EZH2 inhibition decreases neuroblastoma proliferation and in vivo tumor growth

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted.

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Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

Medical Research ◽

10.6913/mr.0304.03 ◽

2021 ◽

Vol 3 (4) ◽

pp. 12-24

Author(s):

Mabao YUAN ◽

Hanjiao HANG ◽

Lubin YAN ◽

Xuanjie HUANG ◽

Ziyang SANG ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Luciferase Reporter ◽

Ubiquitin Protein Ligase ◽

Neuroblastoma Cell Lines ◽

Dual Luciferase Reporter Assay

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

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GX15–070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma

BMC Cancer ◽

10.1186/s12885-019-6195-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 6

Author(s):

Sonia Cournoyer ◽

Anissa Addioui ◽

Assila Belounis ◽

Mona Beaunoyer ◽

Carine Nyalendo ◽

...

Keyword(s):

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Distant Metastases ◽

Caspase Inhibitor ◽

Pediatric Tumor ◽

Apoptotic Protein ◽

Synergistic Cytotoxicity ◽

Neuroblastoma Cell Lines

Abstract Background Neuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor. Methods Five neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ. Results Synergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases. Conclusions Based on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma.

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The role of c-Met inhibition for neuroblastoma treatment.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.10041 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 10041-10041

Author(s):

Peter Zage ◽

Kathy Scorsone ◽

Linna Zhang

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Tumor Growth ◽

Tumor Cells ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Neuroblastoma Tumor ◽

Met Phosphorylation

10041 Background: Neuroblastoma is the most common extra-cranial solid tumor of childhood. Many children present with high-risk disease characterized by rapid tumor growth, resistance to chemotherapy, and widespread metastasis, and novel therapies are needed. Previous studies have identified a role for the HGF/c-Met pathway in the pathogenesis of neuroblastoma. We hypothesized that EMD1214063 would be effective against neuroblastoma tumor cells and tumors in preclinical models via inhibition of HGF/c-Met signaling. Methods: We determined the expression of c-Met in a panel of neuroblastoma tumor cells and neuroblastoma cell viability after treatment with EMD1214063 using MTT assays. Analyses were performed for changes in cell morphology, cell cycle progression, and cell death via apoptosis after EMD1214063 treatment. To investigate the efficacy of EMD1214063 against neuroblastoma tumors in vivo, neuroblastoma cells were injected orthotopically into immunocompromised mice, and the mice in which tumors developed were treated with oral EMD1214063. Results: All neuroblastoma cell lines were sensitive to EMD1214063, and IC50 values ranged from 2.4 - 8.5 mcM. EMD1214063 treatment inhibited HGF-mediated c-Met phosphorylation in neuroblastoma cells. EMD1214063 induced cell cycle arrest in neuroblastoma tumor cells with high c-Met expression, and induced apoptosis in all tested cell lines. In mice with neuroblastoma xenograft tumors, EMD1214063 inhibited tumor growth. Conclusions: Treatment of neuroblastoma tumor cells with EMD1214063 inhibits HGF-induced c-Met phosphorylation and results in cell death. Furthermore, EMD1214063 induces cell cycle arrrest prior to cell death in neuroblastoma tumor cells with high c-Met expression. EMD1214063 treatment is effective in reducing tumor growth in vivo in mice. Inhibition of c-Met represents a potential new therapeutic strategy for neuroblastoma, and further preclinical studies of EMD1214063 are warranted.

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ALT neuroblastoma chemoresistance due to telomere dysfunction–induced ATM activation is reversible with ATM inhibitor AZD0156

Science Translational Medicine ◽

10.1126/scitranslmed.abd5750 ◽

2021 ◽

Vol 13 (607) ◽

pp. eabd5750

Author(s):

Balakrishna Koneru ◽

Ahsan Farooqi ◽

Thinh H. Nguyen ◽

Wan Hsi Chen ◽

Ashly Hindle ◽

...

Keyword(s):

Cell Lines ◽

Ataxia Telangiectasia ◽

De Novo ◽

Clinical Stage ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Telomere Dysfunction ◽

Neuroblastoma Cell Lines

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

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

Oncotarget ◽

10.18632/oncotarget.17879 ◽

2017 ◽

Vol 8 (43) ◽

pp. 73905-73924 ◽

Cited By ~ 12

Author(s):

Bhaskar Saha ◽

Birija Sankar Patro ◽

Mrunesh Koli ◽

Ganesh Pai ◽

Jharna Ray ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Human Neuroblastoma ◽

Neuroblastoma Tumor ◽

Neuroblastoma Cell Lines

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Preclinical Evaluation of Antitumoral and Cytotoxic Properties of Viscum album Fraxini Extract on Pediatric Tumor Cells

Planta Medica ◽

10.1055/a-1013-0382 ◽

2019 ◽

Vol 85 (14/15) ◽

pp. 1150-1159 ◽

Cited By ~ 1

Author(s):

Katrin Menke ◽

Melanie Schwermer ◽

Alexander Schramm ◽

Tycho J. Zuzak

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Migration And Invasion ◽

Tumor Cell Lines ◽

Pediatric Tumor ◽

Mistletoe Extract ◽

Mitochondrial Integrity ◽

Neuroblastoma Cell Lines

AbstractIn Europe, especially in German-speaking countries, administration of mistletoe extracts is the most common and popular complementary and alternative therapy approach reported in oncology. Mistletoe therapy is applied to children with cancer for curative and palliative therapeutic regimes with increasing frequency, but at the same time, there are only a few studies on the effectiveness of this therapy. Therefore, we have investigated the response of various pediatric cell lines (acute myeloid leukemia, Ewingʼs sarcoma, hepatocellular carcinoma, medulloblastoma, neuroblastoma, and osteosarcoma) to mistletoe extract, abnobaVISCUM Fraxini. Effects on cell proliferation, cell cycle distribution as well as on mitochondrial integrity and caspase-mediated apoptosis were investigated in neuroblastoma cell lines, SH-SY5Y and Kelly. Additionally, in vitro tumor cell migration and invasion were studied. In vivo effects of the mistletoe extract were investigated in a syngeneic neuroblastoma mouse model. We could show that tumor cell lines were from 5- to 640-fold more sensitive to abnobaVISCUM Fraxini treatment than non-tumorigenic fibroblasts, whereby neuroblastoma cell lines were the most sensitive. For two neuroblastoma cell lines, SH-SY5Y and Kelly, induction of caspase-9-mediated apoptosis, a decrease of mitochondrial integrity as well as attenuation of migration and invasion were observed. In vivo experiments revealed a reduction of tumor growth and a prolonged survival of tumor-bearing animals. In summary, we can state that these results provide the first preclinical data for cytotoxic activities of abnobaVISCUM Fraxini for a broad panel of pediatric tumor cell lines, in particular, neuroblastoma cells. Thus, it might be a potential remedy for the supportive treatment of neuroblastoma.

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Specific Inhibition of GSK-3β by Tideglusib: Potential Therapeutic Target for Neuroblastoma Cancer Stem Cells

10.1101/2020.02.18.953596 ◽

2020 ◽

Author(s):

Hisham F. Bahmad ◽

Reda M. Chalhoub ◽

Hayat Harati ◽

Jolie Bou-Gharios ◽

Farah Ballout ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Human Neuroblastoma ◽

And Migration ◽

Gsk 3Β ◽

Neuroblastoma Cell Lines

AbstractNeuroblastoma is an embryonic tumor that represents the most common extracranial solid tumor in children. Resistance to therapy is attributed, in part, to the persistence of a subpopulation of slowly dividing cancer stem cells (CSCs) within those tumors. Glycogen synthase kinase (GSK)-3β is an active proline-directed serine/threonine kinase, well-known to be involved in different signaling pathways entangled in the pathophysiology of neuroblastoma. This study aims to assess the potency of an irreversible GSK-3β inhibitor drug, Tideglusib (TDG), in suppressing proliferation, viability, and migration of human neuroblastoma cell lines, as well as its effects on their CSCs subpopulation in vitro and in vivo. Our results showed that treatment with TDG significantly reduced cell proliferation, viability, and migration of SK-N-SH and SH-SY5Y cells. TDG also significantly inhibited neurospheres formation capability in both cell lines, eradicating the self-renewal ability of highly resistant CSCs. Importantly, TDG potently inhibited neuroblastoma tumor growth and progression in vivo. In conclusion, TDG proved to be an effective in vitro and in vivo treatment for neuroblastoma cell lines and may hence serve as a potential adjuvant therapeutic agent for this aggressive nervous system tumor.

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Analysis of Asymmetric Cell Division Using Human Neuroblastoma Cell Lines as a Model System

Symmetry ◽

10.3390/sym13101907 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1907

Author(s):

Hideki Izumi ◽

Yasuhiko Kaneko ◽

Akira Nakagawara

Keyword(s):

Cell Division ◽

Cell Lines ◽

Asymmetric Cell Division ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Model System ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Neuroblastoma Cell Lines

Neuroblastoma is one of the most common childhood solid tumors and develops from neural stem cells that normally comprise the embryonic structure termed the neural crest. Human neuroblastoma cell lines have special properties as they exhibit cell growth and are induced to become mature neurons by drugs such as retinoid. Therefore, we examined asymmetric cell division (ACD) using human neuroblastoma cells as an ACD model, and confirmed that ACD in human cancer cells is evolutionally conserved. Furthermore, we demonstrated that MYCN is involved in cell division fate. We introduce the brief history of ACD study using neuroblastoma cell lines and discuss why human neuroblastoma cells are an ideal model system for clarifying the mechanism of ACD.

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OS5 - 173 Inhibition of eEF2K as a Novel Therapeutic Strategy in Neuroblastoma and Medulloblastoma

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2016.336 ◽

2016 ◽

Vol 43 (S4) ◽

pp. S3-S3

Author(s):

A Delaidelli ◽

D Khan ◽

G Leprivier ◽

SM Pfister ◽

MD Taylor ◽

...

Keyword(s):

Cell Lines ◽

Acute Stress ◽

Neuroblastoma Cell ◽

Survival Rates ◽

Neuroblastoma Cells ◽

Elongation Factor ◽

Metabolic Stress ◽

Genomic Analysis ◽

Human Neuroblastoma ◽

Neuroblastoma Cell Lines

Medulloblastoma and neuroblastoma are aggressive solid pediatric tumors, with 5 year survival rates lower than 50-60%. In addition, more than 80% of the survivors develop permanent neurological impairments. Hence, there is a dire need to identify and validate novel, more effective and less toxic therapeutic approaches. Tumors are continually exposed to acute changes in the micro-environment, including nutrient availability. We previously showed that eukaryotic Elongation Factor-2 Kinase (eEF2K) is a critical regulator of cellular adaptation to acute metabolic stress. Based on those findings, we hypothesize that eEF2K is a marker of outcome and mediates medulloblastoma and neuroblastoma adaptation to acute stress. METHODS – Proprietary gene expression datasets (for medulloblastoma) and the R2 genomic analysis platform (for neuroblastoma) were analyzed for links between eEF2K expression and outcome. Effects of eEF2K knockdown on cell survival were evaluated in BE(2)C neuroblastoma cells. Immunoblotting and immunohistochemistry were performed on neuroblastoma cell lines and tissue microarrays (TMAs) for key molecules in the pathway. Similar studies are underway in medulloblastoma cell lines and TMAs. RESULTS - Low eEF2K mRNA expression is predictive of improved survival in medulloblastoma and neuroblastoma. Low p-eEF2 protein expression, indicative of low eEF2K activity, improves survival in human neuroblastoma. Neuroblastoma cell lines with eEF2K knockdown are more sensitive than controls to nutrient deprivation. CONCLUSIONS - eEF2K may represent a critical mechanism for adaptation to acute metabolic stress in neuroblastoma and medulloblastoma, and is therefore a promising therapeutic target. We are currently exploring the pharmacological inhibition of eEF2K in xenograft tumor models.

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Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/7568287 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Yaoli Pu Yang ◽

Simeng Wang ◽

Xingguo Li ◽

Nina F. Schor

Keyword(s):

Cell Line ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Neurotrophin Receptor ◽

Human Neuroblastoma Cell ◽

P75 Neurotrophin Receptor ◽

Human Neuroblastoma ◽

Neuroblastoma Cell Lines

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinidein vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

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