit ASM cell proliferation. To evaluate the effectsof PDE inhibitors on ASM cell proliferation, ASM cells isolated from bovine tracheae were cultured in the presence of fetal bovine serum (FBS, with or without a non-selective PDE inhibitor (theophylline, a selective PDE III inhibitor (cilostazol, and a selective PDE IV inhibitor (rolipram. The number of ASM cells cultured with 5% FBS was significantly reduced by the presence of theophylline at 10−3 and 3 × 10−4 mol/L, cilostazol at 10−5, 10−6 and 10−7 mol/L, and rolipram at 10−4 and 10−5 mol/L. The release of lactic dehydrogenase from ASM cells cultured with any concentration of these agents was not significantly different from that with medium alone. Inhibitors of PDE III and IV were demonstrated to have an inhibitory effect on ASM cell proliferation induced by FBS. Our results suggest the value of the further development of PDE inhibitors for the treatment of hyperplasia of ASM cells characteristic of airway remodeling, in addition to bronchospasm and airway inflammation, in bronchial asthma.

  14. Gastrokine 1 inhibits gastrin-induced cell proliferation.

    Science.gov (United States)

    Kim, Olga; Yoon, Jung Hwan; Choi, Won Suk; Ashktorab, Hassan; Smoot, Duane T; Nam, Suk Woo; Lee, Jung Young; Park, Won Sang

    2016-04-01

    Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth. We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGS(GKN1) and MKN1(GKN1) gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses. Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGS(mock) and MKN1(mock), but not in HFE-145, AGS(GKN1), and MKN1(GKN1), cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae. These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

  15. Noninvasive Assessment of Tumor Cell Proliferation in Animal Models

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    Matthias Edinger

    1999-10-01

    Full Text Available Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, proliferation of these cells in irradiated severe combined immunodeficiency (SCID mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1×103 cells in the peritoneal cavity, 1×104 cells at subcutaneous sites and 1×106 circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1×103 cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, this method will accelerate development of novel therapeutic

  16. Promotion of stem cell proliferation by vegetable peptone.

    Science.gov (United States)

    Lee, J; Lee, J; Hwang, H; Jung, E; Huh, S; Hyun, J; Park, D

    2009-10-01

    Technical limitations and evolution of therapeutic applications for cell culture-derived products have accelerated elimination of animal-derived constituents from such products to minimize inadvertent introduction of microbial contaminants, such as fungi, bacteria or viruses. The study described here was conducted to investigate the proliferative effect of vegetable peptone on adult stem cells in the absence of serum, and its possible mechanisms of action. Cell viability and proliferation were determined using the MTT assay and Click-iT EdU flow cytometry, respectively. In addition, changes in expression of cytokine genes were analysed using MILLIPLEX human cytokine enzyme-linked immunosorbent assay kit. Viability of cord blood-derived mesenchymal stem cells (CB-MSC) and adipose tissue-derived stem cells (ADSC) increased significantly when treated with the peptone. In addition, median value of the group treated with peptone shifted to the right when compared to the untreated control group. Furthermore, quantitative analysis of the cytokines revealed that production of vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) increased significantly in response to treatment with our vegetable peptone in both CB-MSCs and ADSCs. Our findings revealed that the vegetable peptone promotes proliferation of CB-MSCs and ADSCs. In addition, results of this study suggest that induction of stem cell proliferation by vegetable peptone is likely to be related to its induction of VEGF, TGF-beta1, and IL-6 expression.

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

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    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.

  18. SKP2 Activation by Thyroid Hormone Receptor β2 Bypasses Rb-Dependent Proliferation in Rb-Deficient Cells.

    Science.gov (United States)

    Xu, Xiaoliang L; Li, Zhengke; Liu, Aihong; Fan, Xianqun; Hu, Dan-Ning; Qi, Dong-Lai; Chitty, David W; Jia, Renbing; Qui, Jianping; Wang, Justin Q; Sharaf, Jake; Zou, Jun; Weiss, Rebecca; Huang, Hongyan; Joseph, Walter J; Ng, Lily; Rosen, Richard; Shen, Binghui; Reid, Mark W; Forrest, Douglas; Abramson, David H; Singer, Samuel; Cobrinik, David; Jhanwar, Suresh C

    2017-12-15

    Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRβ2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRβ1. TRβ2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRβ2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRβ1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRβ2 counteracts TRβ1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR. ©2017 American Association for Cancer Research.

  19. Matrix stiffness regulates endothelial cell proliferation through septin 9.

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    Yi-Ting Yeh

    Full Text Available Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa in comparison to those with low stiffness (LSG, 1.72 kPa. ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9, the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin α(vβ(3 caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.

  20. The Retinoblastoma pathway regulates stem cell proliferation in freshwater planarians.

    Science.gov (United States)

    Zhu, Shu Jun; Pearson, Bret J

    2013-01-15

    Freshwater planarians are flatworms of the Lophotrochozoan superphylum and are well known for their regenerative abilities, which rely on a large population of pluripotent adult stem cells. However, the mechanisms by which planarians maintain a precise population of adult stem cells while balancing proliferation and cell death, remain to be elucidated. Here we have identified, characterized, and functionally tested the core Retinoblastoma (Rb) pathway components in planarian adult stem cell biology. The Rb pathway is an ancient and conserved mechanism of proliferation control from plants to animals and is composed of three core components: an Rb protein, and a transcription factor heterodimer of E2F and DP proteins. Although the planarian genome contains all components of the Rb pathway, we found that they have undergone gene loss from the ancestral state, similar to other species in their phylum. The single Rb homolog (Smed-Rb) was highly expressed in planarian stem cells and was required for stem cell maintenance, similar to the Rb-homologs p107 and p130 in vertebrates. We show that planarians and their phylum have undergone the most severe reduction in E2F genes observed thus far, and the single remaining E2F was predicted to be a repressive-type E2F (Smed-E2F4-1). Knockdown of either Smed-E2F4-1 or its dimerization partner Dp (Smed-Dp) by RNAi resulted in temporary hyper-proliferation. Finally, we showed that known Rb-interacting genes in other systems, histone deacetylase 1 and cyclinD (Smed-HDAC1; Smed-cycD), were similar to Rb in expression and phenotypes when knocked down by RNAi, suggesting that these established interactions with Rb may also be conserved in planarians. Together, these results showed that planarians use the conserved components of the Rb tumor suppressor pathway to control proliferation and cell survival. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Phospholipase Cβ interacts with cytosolic partners to regulate cell proliferation.

    Science.gov (United States)

    Scarlata, Suzanne; Singla, Ashima; Garwain, Osama

    2018-01-01

    Phospholipase Cβ (PLCβ) is the main effector of the Gαq signaling pathway relaying different extracellular sensory information to generate intracellular calcium signals. Besides this classic function, we have found that PLCβ plays an important but unknown role in regulating PC12 cell differentiation by interacting with components in the RNA-induced silencing machinery. In trying to understand the role of PLCβ in PC12 cell differentiation, we find that over-expressing PLCβ reduces PC12 cell proliferation while down-regulating PLCβ increases the rate of cell proliferation. However, this behavior is not seen in other cancerous cell lines. To determine the underlying mechanism, we carried out mass spectrometry analysis of PLCβ complexes in PC12 cells. We find that in unsynchronized cells, PLCβ primarily binds cyclin-dependent kinase (CDK)16 whose activity plays a key role in cell proliferation. In vitro studies show a direct association between the two proteins that result in loss in CDK16 activity. When cells are arrested in the G2/M phase, a large population of PLCβ is bound to Ago2 in a complex that contains C3PO and proteins commonly found in stress granules. Additionally, another population of PLCβ complexes with CDK18 and cyclin B1. Fluorescence lifetime imaging microscopy (FLIM) confirms cell cycle dependent associations between PLCβ and these other protein binding partners. Taken together, our studies suggest that PLCβ may play an active role in mediating interactions required to move through the cell cycle. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Petasites japonicus Stimulates the Proliferation of Mouse Spermatogonial Stem Cells.

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    Hye-Ryun Kang

    Full Text Available Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.

  3. Crucial Role for Membrane Fluidity in Proliferation of Primitive Cells

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    Romain Mercier

    2012-05-01

    Full Text Available The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient “L-form” state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.

  4. Targeting Neuroblastoma Cell Surface Proteins: Recommendations for Homology Modeling of hNET, ALK, and TrkB.

    Science.gov (United States)

    Haddad, Yazan; Heger, Zbyněk; Adam, Vojtech

    2017-01-01

    Targeted therapy is a promising approach for treatment of neuroblastoma as evident from the large number of targeting agents employed in clinical practice today. In the absence of known crystal structures, researchers rely on homology modeling to construct template-based theoretical structures for drug design and testing. Here, we discuss three candidate cell surface proteins that are suitable for homology modeling: human norepinephrine transporter (hNET), anaplastic lymphoma kinase (ALK), and neurotrophic tyrosine kinase receptor 2 (NTRK2 or TrkB). When choosing templates, both sequence identity and structure quality are important for homology modeling and pose the first of many challenges in the modeling process. Homology modeling of hNET can be improved using template models of dopamine and serotonin transporters instead of the leucine transporter (LeuT). The extracellular domains of ALK and TrkB are yet to be exploited by homology modeling. There are several idiosyncrasies that require direct attention throughout the process of model construction, evaluation and refinement. Shifts/gaps in the alignment between the template and target, backbone outliers and side-chain rotamer outliers are among the main sources of physical errors in the structures. Low-conserved regions can be refined with loop modeling method. Residue hydrophobicity, accessibility to bound metals or glycosylation can aid in model refinement. We recommend resolving these idiosyncrasies as part of "good modeling practice" to obtain highest quality model. Decreasing physical errors in protein structures plays major role in the development of targeting agents and understanding of chemical interactions at the molecular level.

  5. GLUT1 regulates cell glycolysis and proliferation in prostate cancer.

    Science.gov (United States)

    Xiao, Hengjun; Wang, Jun; Yan, Weixin; Cui, Yubin; Chen, Zheng; Gao, Xin; Wen, Xingqiao; Chen, Jun

    2018-02-01

    Glucose transporter 1 (GLUT1) plays a critical role in tumorigenesis and tumor progression in multiple cancer types. However, the specific function and clinical significance of GLUT1 in prostate cancer (PCa) are still unclear. Therefore, in this study, we investigated the role of GLUT1 in PCa. GLUT1 protein levels in prostate cancer tissue and tumor-adjacent normal tissues were measured and compared. Furthermore, real-time PCR and Western blot analysis were both used to detect GLUT1 expression levels in different PCa cell lines. Flow cytometry and cell-based assays, such as a glucose uptake and lactate secretion assay, CCK-8 assay, and transwell migration and wound healing assay, were used to monitor cancer cell cycle distribution, glycolysis, proliferation, and motility, respectively. Moreover, a mouse tumor xenograft model was used to investigate the role of GLUT1 in tumor progression in vivo. GLUT1 expression levels are higher in PCa tissues than in tumor-adjacent normal tissues. The results from real-time PCR and Western blot analysis revealed a similar increase in the GLUT1 expression levels in PCa cell lines. Moreover, knockdown of GLUT1 inhibits cell glycolysis and proliferation and leads to cell cycle arrest at G2/M phase in the 22RV1 cell line but not in the PC3 cell line. In vivo experiments further confirmed that GLUT1 knockdown inhibits the growth of tumors derived from the 22RV1 cell line. In addition, we also showed that GLUT1 knockdown has no effect on cell migration in vitro. GLUT1 may play an important role in PCa progression via mediating glycolysis and proliferation. Our study also indicated a potential crosstalk between GLUT1-mediated glycolysis and androgen sensitivity in PCa. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2016-06-01

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

  7. Modulation of adenylate cyclase activity by a cytosolic factor following chronic opiate exposure in neuroblastoma x glioma NG108-15 hybrid cells.

    Science.gov (United States)

    Griffin, M T; Law, P Y; Loh, H H

    1983-01-01

    A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

  8. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    Science.gov (United States)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  9. The emerging molecular pathogenesis of neuroblastoma: implications for improved risk assessment and targeted therapy

    OpenAIRE

    Van Roy, Nadine; De Preter, Katleen; Hoebeeck, Jasmien; Van Maerken, Tom; Pattyn, Filip; Mestdagh, Pieter; Vermeulen, Joëlle; Vandesompele, Jo; Speleman, Franki

    2009-01-01

    Neuroblastoma is one of the most common solid tumors of childhood, arising from immature sympathetic nervous system cells. The clinical course of patients with neuroblastoma is highly variable, ranging from spontaneous regression to widespread metastatic disease. Although the outcome for children with cancer has improved considerably during the past decades, the prognosis of children with aggressive neuroblastoma remains dismal. The clinical heterogeneity of neuroblastoma mirrors the biologic...

  10. Nifedipine promotes the proliferation and migration of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Dong-Qing Guo

    Full Text Available Nifedipine is widely used as a calcium channel blocker (CCB to treat angina and hypertension,but it is controversial with respect the risk of stimulation of cancers. In this study, we demonstrated that nifedipine promoted the proliferation and migration of breast cancer cells both invivo and invitro. However, verapamil, another calcium channel blocker, didn't exert the similar effects. Nifedipine and high concentration KCl failed to alter the [Ca2+]i in MDA-MB-231 cells, suggesting that such nifedipine effect was not related with calcium channel. Moreover, nifedipine decreased miRNA-524-5p, resulting in the up-regulation of brain protein I3 (BRI3. Erk pathway was consequently activated and led to the proliferation and migration of breast cancer cells. Silencing BRI3 reversed the promoting effect of nifedipine on the breast cancer. In a summary, nifedipine stimulated the proliferation and migration of breast cancer cells via the axis of miRNA-524-5p-BRI3-Erk pathway independently of its calcium channel-blocking activity. Our findings highlight that nifedipine but not verapamil is conducive for breast cancer growth and metastasis, urging that the caution should be taken in clinic to prescribe nifedipine to women who suffering both hypertension and breast cancer, and hypertension with a tendency in breast cancers.

  11. Voltage-Gated Ion Channels in Cancer Cell Proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Vidhya R.; Perez-Neut, Mathew [Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago 2160 S. 1st Ave, Maywood, IL 60153 (United States); Kaja, Simon [Department of Ophthalmology and Vision Research Center, School of Medicine, University of Missouri-Kansas City, 2411 Holmes St., Kansas City, MO 64108 (United States); Gentile, Saverio, E-mail: sagentile@luc.edu [Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago 2160 S. 1st Ave, Maywood, IL 60153 (United States)

    2015-05-22

    Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K{sup +}, Ca{sup ++}, Cl{sup −}, Na{sup +}. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation.

  12. Comparison of the Side Populations in Pretreatment and Postrelapse Neuroblastoma Cell Lines

    Science.gov (United States)

    2010-08-01

    Acad Sci USA 103, 11154–11159. [32] Wu C, Wei Q, Utomo V, Nadesan P, Whetstone H, Kandel R, Wunder JS, and Alman BA (2007). Side population cells...Okano H, and Miyake J (2005). Functional expression of ABCG2 transporter in human neural stem/progenitor cells. Neurosci Res 52, 75–82. [37] Thomas SK

  13. TRPM7 triggers Ca(2+) sparks and invadosome formation in neuroblastoma cells

    NARCIS (Netherlands)

    Visser, D.; Langeslag, M.; Kedziora, K.M.; Klarenbeek, J.; Kamermans, A.; Horgen, F.D.; Fleig, A.; Leeuwen, F.N. van; Jalink, K.

    2013-01-01

    Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca(2+) signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much

  14. 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.

  15. Hemopoietic stem cells: stochastic differentiation and humoral control of proliferation.

    Science.gov (United States)

    Ogawa, M

    1989-03-01

    The central feature of hemopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The entry into cell cycle of the dormant stem cells may be promoted by such factors as interleukin-1, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Once the stem cells leave G0 and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. While several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a random (stochastic) process. The proliferation of early hemopoietic progenitors requires the presence of interleukin-3 (IL-3), and the intermediate process appears to be supported by granulocyte/macrophage colony-stimulating factor (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin and G-CSF. Synthesis of a hemopoietic factor may take place in different cell types and is regulated by multiple factors. The physiological regulator of erythropoiesis is erythropoietin, which, by a feedback mechanism, provides fine control of erythrocyte production. Feedback mechanisms for leukocyte production have not been identified. It is possible that there is no feedback regulator of leukopoiesis. In this model, leukocyte production in the steady state is maintained at a genetically determined level. When an infection occurs, the bacterial lipopolysaccharides may augment the production of interleukin 1 alpha and beta, tumor necrosis factor, macrophage colony-stimulating factor, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration.

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    Erika Costa de Alvarenga

    Full Text Available The angiotensin-I converting enzyme (ACE plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II. More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet.Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration.We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC, and experimental results in CHO cells, demonstrated that the β3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5 showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein.ACE activation regulates melanoma cell proliferation and migration.

  17. Simulated Hypergravity Alters Vascular Smooth Muscle Cell Proliferation and Motility

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    Hunt, Shameka; Bettis, Barika; Harris-Hooker, Sandra; Sanford, Gary L.

    1997-01-01

    The cellular effects of gravity are poorly understood due to its constancy and nonavailability of altered gravitational models. Such an understanding is crucial for prolonged space flights. In these studies, we assessed the influence of centrifugation at 6G (HGrav) on vascular smooth muscle (SMC) mobility and proliferation. Cells were: (a) plated at low density and subjected to HGrav for 24-72 hr for proliferation studies, or (b) grown to confluency, subjected to HGrav, mechanically denuded and monitored for cell movement into the denuded area. Controls were maintained under normogravity. SMC showed a 50% inhibition of growth under HGrav and 10% serum; HGrav and low serum resulted in greater growth inhibition. The rate of movement of SMC into the denuded area was 2-3-fold higher under HGrav in low serum compared to controls, but similar in 10% serum. These studies show that HGrav has significant effects on SMC growth and mobility, which are dependent on serum levels.

  18. XIAP antagonist embelin inhibited proliferation of cholangiocarcinoma cells.

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    Cody J Wehrkamp

    Full Text Available Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 cells. Assessment of nuclear morphology demonstrated a concentration-dependent increase in nuclear staining. Interestingly, embelin induced nuclear morphology changes as a single agent, independent of the addition of TNF-related apoptosis inducing ligand (TRAIL. However, caspase activity assays revealed that increasing embelin concentrations resulted in slight inhibition of caspase activity, not activation. In addition, the use of a pan-caspase inhibitor did not prevent nuclear morphology changes. Finally, embelin treatment of cholangiocarcinoma cells did not induce DNA fragmentation or PARP cleavage. Apoptosis does not appear to contribute to the effects of embelin on cholangiocarcinoma cells. Instead, embelin caused inhibition of cell proliferation and cell cycle analysis indicated that embelin increased the number of cells in S and G2/M phase. Our results demonstrate that embelin decreased proliferation in cholangiocarcinoma cell lines. Embelin treatment resulted in decreased XIAP protein expression, but did not induce or enhance apoptosis. Thus, in cholangiocarcinoma cells the mechanism of action of embelin may not be dependent on apoptosis.

  19. Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

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    Shunsuke Nojiri

    2014-03-01

    Full Text Available Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP, p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD with appropriate software (ModFit LT; BD. The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore. The mRNA levels of AFP relative to Alb(−: Alb(−, Alb(+, and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−, and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(− and p = 0.004 for Prionex, and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(− and Prionex, and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+. More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+ than in Alb(− (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−, Alb

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Aging and immortality in a cell proliferation model.

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    Antal, T; Blagoev, K B; Trugman, S A; Redner, S

    2007-10-07

    We investigate a model of cell division in which the length of telomeres within a cell regulates its proliferative potential. At each division, telomeres undergo a systematic length decrease as well as a superimposed fluctuation due to exchange of telomere DNA between the two daughter cells. A cell becomes senescent when one or more of its telomeres become shorter than a critical length. We map this telomere dynamics onto a biased branching-diffusion process with an absorbing boundary condition whenever any telomere reaches the critical length. Using first-passage ideas, we find a phase transition between finite lifetime and immortality (infinite proliferation) of the cell population as a function of the influence of telomere shortening, fluctuations, and cell division.

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

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    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.

  3. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

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    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  4. Oxytocin stimulates cell proliferation in vaginal cell line Vk2E6E7.

    Science.gov (United States)

    Kallak, Theodora K; Uvnäs-Moberg, Kerstin

    2017-03-01

    Objective During and after menopause, the symptoms of vaginal atrophy cause great discomfort and necessitate effective treatment options. Currently, vaginally applied oxytocin is being investigated as a treatment for the symptoms of vaginal atrophy in postmenopausal women. To clarify the mechanisms behind oxytocins effects on vaginal atrophy, the present study investigated the effects of oxytocin on cell proliferation in the cells of the Vk2E6E7 line, a non-tumour vaginal cell line. The study also compared the effects of oxytocin with those of estradiol (E2). Study design The effects of both oxytocin and E2 on the proliferation of Vk2E6E7 cells were investigated using Cell Proliferation ELISA BrdU Colorimetric Assay. The expression of both oxytocin and oxytocin receptor was studied in Vk2E6E7 cells using quantitative real-time polymerase chain reaction and immunofluorescent staining. Main outcome measures Cell proliferation and gene expression. Results Oxytocin increased cell proliferation both time dependently and dose dependently. This differed from the effect pattern observed in cells treated with E2. In addition, in oxytocin-treated cells, the oxytocin receptor was found to be co-localized with caveolin-1, indicating pro-proliferative signalling within the cell. Conclusions Oxytocin stimulates cell proliferation and the co-localization of oxytocin receptor with caveolin-1 in oxytocin-treated cells, supporting the role of oxytocin signalling in cell proliferation. In addition, these findings suggest that increased cell proliferation is one mechanism by which local vaginal oxytocin treatment increases vaginal thickness and relieves vaginal symptoms in postmenopausal women with vaginal atrophy.

  5. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People' s Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  6. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

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    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  7. Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

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    Tomizawa M

    2013-11-01

    Full Text Available Minoru Tomizawa,1 Fuminobu Shinozaki,2 Yasufumi Motoyoshi,3 Takao Sugiyama,4 Shigenori Yamamoto,5 Makoto Sueishi,4 Takanobu Yoshida6 1Department of Gastroenterology, 2Department of Radiology, 3Department of Neurology, 4Department of Rheumatology, 5Department of Pediatrics, 6Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Chiba, Japan Background: The Wnt pathway plays an important role in hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. Methods: We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1. Results: Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. Conclusion: Niclosamide is a potential

  8. Effects of Cisplatin in Neuroblastoma Rat Cells: Damage to Cellular Organelles

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    Giada Santin

    2012-01-01

    Full Text Available Cisplatin (cisPt is a chemotherapy agent used as a treatment for several types of cancer. The main cytotoxic effect of cisplatin is generally accepted to be DNA damage. Recently, the mechanism by which cisPt generates the cascade of events involved in the apoptotic process has been demonstrated. In particular it has been shown that some organelles are cisPt target and are involved in cell death. This paper aims to describe the morphological and functional changes of the Golgi apparatus and lysosomes during apoptosis induced in neuronal rat cells (B50 by cisplatin. The results obtained show that the cellular organelles are the target of cisPt, so their damage can induce cell death.

  9. Serglycin in Quiescent and Proliferating Primary Endothelial Cells

    Science.gov (United States)

    Reine, Trine M.; Vuong, Tram T.; Rutkovskiy, Arkady; Meen, Astri J.; Vaage, Jarle; Jenssen, Trond G.; Kolset, Svein O.

    2015-01-01

    Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development. PMID:26694746

  10. Cell topology, geometry, and morphogenesis in proliferating epithelia.

    Science.gov (United States)

    Gibson, William T; Gibson, Matthew C

    2009-01-01

    Epithelia are sheets of tightly adherent cells that line both internal and external surfaces in a vast array of metazoans. During development, an intrinsic consequence of coupling tight adhesion with cellular proliferation is the emergence of an epithelial form characterized by a stereotyped distribution of polygonal cell shapes. Despite the near universality of this constraint on cell shape and tissue organization, very little is known about the possible implications of cell pattern geometry for mechanical properties of tissues or key biological processes, such as planar polarization, tissue remodeling, and cell division. In this chapter, through an examination of increasingly complex models, we highlight what is known about the role of mitotic proliferation in the emergence of epithelial cell geometry, and examine some possible implications for tissue morphogenesis. Ideally, continued progress in this area will address a major conceptual challenge in biology, which is to understand aspects of morphogenesis that are not explicitly directed by genetic control, but instead emerge from the complex interactions between geometric and biomechanical properties of epithelial tissues.

  11. Rnd3 regulates lung cancer cell proliferation through notch signaling.

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    Yongjun Tang

    Full Text Available Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.

  12. SerpinB1 Promotes Pancreatic β Cell Proliferation

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    El Ouaamari, Abdelfattah; Dirice, Ercument; Gedeon, Nicholas; Hu, Jiang; Zhou, Jian-Ying; Shirakawa, Jun; Hou, Lifei; Goodman, Jessica; Karampelias, Christos; Qiang, Guifeng; Boucher, Jeremie; Martinez, Rachael; Gritsenko, Marina A.; De Jesus, Dario F.; Kahraman, Sevim; Bhatt, Shweta; Smith, Richard D.; Beer, Hans-Dietmar; Jungtrakoon, Prapaporn; Gong, Yanping; Goldfine, Allison B.; Liew, Chong Wee; Doria, Alessandro; Andersson, Olov; Qian, Wei-Jun; Remold-O’Donnell, Eileen; Kulkarni, Rohit N.

    2016-01-01

    Compensatory β-cell growth in response to insulin resistance is a common feature in diabetes. We recently reported that liver-derived factors participate in this compensatory response in the liver insulin receptor knockout (LIRKO) mouse, a model of significant islet hyperplasia. Here we show that serpinB1 is a liver-derived secretory protein that controls β-cell proliferation. SerpinB1 is abundant in the hepatocyte secretome and sera derived from LIRKO mice. SerpinB1 and small molecule compounds that partially mimic serpinB1 activity enhanced proliferation of zebrafish, mouse and human β-cells. We report that serpinB1-induced β-cell replication requires protease inhibition activity and mice lacking serpinB1 exhibit attenuated β-cell replication in response to insulin resistance. Finally, SerpinB1-treatment of islets modulated signaling proteins in growth and survival pathways such as MAPK, PKA and GSK3. Together, these data implicate SerpinB1 as a protein that can potentially be harnessed to enhance functional β-cell mass in patients with diabetes.

  13. Effects of drinking desalinated seawater on cell viability and proliferation.

    Science.gov (United States)

    Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

    2017-06-01

    Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

  14. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  15. Rho-associated kinase is a therapeutic target in neuroblastoma.

    Science.gov (United States)

    Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K; Forsberg, David; Herlenius, Eric; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge; Wickström, Malin

    2017-08-08

    Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

  16. CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis.

    Science.gov (United States)

    Baumer, Yvonne; McCurdy, Sara; Alcala, Martin; Mehta, Nehal; Lee, Bog-Hieu; Ginsberg, Mark H; Boisvert, William A

    2017-01-01

    Vascular smooth muscle cells (VSMC) migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. CD98 is a transmembrane protein made of two subunits, CD98 heavy chain (CD98hc) and one of six light chains, and is known to be involved in cell proliferation and survival. Because the influence of CD98hc on atherosclerosis development is unknown, our aim was to determine if CD98hc expressed on VSMC plays a role in shaping the morphology of atherosclerotic plaques by regulating VSMC function. In addition to determining the role of CD98hc in VSMC proliferation and apoptosis, we utilized mice with SMC-specific deletion of CD98hc (CD98hc(fl/fl)SM22αCre(+)) to determine the effects of CD98hc deficiency on VSMC function in atherosclerotic plaque. After culturing for 5 days in vitro, CD98hc(-/-) VSMC displayed dramatically reduced cell counts, reduced proliferation, as well as reduced migration compared to control VSMC. Analysis of aortic VSCM after 8 weeks of HFD showed a reduction in CD98hc(-/-) VSMC proliferation as well as increased apoptosis compared to controls. A long-term atherosclerosis study using SMC-CD98hc(-/-)/ldlr(-/-) mice was performed. Although total plaque area was unchanged, CD98hc(-/-) mice showed reduced presence of VSMC within the plaque (2.1 ± 0.4% vs. 4.3 ± 0.4% SM22α-positive area per plaque area, p < 0.05), decreased collagen content, as well as increased necrotic core area (25.8 ± 1.9% vs. 10.9 ± 1.6%, p < 0.05) compared to control ldlr(-/-) mice. We conclude that CD98hc is required for VSMC proliferation, and that its deficiency leads to significantly reduced presence of VSMC in the neointima. Thus, CD98hc expression in VSMC contributes to the formation of plaques that are morphologically more stable, and thereby protects against atherothrombosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. 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.

  18. 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.

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

    NARCIS (Netherlands)

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

    2016-01-01

    Shugoshin 1 (SGO1) is required for accurate chromosome segregation during mitosis and meiosis; however, its other functions, especially at interphase, are not clearly understood. Here, we found that downregulation of SGO1 caused a synergistic phenotype in cells overexpressing MYCN. Downregulation of

  20. Cyclin C stimulates β-cell proliferation in rat and human pancreatic β-cells

    Science.gov (United States)

    Jiménez-Palomares, Margarita; López-Acosta, José Francisco; Villa-Pérez, Pablo; Moreno-Amador, José Luis; Muñoz-Barrera, Jennifer; Fernández-Luis, Sara; Heras-Pozas, Blanca; Perdomo, Germán; Bernal-Mizrachi, Ernesto

    2015-01-01

    Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional β-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [3H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected β-cell death by TUNEL, β-cell differentiation by RT-PCR, and β-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human β-cell proliferation. This augmented proliferation did not induce β-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing β-cell regeneration. PMID:25564474

  1. Parathyroid hormone dependent T cell proliferation in uremic rats

    DEFF Research Database (Denmark)

    Lewin, E; Ladefoged, Jens; Brandi, L

    1993-01-01

    was normalized. Rat PTH 1-84 stimulated in vitro the PHA-induced proliferation of T cells in a dose dependent manner. This effect was significant in CRF rat lymphocytes, but not in lymphocytes obtained from normal rats. Based upon the present results it is suggested that the secondary hyperparathyroidism......Chronic renal failure (CRF) is combined with an impairment of the immune system. The T cell may be a target for the action of parathyroid hormone (PTH). Rats with CRF have high blood levels of PTH. Therefore, the present investigation examined some aspects of the T cell function in both normal...... and CRF rats before and after parathyroidectomy and after an isogenic kidney transplantation. The T cell proliferative response to phytohemagglutinin (PHA) stimulation was significantly higher in peripheral blood mononuclear cell (PBMC) cultures obtained from CRF rats than from normal rats. After...

  2. Anti-cancer effect of HIV-1 viral protein R on doxorubicin resistant neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Richard Y Zhao

    Full Text Available Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX. To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

  3. Biochemical Characterization of Liver Oil of Echinorhinus brucus (Bramble Shark and Its Cytotoxic Evaluation on Neuroblastoma Cell Lines (SHSY-5Y

    Directory of Open Access Journals (Sweden)

    Vishnu Venugopal

    2016-01-01

    Full Text Available The objective of the present study was to characterize the liver oil extracted from the deep sea shark, Echinorhinus brucus, caught from Central Indian Ocean and to evaluate its cytotoxic effect on neuroblastoma cell line (SHSY-5Y. Characterization of liver oil of Echinorhinus brucus revealed the presence of palmitic acid (15%, oleic acid (12%, stearic acid (8%, docosahexaenoic acid (DHA (18%, and eicosapentaenoic acid (EPA (16%. It was also found to be a good source of squalene (38.5% and fat soluble vitamins such as A, D, and K (vitamin A: 17.08 mg/100 g of oil, vitamin D: 15.04 mg/100 g oil, and vitamin K: 11.45 mg/100 g oil. Since it was found to be rich in essential fatty acids, fat soluble vitamins, and squalene, it can be considered as better dietary supplement. The oil of Echinorhinus brucus also showed high in vitro cytotoxic effect against the human neuroblastoma cell line (SHSY-5Y and the IC50 value laid between 35 and 45 ng.

  4. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

    Directory of Open Access Journals (Sweden)

    Julien Vibert

    2017-03-01

    Full Text Available Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific "signature" that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU data, revealing T cell proliferation heterogeneity and specific signatures.

  5. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

    Science.gov (United States)

    Vibert, Julien; Thomas-Vaslin, Véronique

    2017-03-01

    Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific "signature" that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures.

  6. 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.

  7. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors.

    Science.gov (United States)

    Chen, Jin; Chaurio, Ricardo A; Maueröder, Christian; Derer, Anja; Rauh, Manfred; Kost, Andriy; Liu, Yi; Mo, Xianming; Hueber, Axel; Bilyy, Rostyslav; Herrmann, Martin; Zhao, Yi; Muñoz, Luis E

    2017-01-01

    Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo. The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  8. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Yi Zhao

    2017-04-01

    Full Text Available IntroductionMany antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells.MethodsCultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS in the presence of dead and dying cells, their supernatants (SNs, or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo.ResultsThe stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment.ConclusionInosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  9. Opioid receptor gene expression in human neuroblastoma SH-SY5Y cells following tapentadol exposure.

    Science.gov (United States)

    Caputi, Francesca Felicia; Carretta, Donatella; Tzschentke, Thomas M; Candeletti, Sanzio; Romualdi, Patrizia

    2014-08-01

    Recent studies showed that combination of mu opioid receptor (MOP) agonism and monoamine reuptake inhibition may improve the therapeutic effect of opioids by reducing requirement for MOP activation. Tapentadol, showing such a combined mechanism of action, exhibits delayed analgesic tolerance development compared to pure MOP agonists. Here we investigated how opioid receptors are regulated following different schedules (two ranges of concentrations for 24 and 48 h) of tapentadol exposure in vitro in SH-SY5Y cells. MOP and nociceptin/orphaninFQ (NOP) receptor gene expressions were quantified using qReal-Time PCR. Moreover, studies were performed in U2 cells to assess tapentadol effect on MOP internalization compared with morphine and DAMGO. Ten and 100 nM tapentadol for 48 h induced a significant increase of MOP gene expression; cells exposed to 100 μM tapentadol for 24 and 48 h showed a significant increase of MOP mRNA levels. NOP gene expression showed a significant decrease following tapentadol at all low concentrations used after 24 h and at high concentrations (45 and 60 μM) after 24 h and (60 μM) after 48 h. Differently from DAMGO, tapentadol or morphine showed no effects on MOP internalization. This study suggests that tapentadol affects MOP and NOP gene expression and MOP internalization showing a pattern distinct from classical MOP agonists. Whether these differences can explain the improved therapeutic profile of tapentadol remains to be investigated.

  10. 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.

  11. Inhibitory effects of ameloblastin on epithelial cell proliferation.

    Science.gov (United States)

    Saito, Noriko; Ariyoshi, Wataru; Okinaga, Toshinori; Kamegawa, Mariko; Matsukizono, Miho; Akebiyama, Yasuo; Kitamura, Chiaki; Nishihara, Tatsuji

    2014-08-01

    Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro. Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag-fused human ameloblastin by the HaloTag system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry. The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase. We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

    OpenAIRE

    Wang, Lintao; Peng, Yanyan; Shi, Kaikai; Wang, Haixiao; Lu, Jianlei; Li, Yanli; Ma, Changyan

    2012-01-01

    Abstract Recent studies have revealed that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, a traditional Chinese medicine, possesses anticancer activity. However, its effect on breast cancer cells so far has not been elucidated clearly. In the present study, we evaluated the effects of osthole on the proliferation, cell cycle and apoptosis of human breast cancer cells MDA-MB 435. We demonstrated that osthole is effective in inhibiting the proliferation ...

  13. TORC1 is required to balance cell proliferation and cell death in planarians

    Science.gov (United States)

    Tu, Kimberly C.; Pearson, Bret J.; Alvarado, Alejandro Sánchez

    2012-01-01

    Multicellular organisms are equipped with cellular mechanisms that enable them to replace differentiated cells lost to normal physiological turnover, injury, and for some such as planarians, even amputation. This process of tissue homeostasis is generally mediated by adult stem cells (ASCs), tissue-specific stem cells responsible for maintaining anatomical form and function. To do so, ASCs must modulate the balance between cell proliferation, i.e. in response to nutrients, and that of cell death, i.e. in response to starvation or injury. But how these two antagonistic processes are coordinated remains unclear. Here, we explore the role of the core components of the TOR pathway during planarian tissue homeostasis and regeneration and identified an essential function for TORC1 in these two processes. RNAi-mediated silencing of TOR in intact animals resulted in a significant increase in cell death, whereas stem cell proliferation and stem cell maintenance were unaffected. Amputated animals failed to increase stem cell proliferation after wounding and displayed defects in tissue remodeling. Together, our findings suggest two distinct roles for TORC1 in planarians. TORC1 is required to modulate the balance between cell proliferation and cell death during normal cell turnover and in response to nutrients. In addition, it is required to initiate appropriate stem cell proliferation during regeneration and for proper tissue remodeling to occur to maintain scale and proportion. PMID:22445864

  14. Olfactory neuroblastoma in a horse.

    Science.gov (United States)

    Yamate, Jyoji; Izawa, Takeshi; Ogata, Keiko; Kobayashi, Osamu; Okajima, Ryoko; Kuwamura, Mitsuru; Kotani, Takao; Aoki, Mika

    2006-05-01

    An 11-year-old thoroughbred gelding was euthanatized because of right nasal cavity tumor. The tumor consisted of round to oval cells with a scanty cytoplasm and hyperchromatic nuclei. Homer-Wright rosettes and pseudorosettes, as well as microcysts were seen. Neoplastic cells were immunoreactive to vimentin, S-100 protein, and neuron-specific enolase, glial fibrillary acidic protein and microtube-associated protein in varying degrees, indicating neurogenic nature. Based on these findings, this tumor was diagnosed as an olfactory neuroblastoma. Since this type is an uncommon tumor showing histological variety, the nature is discussed.

  15. Evaluation of the neurotoxic/neuroprotective role of organoselenides using differentiated human neuroblastoma SH-SY5Y cell line challenged with 6-hydroxydopamine.

    Science.gov (United States)

    Lopes, Fernanda Martins; Londero, Giovana Ferreira; de Medeiros, Liana Marengo; da Motta, Leonardo Lisbôa; Behr, Guilherme Antônio; de Oliveira, Valeska Aguiar; Ibrahim, Mohammad; Moreira, José Cláudio Fonseca; Porciúncula, Lisiane de Oliveira; da Rocha, João Batista Teixeira; Klamt, Fábio

    2012-08-01

    It is well established that oxidative stress plays a major role in several neurodegenerative conditions, like Parkinson disease (PD). Hence, there is an enormous effort for the development of new antioxidants compounds with therapeutic potential for the management of PD, such as synthetic organoselenides molecules. In this study, we selected between nine different synthetic organoselenides the most eligible ones for further neuroprotection assays, using the differentiated human neuroblastoma SH-SY5Y cell line as in vitro model. Neuronal differentiation of exponentially growing human neuroblastoma SH-SY5Y cells was triggered by cultivating cells with DMEM/F12 medium with 1% of fetal bovine serum (FBS) with the combination of 10 μM retinoic acid for 7 days. Differentiated cells were further incubated with different concentrations of nine organoselenides (0.1, 0.3, 3, 10, and 30 μM) for 24 h and cell viability, neurites densities and the immunocontent of neuronal markers were evaluated. Peroxyl radical scavenging potential of each compound was determined with TRAP assay. Three organoselenides tested presented low cytotoxicity and high antioxidant properties. Pre-treatment of cells with those compounds for 24 h lead to a significantly neuroprotection against 6-hydroxydopamine (6-OHDA) toxicity, which were directly related to their antioxidant properties. Neuroprotective activity of all three organoselenides was compared to diphenyl diselenide (PhSe)₂, the simplest of the diaryl diselenides tested. Our results demonstrate that differentiated human SH-SY5Y cells are suitable cellular model to evaluate neuroprotective/neurotoxic role of compounds, and support further evaluation of selected organoselenium molecules as potential pharmacological and therapeutic drugs in the treatment of PD.

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

    OpenAIRE

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

    2015-01-01

    There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to in...

  17. Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Alan M. [School of Applied Sciences, University of Huddersfield, Huddersfield HD1 3DH (United Kingdom); Paxton, Jennifer Z.; Hung, Yi-Pei; Hadley, Martin J.; Bowen, James; Williams, Richard L. [School of Chemical Engineering, University of Birmingham, Edgbaston, B15 2TT (United Kingdom); Grover, Liam M., E-mail: l.m.grover@bham.ac.uk [School of Chemical Engineering, University of Birmingham, Edgbaston, B15 2TT (United Kingdom)

    2015-03-01

    Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40 years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macro-scale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation. - Highlights: • Crystallinity of HA at the nano-scale was dominant over macro-scale topography. • Lower crystallinity caused rapid cell attachment and proliferation rate. • Crystallinity could be easily adjusted by without compromising coating purity.

  18. Lysyl Oxidase Propeptide Inhibits Smooth Muscle Cell Signaling and Proliferation

    Science.gov (United States)

    Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva; Yang, Dan; St. Hilaire, Cynthia; Guo, Ying; Palamakumbura, Amitha H.; Schreiber, Barbara M.; Ravid, Katya; Trackman, Philip C.

    2008-01-01

    Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology. PMID:18060869

  19. Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

    Science.gov (United States)

    Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

    2014-01-01

    Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

  20. 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

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

    Directory of Open Access Journals (Sweden)

    Rosasco Lorenzo

    2009-10-01

    Full Text Available Abstract Background 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. Results 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 ε equal to 100. This signature is composed by 11 probesets representing 8 well characterized genes known to be modulated by hypoxia. Conclusion We demonstrate that l1-l2 regularization

  2. CD4 + T cells promote renal cell carcinoma proliferation via modulating YBX1.

    Science.gov (United States)

    Wang, Yong; Wang, Yiting; Xu, Liang; Lu, Xianqi; Fu, Donghe; Su, Jing; Geng, Hua; Qin, Guoxuan; Chen, Ruibing; Quan, Changyi; Niu, Yuanjie; Yue, Dan

    2018-02-01

    Renal cell carcinoma (RCC) is a common urologic tumor and the third leading cause of death among urological tumors. Recent studies demonstrate that RCC tumors are more heavily infiltrated by lymphocytes than other cancers. However, the exact roles played by CD4 + T cells in RCC proliferation remain unknown. In this study, we cocultured RCC cells with CD4 + T cells. Stable knockdown of YBX1 in RCC cells was constructed. The effects of CD4 + T cells, TGFβ1 and YBX1 on RCC cells were investigated using cell viability assays. In situ RCC nude mouse model was used to observe the tumor growth. The potential mechanisms of CD4 + T cells and YBX1 in RCC cells proliferation were explored by qRT-PCR and