In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.

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Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models

eLife ◽

10.7554/elife.17137 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 14

Author(s):

Hui Qin Wang ◽

Ensar Halilovic ◽

Xiaoyan Li ◽

Jinsheng Liang ◽

Yichen Cao ◽

...

Keyword(s):

Antitumor Activity ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Human Neuroblastoma Cell ◽

Wild Type ◽

Combination Strategy ◽

Human Neuroblastoma ◽

Xenograft Models ◽

Neuroblastoma Cell Lines

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

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The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

International Journal of Molecular Sciences ◽

10.3390/ijms22126565 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6565

Author(s):

Jennifer H. Foster ◽

Eveline Barbieri ◽

Linna Zhang ◽

Kathleen A. Scorsone ◽

Myrthala Moreno-Smith ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Wild Type ◽

Tumor Weight ◽

Cycle Arrest ◽

P53 Status ◽

Neuroblastoma Cell Lines ◽

Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

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Activation of sonic hedgehog signaling pathway in S-type neuroblastoma cell lines

Journal of Huazhong University of Science and Technology [Medical Sciences] ◽

10.1007/s11596-010-0342-7 ◽

2010 ◽

Vol 30 (3) ◽

pp. 271-277 ◽

Cited By ~ 2

Author(s):

Yunan Zhou ◽

Ruolian Dai ◽

Ling Mao ◽

Yuanpeng Xia ◽

Yufang Yao ◽

...

Keyword(s):

Signaling Pathway ◽

Cell Lines ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Neuroblastoma Cell ◽

Hedgehog Signaling Pathway ◽

Sonic Hedgehog Signaling ◽

Sonic Hedgehog Signaling Pathway ◽

Neuroblastoma Cell Lines

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Purification, activity, and expression levels of two uridine-cytidine kinase isoforms in neuroblastoma cell lines

Nucleosides Nucleotides & Nucleic Acids ◽

10.1080/15257770.2015.1124998 ◽

2016 ◽

Vol 35 (10-12) ◽

pp. 613-618 ◽

Cited By ~ 2

Author(s):

R. Meinsma ◽

A.B.P. van Kuilenburg

Keyword(s):

Cell Lines ◽

Neuroblastoma Cell ◽

Expression Levels ◽

Neuroblastoma Cell Lines

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A Systematic Analysis Revealed the Potential Gene Regulatory Processes of ATRA-Triggered Neuroblastoma Differentiation and Identified a Novel RA Response Sequence in the NTRK2 Gene

BioMed Research International ◽

10.1155/2020/6734048 ◽

2020 ◽

Vol 2020 ◽

pp. 1-19

Author(s):

Liyuan Guo ◽

Wei Lin ◽

Yidan Zhang ◽

Jing Wang

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Western Blotting ◽

Chromatin Immunoprecipitation ◽

Neuroblastoma Cell ◽

Specific Gene ◽

A Cells ◽

Functional Genomic Analysis ◽

Gene Regulatory ◽

Neuroblastoma Cell Lines

Retinoic acid- (RA-) triggered neuroblastoma cell lines are widely used cell modules of neuronal differentiation in neurodegenerative disease studies, but the gene regulatory mechanism underlying differentiation is unclear now. In this study, system biological analysis was performed on public microarray data from three neuroblastoma cell lines (SK-N-SH, SH-SY5Y-A, and SH-SY5Y-E) to explore the potential molecular processes of all-trans retinoic acid- (ATRA-) triggered differentiation. RT-qPCR, functional genomics analysis, western blotting, chromatin immunoprecipitation (ChIP), and homologous sequence analysis were further performed to validate the gene regulation processes and identify the RA response element in a specific gene. The potential disturbed biological pathways (111 functional GO terms in 14 interactive functional groups) and gene regulatory network (10 regulators and 71 regulated genes) in neuroblastoma differentiation were obtained. 15 of the 71 regulated genes are neuronal projection-related. Among them, NTRK2 is the only one that was dramatically upregulated in the RT-qPCR test that we performed on ATRA-treated SH-SY5Y-A cells. We further found that the overexpression of the NTRK2 gene can trigger differentiation-like changes in SH-SY5Y-A cells. Functional genomic analysis and western blotting assay suggested that, in neuroblastoma cells, ATRA may directly regulate the NTRK2 gene by activating the RA receptor (RAR) that binds in its promoter region. A novel RA response DNA element in the NTRK2 gene was then identified by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. The novel element is sequence conservation and position variation among different species. Our study systematically provided the potential regulatory information of ATRA-triggered neuroblastoma differentiation, and in the NTRK2 gene, we identified a novel RA response DNA element, which may contribute to the differentiation in a human-specific manner.

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A Context-Dependent Role for MiR-124-3p on Cell Phenotype, Viability and Chemosensitivity in Neuroblastoma in vitro

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.559553 ◽

2020 ◽

Vol 8 ◽

Author(s):

John C. Nolan ◽

Manuela Salvucci ◽

Steven Carberry ◽

Ana Barat ◽

Miguel F. Segura ◽

...

Keyword(s):

Cell Lines ◽

Epithelial To Mesenchymal Transition ◽

Neuroblastoma Cell ◽

Chemotherapy Resistance ◽

Cellular Transformation ◽

Cell Phenotype ◽

Drug Resistant ◽

Mesenchymal Transition ◽

Neuroblastoma Cell Lines

Neuroblastoma (NB) is a neural crest-derived tumor, which develops before birth or in early childhood, with metastatic dissemination typically preceding diagnosis. Tumors are characterized by a highly heterogeneous combination of cellular phenotypes demonstrating varying degrees of differentiation along different lineage pathways, and possessing distinct super-enhancers and core regulatory circuits, thereby leading to highly varied malignant potential and divergent clinical outcomes. Cytoskeletal reorganization is fundamental to cellular transformations, including the processes of cellular differentiation and epithelial to mesenchymal transition (EMT), previously reported by our lab and others to coincide with chemotherapy resistance and enhanced metastatic ability of tumor cells. This study set out to investigate the ability of the neuronal miR-124-3p to reverse the cellular transformation associated with drug resistance development and assess the anti-oncogenic role of this miRNA in in vitro models of drug-resistant adrenergic (ADRN) and mesenchymal (MES) neuroblastoma cell lines. Low expression of miR-124-3p in a cohort of neuroblastomas was significantly associated with poor overall and progression-free patient survival. Over-expression of miR-124-3p in vitro inhibited cell viability through the promotion of cell cycle arrest and induction of apoptosis in addition to sensitizing drug-resistant cells to chemotherapeutics in a panel of morphologically distinct neuroblastoma cell lines. Finally, we describe miR-124-3p direct targeting and repression of key up-regulated cytoskeletal genes including MYH9, ACTN4 and PLEC and the reversal of the resistance-associated EMT and enhanced invasive capacity previously reported in our in vitro model (SK-N-ASCis24).

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