scholarly journals Effects of hypoxic preconditioning on neuroblastoma tumour oxygenation and metabolic signature in a chick embryo model

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Yousef K. Al-Mutawa ◽  
Anne Herrmann ◽  
Catriona Corbishley ◽  
Paul D. Losty ◽  
Marie Phelan ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Chorioallantoic Membrane ◽  
Hypoxic Preconditioning ◽  
Oxygen Levels ◽  
Tumour Oxygenation ◽  
Chick Embryo Model

Hypoxia episodes and areas in tumours have been associated with metastatic dissemination and poor prognosis. Given the link between tumour tissue oxygen levels and cellular metabolic activity, we hypothesised that the metabolic profile between metastatic and non-metastatic tumours would reveal potential new biomarkers and signalling cues. We have used a previously established chick embryo model for neuroblastoma growth and metastasis, where the metastatic phenotype can be controlled by neuroblastoma cell hypoxic preconditioning (3 days at 1% O2). We measured, with fibre-optic oxygen sensors, the effects of the hypoxic preconditioning on the tumour oxygenation, within tumours formed by SK-N-AS cells on the chorioallantoic membrane (CAM) of chick embryos. We found that the difference between the metastatic and non-metastatic intratumoural oxygen levels was small (0.35% O2), with a mean below 1.5% O2 for most tumours. The metabolomic profiling, using NMR spectroscopy, of neuroblastoma cells cultured in normoxia or hypoxia for 3 days, and of the tumours formed by these cells showed that the effects of hypoxia in vitro did not compare with in vivo tumours. One notable difference was the high levels of the glycolytic end-products triggered by hypoxia in vitro, but not by hypoxia preconditioning in tumours, likely due to the very high basal levels of these metabolites in tumours compared with cells. In conclusion, we have identified high levels of ketones (3-hydroxybutyrate), lactate and phosphocholine in hypoxic preconditioned tumours, all known to fuel tumour growth, and we herein point to the poor relevance of in vitro metabolomic experiments for cancer research.

scholarly journals KDM6B promotes oncogenic CDK4/6-pRB-E2F pathway via maintaining enhancer activation in high-risk neuroblastoma

2020 ◽  
Author(s):  
Alexandra D’Oto ◽  
Jie Fang ◽  
Hongjian Jin ◽  
Beisi Xu ◽  
Shivendra Singh ◽  
...  
Keyword(s):  
High Risk ◽  
Target Genes ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Gene Signature ◽  
Chromatin Accessibility ◽  
Chromatin Interaction ◽  
Pharmacologic Inhibition

ABSTRACTThe H3K27me2/me3 histone demethylase KDM6B is over-expressed in neuroblastoma and essential to neuroblastoma cell survival. While the KDM6B inhibitor, GSK-J4, has shown activity in in vitro and in vivo preclinical models, the mechanism of action remains poorly defined. We demonstrate that genetic and pharmacologic inhibition of KDM6B downregulate the pRB-E2F transcriptome and MYCN expression. Chemical genetics analyses show that a high E2F transcriptome is positively correlated with sensitivity of cancer cells to the KDM6 inhibitor GSK-J4. Mechanistically, inhibition of KDM6B activity reduces the chromatin accessibility of E2F target genes and MYCN. GSK-J4 alters distribution of H3K27me3 and broadly represses the enhancer mark H3K4me1, which may consequently disrupt the long-range chromatin interaction of E2F target genes. KDM6B inhibition phenocopies the transcriptome induced by the specific CDK4/6 inhibitor palbociclib. Overexpression of CDK4/6 or Rb1 knockout not only confers neuroblastoma cell resistance to palbociclib but also to GSK-J4. A gene signature targeted by KDM6B inhibition is associated with poor survival of patients with neuroblastoma regardless of the MYCN status. These data indicate that KDM6B activity promotes an oncogenic CDK4/6-pRB-E2F pathway in neuroblastoma cells via H3K27me3-dependent enhancer-promoter interactions, providing a rationale to target KDM6B for high-risk neuroblastoma.

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

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.

scholarly journals Silencing of AURKA augments the antitumor efficacy of the AURKA inhibitor MLN8237 on neuroblastoma cells

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yan Yang ◽  
Lili Ding ◽  
Qi Zhou ◽  
Li Fen ◽  
Yuhua Cao ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Inhibitory Effect ◽  
Neuroblastoma Cell Line ◽  
Inhibitory Effects ◽  
Rna Transfection ◽  
Interfering Rna

Abstract Background Aurora kinase A (AURKA) has been implicated in the regulation of cell cycle progression, mitosis and a key number of oncogenic signaling pathways in various malignancies including neuroblastoma. Small molecule inhibitors of AURKA have shown potential, but still not as good as expected effects in clinical trials. Little is known about this underlying mechanism. Here, we evaluated the inhibitory effects of AURKA inhibitor MLN8237 on neuroblastoma cells to understand the potential mechanisms responsible for tumor therapy. Methods MLN8237 treatment on neuroblastoma cell line IMR32 was done and in vivo inhibitory effects were investigated using tumor xenograft model. Cellular senescence was evaluated by senescence-associated β-gal Staining assay. Flow cytometry was used to tested cell cycle arrest and cell apoptosis. Senescence-associated signal pathways were detected by western blot. CD133 microbeads and microsphere formation were used to separate and enrich CD133+ cells. AURKA small interfering RNA transfection was carried to downregulate AURKA level. Finally, the combination of MLN8237 treatment with AURKA small interfering RNA transfection were adopted to evaluate the inhibitory effect on neuroblastoma cells. Results We demonstrate that MLN8237, an inhibitor of AURKA, induces the neuroblastoma cell line IMR32 into cellular senescence and G2/M cell phase arrest. Inactivation of AURKA results in MYCN destabilization and inhibits cell growth in vitro and in a mouse model. Although MLN8237 inhibits AURKA kinase activity, it has almost no inhibitory effect on the AURKA protein level. By contrast, MLN8237 treatment leads to abnormal high expression of AURKA in vitro and in vivo. Knockdown of AURKA reduces cell survival. The combination of MLN8237 with AURKA small interfering RNA results in more profound inhibitory effects on neuroblastoma cell growth. Moreover, MLN8237 treatment followed by AURKA siRNA forces senescent cells into apoptosis via suppression of the Akt/Stat3 pathway. Conclusions The effect of AURKA-targeted inhibition of tumor growth plays roles in both the inactivation of AURKA activity and the decrease in the AURKA protein expression level.

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

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.

scholarly journals Magnetic Resonance Imaging for Characterization of a Chick Embryo Model of Cancer Cell Metastases

2018 ◽  
Vol 17 ◽  
pp. 153601211880958 ◽  
Author(s):  
Anne Herrmann ◽  
Arthur Taylor ◽  
Patricia Murray ◽  
Harish Poptani ◽  
Violaine Sée
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Chick Embryo ◽  
Neuroblastoma Cells ◽  
In Vivo Model ◽  
Resonance Imaging ◽  
Human Neuroblastoma ◽  
In Vivo Models ◽  
Chick Embryo Model

Metastasis is the most common cause of death for patients with cancer. To fully understand the steps involved in metastatic dissemination, in vivo models are required, of which murine ones are the most common. Therefore, preclinical imaging methods such as magnetic resonance imaging (MRI) have mainly been developed for small mammals and their potential to monitor cancer growth and metastasis in nonmammalian models is not fully harnessed. We have here used MRI to measure primary neuroblastoma tumor size and metastasis in a chick embryo model. We compared its sensitivity and accuracy to end-point fluorescence detection upon dissection. Human neuroblastoma cells labeled with green fluorescent protein (GFP) and micron-sized iron particles were implanted on the extraembryonic chorioallantoic membrane of the chick at E7. T2 RARE, T2-weighted fast low angle shot (FLASH) as well as time-of-flight MR angiography imaging were applied at E14. Micron-sized iron particle labeling of neuroblastoma cells allowed in ovo observation of the primary tumor and tumor volume measurement noninvasively. Moreover, T2 weighted and FLASH imaging permitted the detection of small metastatic deposits in the chick embryo, thereby reinforcing the potential of this convenient, 3R compliant, in vivo model for cancer research.

scholarly journals Ginsenoside Compound K Induces Ros-Mediated Apoptosis and Autophagic Inhibition in Human Neuroblastoma Cells In Vitro and In Vivo

2019 ◽  
Vol 20 (17) ◽  
pp. 4279 ◽  
Author(s):  
Jung-Mi Oh ◽  
Eunhee Kim ◽  
Sungkun Chun
Keyword(s):  
Cell Death ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Autophagic Flux ◽  
Ros Scavenging ◽  
Compound K ◽  
Human Neuroblastoma ◽  
Ginsenoside Compound K

Autophagy can result in cellular adaptation, as well as cell survival or cell death. Modulation of autophagy is increasingly regarded as a promising cancer therapeutic approach. Ginsenoside compound K (CK), an active metabolite of ginsenosides isolated from Panax ginseng C.A. Meyer, has been identified to inhibit growth of cancer cell lines. However, the molecular mechanisms of CK effects on autophagy and neuroblastoma cell death have not yet been investigated. In the present study, CK inhibited neuroblastoma cell proliferation in vitro and in vivo. Treatment by CK also induced the accumulation of sub-G1 population, and caspase-dependent apoptosis in neuroblastoma cells. In addition, CK promotes autophagosome accumulation by inducing early-stage autophagy but inhibits autophagic flux by blocking of autophagosome and lysosome fusion, the step of late-stage autophagy. This effect of CK appears to be mediated through the induction of intracellular reactive oxygen species (ROS) and mitochondria membrane potential loss. Moreover, chloroquine, an autophagy flux inhibitor, further promoted CK-induced apoptosis, mitochondrial ROS induction, and mitochondria damage. Interestingly, those promoted phenomena were rescued by co-treatment with a ROS scavenging agent and an autophagy inducer. Taken together, our findings suggest that ginsenoside CK induced ROS-mediated apoptosis and autophagic flux inhibition, and the combination of CK with chloroquine, a pharmacological inhibitor of autophagy, may be a novel therapeutic potential for the treatment of neuroblastoma.

scholarly journals Ring-Fused Diphenylchlorins as Potent Photosensitizers for Photodynamic Therapy Applications: In Vitro Tumor Cell Biology and in Vivo Chick Embryo Chorioallantoic Membrane Studies

ACS Omega ◽  
2019 ◽  
Vol 4 (17) ◽  
pp. 17244-17250 ◽  
Author(s):  
Bruno F. O. Nascimento ◽  
Mafalda Laranjo ◽  
Nelson A. M. Pereira ◽  
João Dias-Ferreira ◽  
Marta Piñeiro ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Chick Embryo ◽  
Tumor Cell ◽  
Cell Biology ◽  
Chorioallantoic Membrane ◽  
Tumor Cell Biology ◽  
Chick Embryo Chorioallantoic Membrane

scholarly journals The Potential Use of Electrochemotherapy in the Treatment of Uveal Melanoma: In Vitro Results in 3D Tumor Cultures and In Vivo Results in a Chick Embryo Model

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1344 ◽  
Author(s):  
Miltiadis Fiorentzis ◽  
Arne Viestenz ◽  
Udo Siebolts ◽  
Berthold Seitz ◽  
Sarah E. Coupland ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Uveal Melanoma ◽  
Chorioallantoic Membrane ◽  
In Vivo Model ◽  
Metastatic Lesions ◽  
Diphenyltetrazolium Bromide ◽  
Chick Embryo Chorioallantoic Membrane ◽  
Tumors Of The Liver

Uveal melanoma (UM) is the most common primary intraocular tumor that arises from neoplastic melanocytes in the choroid, iris, and ciliary body. Electrochemotherapy (ECT) has been successfully established for the treatment of skin and soft tissue metastatic lesions, deep-seated tumors of the liver, bone metastases, and unresectable pancreas lesions. The aim of this study was to evaluate the effect of ECT in vitro in 3D spheroid culture systems in primary and metastatic UM cell lines. We also investigated the chick embryo chorioallantoic membrane (CAM) as an in vivo model system for the growth and treatment of UM tumors using ECT. The cytotoxic effect of ECT in 3D spheroids was analyzed seven days following treatment by assessment of the size and MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction] assay. The cytotoxicity of ECT after intratumoral or intraarterial administration was evaluated histologically. In vitro and in vivo ECT caused a significant reduction in tumor size and viability compared to electroporation or chemotherapy in both sections of our study. The current results underline the effectiveness of ECT in the treatment of UM and prepare the way for further investigation of its potential application in UM.

scholarly journals Stathmin levels alter PTPN14 expression and impact neuroblastoma cell migration

2019 ◽  
Vol 122 (3) ◽  
pp. 434-444 ◽  
Author(s):  
Sela T. Po’uha ◽  
Marion Le Grand ◽  
Miriam B. Brandl ◽  
Andrew J. Gifford ◽  
Gregory J. Goodall ◽  
...  
Keyword(s):  
Cell Migration ◽  
Neuroblastoma Cell ◽  
Gene Array ◽  
Neuroblastoma Cells ◽  
Migration And Invasion ◽  
Invasion Pathways ◽  
Cell Migration And Invasion ◽  
Regulatory Loop

Abstract Background Stathmin mediates cell migration and invasion in vitro, and metastasis in vivo. To investigate stathmin’s role on the metastatic process, we performed integrated mRNA–miRNA expression analysis to identify pathways regulated by stathmin. Methods MiRNA and gene arrays followed by miRNA-target-gene integration were performed on stathmin-depleted neuroblastoma cells (CtrlshRNA vs. Stmn Seq2shRNA). The expression of the predicted target PTPN14 was evaluated by RT-qPCR, western blot and immunohistochemistry. Gene-silencing technology was used to assess the role of PTPN14 on proliferation, migration, invasion and signalling pathway. Results Stathmin levels modulated the expression of genes and miRNA in neuroblastoma cells, leading to a deregulation of migration and invasion pathways. Consistent with gene array data, PTPN14 mRNA and protein expression were downregulated in stathmin- depleted neuroblastoma cells and xenografts. In two independent neuroblastoma cells, suppression of PTPN14 expression led to an increase in cell migration and invasion. PTPN14 and stathmin expression did not act in a feedback regulatory loop in PTPN14- depleted cells, suggesting a complex interplay of signalling pathways. The effect of PTPN14 on YAP pathway activation was cell-type dependent. Conclusions Our findings demonstrate that stathmin levels can regulate PTPN14 expression, which can modulate neuroblastoma cell migration and invasion.

scholarly journals Down-Regulation of Phosphoribosyl Pyrophosphate Synthetase 1 Inhibits Neuroblastoma Cell Proliferation

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 955
Author(s):  
Jifu Li ◽  
Junhong Ye ◽  
Shunqin Zhu ◽  
Hongjuan Cui
Keyword(s):  
Cell Proliferation ◽  
De Novo ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Phosphoribosyl Pyrophosphate ◽  
Down Regulation ◽  
Nucleotide Synthesis ◽  
Phosphoribosyl Pyrophosphate Synthetase

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

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