Response Of Neuroblastoma Cells Research Articles

Integrated re-analysis of transcriptomic and proteomic datasets reveals potential mechanisms for Zika viral-based oncolytic therapy in neuroblastoma

Background Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) can access the nervous system and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. Methods Here, we perform extensive data mining, integration and re-analysis of ZIKV infection datasets to highlight molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We collate infection data of multiple neuroblastoma cell lines by different ZIKV strains from a body of published literature to inform the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating published transcriptomics, interaction proteomics, dependency factor and compound datasets we propose the involvement of multiple host systems during ZIKV infection. Results Through data mining of published literature, we observed most paediatric neuroblastoma cell lines to be highly susceptible to ZIKV infection and propose the PRVABC59 ZIKV strain to be the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV infection is dependent on sterol regulatory element binding protein (SREBP)-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV non-structural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. Conclusions Our work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.

Silencing of the PHLDA1 leads to global proteome changes and differentiation pathways of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.

A Novel Druggable Dual-Specificity tYrosine-Regulated Kinase3/Calmodulin Kinase-like Vesicle-Associated Signaling Module with Therapeutic Implications in Neuroblastoma.

High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca2+/calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer.

Integrated re-analysis of transcriptomic and proteomic datasets reveals potential mechanisms for Zika viral-based oncolytic therapy in neuroblastoma

Background Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) can access the nervous system and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. Methods Here, we perform extensive data mining, integration and re-analysis of ZIKV infection datasets to highlight molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We collate infection data of multiple neuroblastoma cell lines by different ZIKV strains from a body of published literature to inform the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating published transcriptomics, interaction proteomics, dependency factor and compound datasets we propose the involvement of multiple host systems during ZIKV infection. Results Through data mining of published literature, we observed most paediatric neuroblastoma cell lines to be highly susceptible to ZIKV infection and propose the PRVABC59 ZIKV strain to be the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV infection is dependent on sterol regulatory element binding protein (SREBP)-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV non-structural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. Conclusions Our work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.

UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL polyubiquitination and enhanced neuroblastoma apoptosis.

Expression of the UBE4B ubiquitin ligase is strongly associated with neuroblastoma patient outcomes, but the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We evaluated interactions of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression on Ku70 and c-FLIPL ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in apoptosis induced by histone deacetylase (HDAC) inhibition using Western blots. UBE4B binding to ITCH was mediated by WW domains in the ITCH protein. ITCH activation led to ITCH-UBE4B complex formation and recruitment of Ku70 and c-FLIPL via ITCH WW domains, followed by Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination and proteasomal degradation. HDAC inhibition induced Ku70 acetylation, leading to release of c-FLIPL and Bax from Ku70, increased Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination via the ITCH-UBE4B complex, and induction of apoptosis. UBE4B depletion led to reduced polyubiquitination and increased levels of Ku70 and c-FLIPL and to reduced apoptosis induced by HDAC inhibition via stabilization of c-FLIPL and Ku70 and inhibition of caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role for the ITCH-UBE4B complex in responses of neuroblastoma cells to HDAC inhibition, suggesting that the ITCH-UBE4B complex plays a critical role in responses of neuroblastoma to therapy and identifying a potential mechanism underlying the association of UBE4B expression with neuroblastoma patient outcomes.

Integrative transcriptomic and proteomic study of Zika viral infection reveals potential mechanisms for oncolytic therapy in neuroblastoma

Background: Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) is neurotropic and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. Methods: Here, we perform extensive analysis of ZIKV infection studies to identify molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We summarise the neuroblastoma cell lines and ZIKV strains utilised and re-evaluate the infection data to deduce the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating transcriptomics, interaction proteomics, dependency factor and compound datasets we show the involvement of multiple host systems during ZIKV infection. Results: We identified that most paediatric neuroblastoma cell lines are highly susceptible to ZIKV infection and that the PRVABC59 ZIKV strain is the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV is dependent on sterol regulatory element binding protein (SREBP)-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV non-structural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. Conclusions: Our work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.

The Involvement of Hypoxia in the Response of Neuroblastoma Cells to the Exposure of Atorvastatin

Cancer is a set of complex diseases, being one of the leading causes of death worldwide. Despite a lot of research on the molecular pathways and effective treatments, there are still huge gaps. Indeed, the development of new anti-cancer drugs is a complex process. To face this problem, drug repurposing is being increasingly applied. This approach aims to identify new indications for already approved drugs. In this regard, statins (clinically used for reducing cholesterol levels) are reported to induce anti-cancer effects, particularly by inducing apoptosis and altering the tumor microenvironment. Atorvastatin is a type of statin with several potentialities as an anti-cancer agent, supported by several studies. Our study aimed to explore the effect of this drug in SH-SY5Y human neuroblastoma cells. Additionally, we also aimed to understand how this drug acts under hypoxia and the inhibition of hypoxia-inducible factor-1 (HIF-1). For that purpose, we assessed cellular viability/morphology after exposure to different concentrations of atorvastatin, with or without chemically induced hypoxia with chloride cobalt (CoCl2) and with or without echinomycin (HIF-1α inhibitor). Our results supported the cytotoxic effects of atorvastatin. Additionally, we also revealed that besides these effects, under hypoxia, this drug induced proliferation of the neuroblastoma cells, supporting the importance of different stimuli and environment on the effect of drugs on cancer cells.

Transcriptional landscape of human neuroblastoma cells in response to SARS-CoV-2

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and the neurological symptoms of SARS-CoV-2 infection have already been reported. However, the mechanisms underlying the effect of SARS-CoV-2 infection on patients with central nervous system injuries remain unclear.MethodsThe high-throughput RNA sequencing was applied to analyze the transcriptomic changes in SK-N-SH cells after SARS-CoV-2 infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the functions of differentially expressed genes and related pathways.ResultsA total of 820 mRNAs were significantly altered, including 671 upregulated and 149 downregulated mRNAs (showing an increase of ≥ 2-fold or decrease to ≤ 0.5-fold, respectively; p ≤ 0.05). Moreover, we verified the significant induction of cytokines, chemokines, and their receptors, as well as the activation of NF-κB, p38, and Akt signaling pathways, in SK-N-SH by SARS-CoV-2.ConclusionsTo our knowledge, this is the first time the transcriptional profiles of the host mRNAs involved in SARS-CoV-2 infection of SK-N-SH cells have been reported. These findings provide novel insight into the pathogenic mechanism of SARS-CoV-2 and might constitute a new approach for future prevention and treatment of SARS-CoV-2-induced central nervous system infection.

Abstract 2957: Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma

Abstract Controlling epigenetic factors such as histone methyltransferases and demethylases has recently emerged as an attractive therapeutic approach for different cancers. KMT2 family protein KMT2A or MLL1 is a H3K4 methyltransferase, which is known to play an essential role in cancer development. MLL1 forms a COMPASS complex to function as an epigenetic regulator and Menin is one of the specific and required binding partner for the activation and function of this epigenetic enzyme. Menin acts as a tumor suppressor protein, and inhibition of Menin-MLL1 interaction has been shown to destabilize the MLL1 epigenetic complex, inhibits H3K4 methyltransferase activity, and hinder the oncogenic potential of various cancers. High-risk neuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for almost 15% of all pediatric cancer-related deaths. Recent reports indicate that aberrant activation of epigenetic regulators may drive NB progression and relapse. Therefore, in the present study, we used a specific small-molecule inhibitor, MI-503 to disrupt Menin-MLL1 interaction, and to further understand the role of Menin in pediatric NB. We used different NB cell lines including MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) and MYCN-amplified (NGP, LAN-5, IMR-32), and performed cytotoxicity and Colonogenic assays to determine the effect of Menin inhibition on NB proliferation. Results showed that MI-503 significantly inhibits NB proliferation and colony formation capacity in a dose-dependent manner in contrast to controls. Further, we performed apoptosis and cell cycle assays and observed that MI-503 significantly induces apoptosis as detected by the increase of Annexin-V positive early apoptotic cells, and blocks cell cycle progression at the S phase in all NB cell lines tested. Additionally, Western blot assays demonstrated an overall inhibition of H3K3me3 levels in NB cells in response to MI-503. To further determine the effects of Menin inhibition on NB tumor growth, we developed a NB 3D spheroid tumor model that recapitulate in vivo tumor growth for solid tumors. Treatment with MI-503 significantly inhibits spheroidal tumor growth by inducing tumor cell death in a dose-dependent manner. Overall, these data highlight the role of epigenetic regulation, MLL1 complex, and of Menin in NB growth and tumorigenicity. In our future efforts, we will further elucidate the effects of MI-503 and the role of Menin in NB by using in vivo tumor models. Epigenetic inhibitor such as MI-503 holds significant potential for further clinical development and as a novel therapeutic approach for NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2957.

Abstract 3702: UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL K63/K48 branched polyubiquitination and enhanced neuroblastoma HDACi-mediated apoptosis

Abstract Expression of the UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes, but the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We evaluated interactions of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression on Ku70, c-FLIPL, and p53 ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in apoptosis induced by HDAC inhibition in our neuroblastoma cell lines model using Western blots. UBE4B was found in a complex with ITCH, with binding mediated by WW domains in the ITCH protein. ITCH activation led to ITCH-UBE4B E3-E4 ubiquitin ligase complex formation and recruitment of Ku70 and c-FLIPL via ITCH WW domains, followed by Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination and proteasomal degradation. Histone deacetylase (HDAC) inhibition induced Ku70 and c-FLIPL acetylation, leading to release of c-FLIPL and Bax from Ku70, increased Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination via the ITCH-UBE4B complex, and induction of apoptosis. UBE4B depletion in our neuroblastoma cell lines model led to reduced polyubiquitination and increased levels of Ku70 and c-FLIPL proteins leading to the massive reduction of apoptosis induced by HDAC inhibition via stabilization of Ku70 and c-FLIPL proteins allowing the inhibition of the full caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role of the ITCH-UBE4B complex in responses of neuroblastoma cells to HDAC inhibition, suggesting that the ITCH-UBE4B complex plays a critical role in responses of neuroblastoma to therapy and suggesting a potential mechanism underlying the association of UBE4B expression with neuroblastoma patient outcomes. Citation Format: Christophe Le Clorennec, Divya Subramonian, Yuchen Huo, Peter E. Zage. UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL K63/K48 branched polyubiquitination and enhanced neuroblastoma HDACi-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3702.

In Vitro Neurotoxicity of Flumethrin Pyrethroid on SH-SY5Y Neuroblastoma Cells: Apoptosis Associated with Oxidative Stress.

Pyrethroids are neurotoxicants for animals, showing a pattern of toxic action on the nervous system. Flumethrin, a synthetic pyrethroid, is used against ectoparasites in domestic animals, plants, and for public health. This compound has been shown to be highly toxic to bees, while its effects on other animals have been less investigated. However, in vitro studies to evaluate cytotoxicity are scarce, and the mechanisms associated with this effect at the molecular level are still unknown. This study aimed to investigate the oxidative stress and cell death induction in SH-SY5Y neuroblastoma cells in response to flumethrin exposure (1–1000 µM). Flumethrin induced a significant cytotoxic effect, as evaluated by MTT and LDH leakage assays, and produced an increase in the biomarkers of oxidative stress as reactive oxygen species and nitric oxide (ROS and NO) generation, malondialdehyde (MDA) concentration, and caspase-3 activity. In addition, flumethrin significantly increased apoptosis-related gene expressions (Bax, Casp-3, BNIP3, APAF1, and AKT1) and oxidative stress and antioxidative (NFκB and SOD2) mediators. The results demonstrated, by biochemical and gene expression assays, that flumethrin induces oxidative stress and apoptosis, which could cause DNA damage. Detailed knowledge obtained about these molecular changes could provide the basis for elucidating the molecular mechanisms of flumethrin-induced neurotoxicity.

SIRPα-specific monoclonal antibody enables antibody-dependent phagocytosis of neuroblastoma cells.

Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting.Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

BackgroundAmplification of the MYCN oncogene is a molecular hallmark of aggressive neuroblastoma (NB), a childhood cancer of the sympathetic nervous system. There is evidence that MYCN promotes a non-inflamed and...

NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS-MAPK Pathway in Neuroblastoma.

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. SIGNIFICANCE: These findings suggest that conventional therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

Exogenous HSP70 and Signaling Pathways Involved in the Inhibition of LPS-Induced Neurotoxicity of Neuroblastoma Cells

Neuroinflammation plays a key role in the pathogenesis of neurodegenerative diseases. Microglial cells are the main immune cells of the central nervous system. On exposure to lipopolysaccharides (LPS, components of the cell wall of Gram-negative enterobacteria), microglia is activated to produce reactive oxygen species (ROS), cytokines, and inflammatory mediators, which may cause neuron death. Exogenous recombinant human heat shock protein 70 (HSP70) was tested for effect on the activation of human microglial and neuroblastoma cells in response to LPS from Escherichia coli. Experiments included cell cultivation separately and transferring the conditioned medium from A-172 microglial cells to SK-N-SH neuroblastoma cells to simulate the effect of microglia treated with LPS and/or HSP70. The levels of ROS, TNFα, and apoptosis in LPS-treated cells were estimated in the presence or absence of HSP70. HSP70 was found to reduce the LPS-induced ROS generation, TNFα production, apoptosis, and necrosis, in both separate cell cultures and neuroblastoma cells grown in the conditioned medium from microglial cells. Signaling pathways involving protein kinases p38MAPK, JNK, and PI3K were demonstrated to play an important role in HSP70-mediated protection of microglial and neuroblastoma cells from LPS-induced apoptosis and ROS production.

Neurotoxicity mediated by oxidative stress caused by titanium dioxide nanoparticles in human neuroblastoma (SH-SY5Y) cells

BackgroundTitanium is widely used in biomedicine. Due to biotribocorrosion, titanium dioxide (TiO2) nanoparticles (NPs) can be released from the titanium implant surface, enter the systemic circulation, and migrate to various organs and tissues including the brain. A previous study showed that 5 nm TiO2 NPs reached the highest concentration in the brain. Even though TiO2 NPs are believed to possess low toxicity, little is known about their neurotoxic effects. The aim of the study was to evaluate in vitro the effects of 5 nm TiO2 NPs on a human neuroblastoma (SH-SY5Y) cell line. MethodsCell cultures were divided into non-exposed and exposed to TiO2 NPs for 24 h. The following were evaluated: reactive oxygen species (ROS) generation, apoptosis, cellular antioxidant response, endoplasmic reticulum stress and autophagy. ResultsExposure to TiO2 NPs induced ROS generation in a dose dependent manner, with values reaching up to 10 fold those of controls (p < 0.001). Nrf2 nuclear localization and autophagy, also increased in a dose dependent manner. Apoptosis increased by 4- to 10-fold compared to the control group, depending on the dose employed. ConclusionsOur results show that TiO2 NPs cause ROS increase, induction of ER stress, Nrf2 cytoplasmic translocation to the nucleus and apoptosis. Thus, neuroblastoma cell response to TiO2 NPs may be associated with an imbalance of the oxidative metabolism where endoplasmic reticulum-mediated signal pathway seems to be the main neurotoxic mechanism.

Response of neuroblastoma cells to RF currents as a function of the signal frequency

BackgroundCapacitive-resistive electric transfer (CRET) is a non-invasive therapeutic strategy that applies radiofrequency electric currents within the 400–600 kHz range to tissue repair and regeneration. Previous studies by our group have shown that 48 h of intermittent exposure to a 570 kHz CRET signal at a subthermal density of 50 μA/mm2 causes significant changes in the expression and activation of cell cycle control proteins, leading to cycle arrest in human cancer cell cultures. The present study investigates the relevance of the signal frequency in the response of the human neuroblastoma cell line NB69 to subthermal electric treatment with four different signal frequency currents within the 350–650 kHz range.MethodsTrypan blue assay, flow cytometry, immunofluorescence and immunoblot were used to study the effects of subthermal CRET currents on cell viability, cell cycle progression and the expression of several marker proteins involved in NB69 cell death and proliferation.ResultsThe results reveal that among the frequencies tested, only a 448 kHz signal elicited both proapoptotic and antiproliferative, statistically significant responses. The apoptotic effect would be due, at least in part, to significant changes induced by the 448 kHz signal in the expression of p53, Bax and caspase-3. The cytostatic response was preceded by alterations in the kinetics of the cell cycle and in the expression of proteins p-ERK1/2, cyclin D1 and p27, which is consistent with a potential involvement of the EGF receptor in electrically induced changes in the ERK1/2 pathway. This receives additional support from results indicating that the proapototic and antiproliferative responses to CRET can be transiently blocked when the electric stimulus is applied in the presence of PD98059, a chemical inhibitor of the ERK1/2 pathway.ConclusionThe understanding of the mechanisms underlying the ability of slowing down cancer cell growth through electrically-induced changes in the expression of proteins involved in the control of cell proliferation and apoptosis might afford new insights in the field of oncology.

Prediction of neuroblastoma cell response to treatment with natural or synthetic retinoids using selected protein biomarkers.

Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.

Exploring Cancer Cell Behavior In Vitro in Three-Dimensional Multicellular Bioprintable Collagen-Based Hydrogels.

In vitro cancer 3D models are valuable tools to provide mechanistic insight into solid tumor growth, invasion, and drug delivery. The 3D spheroid model of solid tumors has been the most popular cancer model in use until now. However, previous studies have shown that these spheroid models lack sufficient morphological parameters, which may affect their response to chemicals. In this work, we proposed the fabrication of miniaturized 3D cancer models using collagen type I-based bioprintable bioinks. In the context of a mimicking model for advanced neuroblastoma studies, we showed that cancer cells contained in bioprintable bioinks formed Homer Wright-like rosettes, maintained their proliferative capacities and produced an equivalent Vimentin-rich matrix unlike that of non-bioprintable bioinks which made for poorer models. In addition, bioprintable bioinks were successfully bioprinted as compartmentalized 3D models in the centimeter scale, which was not feasible using non-bioprintable bioinks. In contrast to non-bioprintable hydrogels, we did not observe contraction in their bioprintable counterparts, which is an advantage for prospective 3D bioprinted models that should attain stable rheological and mechanical properties after bioprinting. By adopting this proposed system for the use of patient-derived primary tumor cells, the approach could be introduced as a first line strategy in precision medicine for testing the response of neuroblastoma cells to drugs, especially when disease progresses rapidly or patients do not respond to actual therapy regimens.

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac-mediated multicellular invasion.

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion.