Properties Of Neuroblastoma Cells Research Articles

The dual HDAC and PI3K inhibitor, CUDC‑907, inhibits tumor growth and stem‑like properties by suppressing PTX3 in neuroblastoma.

Neuroblastoma (NB) is one of the common solid tumors in childhood and poses a threat to the lives of children. Patients with advanced‑stage or recurrent NB have a poor prognosis. CUDC‑907, as a novel dual‑target inhibitor of histone deacetylase (HDAC) and phosphatidylinositol‑3‑kinase (PI3K), has been proven to play an antitumor role in several types of tumors. However, the exact role of CUDC‑907 in NB remains unclear. In the present study, invivo and invitro assays were performed to investigate the anti‑NB activity of CUDC‑907. Pentraxin 3 (PTX3) small interfering RNA (siRNA) and PTX3 overexpression plasmid were transfected into cells to define the underlying mechanisms of CUDC‑907. Tumor tissues and clinical information were collected and immunohistochemistry (IHC) was conducted to analyze the association between the expression of HDAC1, HDAC2, HDAC3 and CD44, and the prognosis of patients with NB. The results indicated that CUDC‑907 significantly inhibited the proliferation and migration, and induced the apoptosis of NB cells, downregulating the expression level of MYCN, and suppressing the PI3K/AKT and MAPK/ERK pathways. Furthermore, CUDC‑907 suppressed the stem‑like properties of NB cells by inhibiting PTX3, a ligand and upstream protein of CD44. IHC revealed that the high expression of HDAC1, 2, 3 and CD44 was associated with a poor prognosis of patients with NB. On the whole, these findings indicate that CUDC‑907 may be developed into a possible therapeutic approach for patients with NB.

Cancer Stem Cells and Neuroblastoma: Characteristics and Therapeutic Targeting Options.

The majority of embryonal tumors or childhood blastomas derive from pluripotent progenitors or fetal stem cells that acquire cancer stem cell (CSC) properties: multipotency, self-renewal ability, metastatic potential, chemoresistance, more pronounced levels of drug transporters, enhanced DNA-damage repair mechanisms, and a quiescent state. Neuroblastoma (NB) is considered a neuroendocrine tumor and is the most common extracranial neoplasm in children. NB pathogenesis has frequently been associated with epigenetic dysregulation and a failure to implement a differentiation program. The origin, characteristics, and isolation of the CSC subpopulation in NB are still incompletely understood, despite the evidence that this cell subset contributes to disease recurrence and acquired resistance to standard therapies. Here, we summarize the literature regarding the isolation and characterization of CSCs in NB over the past decades, from the early recognition of the expression of stem cell factor (SCF) or its receptor c-KIT to more recent studies identifying the ability of G-CSF and STAT3 to support stem cell-like properties in NB cells. Additionally, we review the morphological variants of NB tumors whose recent epigenetic analyses have shed light on the tumor heterogeneity so common in NB. NB-derived mesenchymal stem cells have recently been isolated from primary tumors of NB patients and associated with a pro-tumorigenic role in the tumor microenvironment, enabling immune escape by tumors, and contributing to their invasive and metastatic capabilities. In particular, we will focus on epigenetic reprogramming in the CSC subpopulation in NB and strategies to target CSCs in NB.

<p>IGF1R facilitates epithelial-mesenchymal transition and cancer stem cell properties in neuroblastoma via the STAT3/AKT axis</p>

BackgroundNeuroblastoma (NB) displays the most heterogeneity in clinical manifestation. The insulin-like growth factor 1 receptor (IGF1R) has long been recognized for its role in tumourigenesis and growth. The IGF/IGF1R pathway is important in maintaining cell survival. It is reported that IGF1R participates in the occurrence of NB, but the mechanism is still unclear.MethodsHuman NB cell lines IMR-32 and SH-SY5Y were recruited in this study. IGF1R was knocked down by transfection with short hairpin RNA. Signal transducer and activator of transcription 3 (STAT3) expression was inhibited by Cryptotanshinone treatment. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The cancer stem cell properties were characterized by tumour sphere formation assay and colony formation assay. The mRNA and protein expression levels of related proteins were detected by RT-PCR and Western blot, respectively.ResultsThe knockdown of IGF1R inhibits NB cell tumourigenesis and the epithelial-mesenchymal transition (EMT) of NB cells. Additionally, IGF1R was found to stimulate cancer stem cell-like properties in NPC cells. The knockdown of IGF1R significantly reduced the phosphorylation of AKT, and STAT3, indicating that the activation of the AKT and STAT3 pathways was inhibited by IGF1R knockdown. Furthermore, IGF1R was demonstrated to stimulate cancer stem cell-like properties in NB cells via the regulation of the STAT3/AKT axis.ConclusionIGF1R promotes cancer stem cell properties to facilitate EMT in neuroblastoma via the STAT3/AKT axis.

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

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

Abstract B037: Improved CD8+ T-cell recognition of neuroblastoma after innate immune attack by natural killer cells

Abstract Neuroblastoma is the most common solid tumor in children, with mortality rates after conventional treatments still being 80% for high-risk (stage 4) disease. There is a clear medical need for development of new treatment strategies. Recently, immunotherapy has gained traction. However, restrained MHC I expression evades recognition of neuroblastoma by immune cells, for a suppressed immunogenic status of neuroblastoma tumors. We here describe a preclinical study aimed to improve neuroblastoma immunogenicity. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells and thereby readily sensitize neuroblastoma cells for recognition by cytotoxic T lymphocytes (CTLs). We show that oncoprotein PRAME serves as a T-cell antigen in neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cell induced MHC I upregulation in neuroblastoma cells requires IFNγ production by NK cells that is triggered by neuroblastoma contact. Our results demonstrate remarkable plasticity in the immunogenic properties of neuroblastoma cells, which are exposed when neuroblastoma cells attempt to escape innate immune attack. These findings support the exploration of NK cells in combined immunotherapy to enforce tumor-specific CTL responses, and thereby strengthen immune-mediated tumor reactivity. Citation Format: Lotte Spel, Jaap J. Boelens, Niek van Til, Dirk M. van der Steen, Nina J.G. Blokland, Max M. van Noesel, Jan J. Molenaar, Mirjam H.M. Heemskerk, Marianne Boes, Stefan Nierkens. Improved CD8+ T-cell recognition of neuroblastoma after innate immune attack by natural killer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B037.

Abstract 842: Role of Galectin-3 in invasive and apoptotic properties of neuroblastoma cells.

Abstract Background: Neuroblastoma (NB) is a childhood cancer derived from the neural crest stem cells. It generally progresses to an aggressive therapy resistant, fatal metastatic tumor. MYCN over-expression and amplification, and increased BDNF/TrkB signaling are used as signatures of high-risk NB. However, only about 25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Up-regulation of Galectin 3 (Gal-3), a multifunctional, carbohydrate binding cytokine has been extensively correlated with highly malignant behavior and poor prognosis of many cancers. However, the role of Gal-3 in NB is largely unknown. In this study, we investigated the expression pattern of Gal-3 in a panel of NB cell lines and examined whether silencing of Gal-3 expression by means of a siRNA approach decreases their invasive properties, and increases their sensitivity to pro-apoptotic chemotherapeutic drug, fenretinide (4-HPR), a synthetic retinoid derivative. Materials and Methods: We utilized RT-PCR gene expression analysis, RNA interference approaches, western blot and immunofluorescence analysis, scratch technique, transwell Boyden chamber migration assay, colony forming assay, and cytokine antibody array screening to address these studies. Results: Gal-3 expression is differentially regulated in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN-amplified (SK-N-AS, SK-N-SH, and SH-SY5Y) NB cell lines. Gal-3 expression was significantly higher in SK-N-AS cells that are of glial sub-lineage. Gal-3 was expressed both in cytoplasm and membrane. On the other hand, Gal-3 expression was relatively lower in less invasive, non-tumorigenic SK-N-SH cells. Silencing of Gal-3 by a small-interfering RNA against Gal-3 (Gal-3-SiRNA) decreased the colony forming potential, in vitro migratory and angiogenic abilities of SK-N-AS cells. Pre-treatment with Gal-3 SiRNA increased the sensitivity of SK-N-AS cells to 4-HPR induced apoptosis. Furthermore, exogenous Gal-3 treatment in neuroblast like Neuro-2a cells resulted in the up-regulation of IL-6 and phosphorylation of the pro-survival molecule AKT. Discussion: Our data suggest that Gal-3 mediates migratory and invasive phenotype of non-MYCN amplified NB derived cells possibly via regulating the IL-6 signaling and Akt phosphorylation. Silencing of Gal-3 may be potentially useful to suppress the resistance of NB cells to apoptosis. Citation Format: Umadevi V. Wesley, Esat Resad, Robert J. Dempsey. Role of Galectin-3 in invasive and apoptotic properties of neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2013-842

Acquisition of neuron‐like electrophysiological properties in neuroblastoma cells by controlled expression of NDM29 ncRNA

Neuroblastoma is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumor nodules. It has been previously shown that the over-expression of a specific non-coding RNA, NDM29, reduces neuroblastoma development promoting cell differentiation. We have used neuroblastoma cells expressing NDM29 at its basal level (Mock cells) or at 5.4-fold higher levels (S1 cells) to investigate whether a functional differentiation correlates with morphological and biochemical development induced by NDM29 expression. First, analyzing the expression of specific markers we demonstrated that NDM29 expression is accompanied by a well coordinated differentiation process toward a neuron-like, rather than toward a glial-like, phenotype. Next, we defined the neuron-like traits of S1 in terms of secretion of cytokines involved in axon guidance, synapse formation and neurite outgrowth. Finally, we characterized the ionic channel apparatus of S1 cells by patch-clamp technique and compared with the Mock counterpart. S1 cells showed much higher levels of fast inactivating Na(+) current and were able to generate mature action potentials. Moreover, they developed expression of functional GABA(A) receptors on their membrane. In contrast, the two cell lines shared very similar pools of functional K(+) channels, although slight quantitative differences can be described. Our results suggest that a maturation occurs in neuroblastoma as a consequence of NDM29 expression, inducing the appearance of neuronal-like properties. In this context, S1 cells may represent a novel in vitro tool for electrophysiological and pharmacological studies of human cells of the neural lineage.

Involvement of the YIGSR sequence of laminin in protein tyrosine phosphorylation

We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR) 3-NH 2 peptide amide is of intermediate affinity (1.5×10 −7 M). Cross-linking experiments with sulfo-MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR) 3-NH 2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.

The cannabinoid receptor: Biochemical and cellular properties in neuroblastoma cells

The cannabinoid receptor that has been pharmacologically characterized for hypothermia, spontaneous activity, analgesia and catalepsy in rodents is the same pharmacological receptor that inhibits adenylate cyclase in vitro. The inhibition of adenylate cyclase by the cannabinoid receptor results from an interaction with G i, based on the biochemical kinetic properties of the response, the sensitivity to pertussis toxin ADP-ribosylation, and the thermodynamic characteristics of the response. From precedents based on studies of the well-characterized G protein coupled receptors, rhodopsin and the β-adrenergic receptor, we can predict the tertiary structure of the cannabinoid receptor. Three sites of potential glycosylation are present on the receptor. However, treatment of N18TG2 neuroblastoma cells with tunicamycin to prevent glycosylation of newly synthesized receptors failed to alter cannabinoid-induced inhibition of cyclic AMP accumulation. The cannabinoid response was rapidly desensitized (within 1 2 h ). Treatment of cells with tunicamycin failed to alter agonist-induced desensitization processes. These findings can be more veraciously interpreted as we gain a better understanding of the cellular dynamics of the cannabinoid receptor.

Identification of renin in cloned neuroblastoma cells and its properties.

The existence of renin(R)-angiotensin system in the brain has long been considered. In this study, we have screened several neuroblastoma cells(NbC) of mouse and rat origin in order to investigate the existence of R. In some NbC of both origins, we could identify intracellular R biochemically and immunohisto-chemically. Some properties of NbC R were also determined.