Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.740
Filtrar
1.
Anticancer Res ; 40(9): 5151-5158, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878803

RESUMO

BACKGROUND/AIM: Magnetic stimulation is used in the treatment of a diversity of diseases, but a complete understanding of the underlying mechanisms of action requires further investigation. We examined the effect of static magnetic stimulation (SMS) in different cell lines. MATERIALS AND METHODS: A culture plate holder with attached NeFeB magnets was developed. Different magnetic field intensities and periods were tested in tumoral and non-tumoral cell lines. To verify the cellular responses to SMS, cell viability, cell death, cell cycle and BDNF expression were evaluated. RESULTS: Exposure of SH-SY5Y cells to SMS for 24 hours led to a decrease in cell viability. Analysis 24 h after stimulation revealed a decrease in apoptotic and double-positive cells, associated with an increase in the number of necrotic cells. CONCLUSION: The effects of SMS on cell viability are cell type-specific, inducing a decrease in cell viability in SH-SY5Y cells. This suggests that SMS may be a potential tool in the treatment of neuronal tumors.


Assuntos
Sobrevivência Celular/efeitos da radiação , Fenômenos Magnéticos , Apoptose/efeitos da radiação , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Especificidade de Órgãos/efeitos da radiação
2.
Proc Natl Acad Sci U S A ; 117(28): 16516-16526, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601179

RESUMO

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7-independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis.


Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/fisiopatologia , Ligação Proteica , Proteínas de Ligação a RNA/genética , Transativadores/genética , Peixe-Zebra
3.
Medicine (Baltimore) ; 99(25): e20853, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32569234

RESUMO

INTRODUCTION: Neuroblastoma (NB) with MYCN amplification has a poor prognosis and high mortality. The potential molecular biological relationship between clinical features and MYCN amplification should be explored. METHODS: NB patients were examined by fluorescence in situ hybridization (FISH) for MYCN amplification in the tumor mass or bone marrow samples to determine whether MYCN was amplified. A series of eleven MYCN-amplified NB patients were included. The age, primary site, tumor size, specific biomarkers, and invaded organs were analyzed. All patients accepted standardized treatment of surgery, chemotherapy, and radiotherapy. Progression-free survival (PFS) and overall survival (OS) were evaluated. RESULTS: The median age at diagnosis was 24 months. Nine patients (81.8%) were in stage IV, with high serum neuron-specific enolase (NSE) expression, normal urine vanillylmandelic acid (VMA) level and extensive metastases. All patients accepted a chemotherapy protocol with 8 to 10 cycles, and 9 patients (81.8%) were sensitive to the initial chemotherapy protocol. At the end of follow-up, four patients (36.3%) died with a median OS of 15 months. Five patients (45%) survived with a median PFS of 13 months. Two patients were still receiving chemotherapy. CONCLUSION: Given the effect of MYCN amplification on poor outcome in NB, novel treatments targeting MYCN should be developed for patients with NB.


Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Pré-Escolar , Terapia Combinada , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/terapia , Análise de Sobrevida
4.
Cancer Sci ; 111(7): 2431-2439, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32415892

RESUMO

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and nonamplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.


Assuntos
Dano ao DNA , Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Quinazolinas/farmacologia , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Humanos , Redes e Vias Metabólicas , Neuroblastoma/metabolismo
5.
Chem Biol Interact ; 326: 109134, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464120

RESUMO

Montelukast is a cysteinyl leukotriene (CysLT) receptor antagonist with efficacy against a variety of diseases, including asthma and inflammation-related conditions. However, various neuropsychiatric events (NEs) suspected to be related to montelukast have been reported recently, with limited understanding on their association and underlying mechanisms. This study aimed to investigate whether montelukast can induce neuroinflammation and neurotoxicity in microglial HAPI cells and neural SH-SY5Y cells. The present study also compared the effects of montelukast with a 5-lipoxygenase inhibitor (zileuton) and a cyclooxygenase-2 inhibitor (celecoxib) to better understand modulation of related pathways. HAPI or SH-SY5Y cells were treated with the indicated drugs (3.125 µM-100 µM) for 24 h to investigate drug-induced neuroinflammation and neurotoxicity. Montelukast induced cytotoxicity in HAPI cells (50-100 µM), accompanied with caspase-3/7 activation, prostaglandin E2 (PGE2) release, and reactive oxygen species (ROS) production. Whilst both montelukast and zileuton down-regulated CysLT release in HAPI cells, zileuton did not significantly affect cell viability or inflammatory and oxidative factors. Celecoxib decreased HAPI cell viability (6.25-100 µM), accompanied with increasing caspase-3/7 activation and ROS production, but in contrast to montelukast increased CysLT release and decreased PGE2 production. Similar to observations in HAPI cells, both montelukast and celecoxib (50-100 µM) but not zileuton produced toxicity in SH-SY5Y neuroblastoma cells. Similarly, CM from HAPI cells treated with either montelukast or zileuton produced toxicity in SH-SY5Y cells. The results of the current study show the capability of montelukast to directly induce toxicity and inflammation in HAPI cells, possibly through the involvement of PGE2 and ROS, and toxicity in undifferentiated SH-SY5Y neuroblastoma cells. The current study highlights the importance of consideration between benefit and risk of montelukast usage and provides references for future investigation on decreasing montelukast-related NEs.


Assuntos
Acetatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Humanos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo
6.
J Vis Exp ; (159)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32421003

RESUMO

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 µg/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 µg/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was <3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.


Assuntos
Eletrólitos/metabolismo , Eletroforese Capilar/métodos , Ferro/análise , Espectrometria de Massas/métodos , Neuroblastoma/metabolismo , Humanos , Oxirredução , Células Tumorais Cultivadas
7.
Pediatr Blood Cancer ; 67(6): e28267, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32307821

RESUMO

BACKGROUND: The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. PROCEDURE: Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. RESULTS: Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. CONCLUSIONS: Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Proteínas/antagonistas & inibidores , Piridonas/farmacologia , Pirimidinonas/farmacologia , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos SCID , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Environ Toxicol ; 35(9): 922-929, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32293791

RESUMO

Excessive fluoride exposure contributes to neurotoxic effects. Emodin exhibits antioxidative functions in the central nervous system (CNS); however, its neuroprotective mechanism against fluoride remains to be elucidated. Our aim was to explore the neuroprotective efficacy and the possible mechanisms of emodin. In our study, synaptic proteins and oxidative stress damage were examined after human neuroblastoma SH-SY5Y cells were treated with high doses of NaF for 24 hours. Moreover, pretreatment with emodin was used to shed light on the neuroprotective effects in NaF-induced toxicity in SH-SY5Y cells. We found that NaF significantly lowered the protein expressions of SNAP 25, synaptophysin and PSD 95 in SH-SY5Y cells. In addition, NaF exposure increased the protein expression of p-ERK1/2 and decreased the protein expressions of Nrf2 and HO-1, as well as facilitated increasing ROS, 4-hydroxynonenal (4-HNE), and 8-Hydroxy-2'-deoxyguanosine (8-OHdG). Pretreatment with emodin significantly recovered these alterations caused by NaF. These data implied that the neuroprotective effects of emodin and pointed to the promising utilization for protecting against neurotoxicity induced by fluoride.


Assuntos
Emodina/farmacologia , Fluoretos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neuroblastoma/metabolismo , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Sinaptofisina/metabolismo
9.
Mol Carcinog ; 59(7): 724-735, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32333465

RESUMO

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Humanos , Neuroblastoma/metabolismo , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/fisiologia
10.
Mol Biol (Mosk) ; 54(1): 128-136, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163396

RESUMO

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.


Assuntos
Meios de Cultivo Condicionados/química , Proteínas de Choque Térmico HSP70/farmacologia , Lipopolissacarídeos/toxicidade , Neuroblastoma/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Lipopolissacarídeos/imunologia , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo
11.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218156

RESUMO

This study examined the biological activities of copaiba essential oil via measurement of its effects on signaling pathways in the SH-SY5Y neuronal cell line. Nanofluidic proteomic technologies were deployed to measure the phosphorylation of biomarker proteins within the signaling cascades. Interestingly, copaiba essential oil upregulated the pI3K/Akt/mTOR, MAPK, and JAK/STAT signaling pathways in neuronal cells. The effects of copaiba essential oil peaked at 30 min post-treatment, with a half-maximal effective concentration (EC50) of approximately 80 ng/mL. Treatment with cannabinoid receptor 2 (CB2) agonist AM1241 or the inverse agonist BML190 abrogated the regulatory effects of copaiba essential oil on the pI3K/Akt/mTOR signaling pathway. Surprisingly, copaiba essential oil also activated the apoptosis signaling pathway and reduced the viability of SH-SY5Y cells with an EC50 of approximately 400 ng/mL. Furthermore, ß-caryophyllene, a principal constituent of copaiba essential oil, downregulated the pI3K/Akt/mTOR signaling pathway. Taken together, the findings indicated that copaiba essential oil upregulated signaling pathways associated with cell metabolism, growth, immunity, and apoptosis. The biological activities of copaiba essential oil were determined to be fast acting, CB2 mediated, and dependent on multiple chemical constituents of the oil. Nanofluidic proteomics provided a powerful means to assess the biological activities of copaiba essential oil.


Assuntos
Fabaceae/química , Neuroblastoma/metabolismo , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo
12.
Arch Med Res ; 51(2): 180-184, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32111494

RESUMO

BACKGROUND: Detrimental effects of high glucose content (HGC) were proved in different tissues such as the central nervous system. It seems that diabetic conditions could also alter the functional behavior of stem cells residing in the context of the nervous system. METHODS: The possible effects of 40 and 70 mmol glucose were examined on HSP70 signaling pathways with a specific focus on protein translation, folding values of human neuroblastoma cell line SHSY-5Y after 72 h. Human neuroblastoma cells were exposed to 5, 40 and 70 mmol glucose doses. The transcription level of genes related to HSP70 signaling was also evaluated by PCR array. RESULTS: The data from PCR array showed high glucose especially 70 mmol could potentially modulate the normal function of protein folding, endoplasmic reticulum derived protein folding and synthesis in neuroblastoma cells (p <0.05). CONCLUSIONS: Data showed that high glucose condition makes neuroblastoma cells prone to biochemical insufficiency by affecting the function of HSP70 signaling pathway and protein synthesis.


Assuntos
Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Glucose/farmacologia , Glucose/fisiologia , Humanos , Transdução de Sinais
13.
Biomed Res Int ; 2020: 6734048, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149119

RESUMO

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.


Assuntos
Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptor trkB/genética , Tretinoína/metabolismo , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Humanos , Glicoproteínas de Membrana/metabolismo , Neuritos/metabolismo , Receptor trkB/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transcriptoma
14.
Nat Commun ; 11(1): 913, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060267

RESUMO

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Pré-Escolar , Estudos de Coortes , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Nuclear Ligada ao X/metabolismo
15.
Life Sci ; 246: 117399, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32032648

RESUMO

AIMS: Glioblastomas are highly aggressive brain tumors with a very poor survival rate. EEF1A2, the proto-oncogenic isoform of the EEF1A translation factor family, has been found to be overexpressed and promoting tumorigenesis in multiple cancers. Interestingly, recent studies reported reduced expression of this protein in brain tumors, drawing our attention to find the functional role and mechanism of this protein in brain tumor progression. MAIN METHODS: Using representative cell line as models, the role of EEF1A2 in cell proliferation, migration and invasion were assessed using MTS assay, scratch wound-healing assay, transwell migration and invasion assay, respectively. Activation of key signaling pathways was assessed using western blots and real-time PCR. Finally, using immunohistochemistry we checked the protein levels of EEF1A2 in CNS tumors. KEY FINDINGS: EEF1A2 was found to increase the proliferative, migratory and invasive properties of cell lines of both glial and neuronal origin. PI3K activation directly correlated with EEF1A2 levels. Protein levels of key EMT markers viz. Twist, Snail, and Slug were increased upon ectopic EEF1A2 expression. Furthermore, EEF1A2 was found to affect the expression levels of key inflammatory cytokines, growth factors and matrix metalloproteases. IHC analysis showed that EEF1A2 is upregulated in tumor tissues compared to normal tissue. SIGNIFICANCE: EEF1A2 acts as an oncogene in both neuronal and glial cells and triggers an EMT program via PI3K pathway. However, it shows enhanced expression in neuronal cells of the brain than the glial cells, which could explain the previously reported anomaly.


Assuntos
Neoplasias Encefálicas/metabolismo , Transição Epitelial-Mesenquimal , Fator 1 de Elongação de Peptídeos/metabolismo , Western Blotting , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fator 1 de Elongação de Peptídeos/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
16.
J Mol Biol ; 432(7): 2080-2098, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32061932

RESUMO

The self-assembly of the 42-residue amyloid-ß peptide, Aß42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aß42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aß42 oligomers. These structures exhibit low ß-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, ß-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aß42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aß42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aß42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, ß-sheet-rich Aß42 fibrils in AD, while structures with low ß-sheet content may be less relevant.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/antagonistas & inibidores , Benzopiranos/farmacologia , Proliferação de Células , Neuroblastoma/tratamento farmacológico , Fragmentos de Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células PC12 , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em Folha beta , Ratos , Células Tumorais Cultivadas
17.
J Exp Clin Cancer Res ; 39(1): 41, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087738

RESUMO

BACKGROUND: The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS: By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS: We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS: Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.


Assuntos
Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia
18.
Nat Commun ; 11(1): 71, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900415

RESUMO

Despite advances in the molecular exploration of paediatric cancers, approximately 50% of children with high-risk neuroblastoma lack effective treatment. To identify therapeutic options for this group of high-risk patients, we combine predictive data mining with experimental evaluation in patient-derived xenograft cells. Our proposed algorithm, TargetTranslator, integrates data from tumour biobanks, pharmacological databases, and cellular networks to predict how targeted interventions affect mRNA signatures associated with high patient risk or disease processes. We find more than 80 targets to be associated with neuroblastoma risk and differentiation signatures. Selected targets are evaluated in cell lines derived from high-risk patients to demonstrate reversal of risk signatures and malignant phenotypes. Using neuroblastoma xenograft models, we establish CNR2 and MAPK8 as promising candidates for the treatment of high-risk neuroblastoma. We expect that our method, available as a public tool (targettranslator.org), will enhance and expedite the discovery of risk-associated targets for paediatric and adult cancers.


Assuntos
Antineoplásicos/administração & dosagem , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Neuroblastoma/metabolismo , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
19.
J Photochem Photobiol B ; 203: 111748, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31918235

RESUMO

Nanotechnology is an emerged field to develop the plant mediated metal based nanodrugs by green method. In this current study, the zinc oxide metal based nanoparticles were developed using (Clausena lansium (Lour.) Skeels) Peel aqueous extracts and zinc nitrate. The C.L extract zinc nanoparticleswere indicated by the sharp peak seen at 350 nm utilizing the Ultraviolet-Visible spectroscopy (UV-Vis). The high peaks indicate the presence of phytochemicals and its functional groups in ZnONPs were studied by the Fourier Transform Infrared Spectroscopy (FT-IR). The X-Ray Diffraction analysis (XRD) explores the pattern and structure of ZnONPs as spherical and base-centered monoclinic crystalline shapes. The C.L extract with Zn nanoparticles were spherical in nature and the size of the synthesized particles were about 28.42 nm respectively. The autophagy (Beclin-1, LC3-I, LC3-II and ATG4B) and apoptotic (Bax, Bcl-2 and Caspase-3) proteins were regulated by the treatment with ZnONPs in SH-SY5Y neuroblastoma cells. The DNA loss or damage was occurred in the ZnONPs treatment and it was performed using Comet assay. The ZnONPs treatment generates the ROS in the cells and decreased its stability and viability. Addition of NAC prevents ROS in the cultured SH-SY5Y cells and prevents the cells from the apoptosis. We concluded that the ZnONPs potentially kills the neuroblastoma cells by producing the intracellular ROS.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Nanopartículas Metálicas/química , Óxido de Zinco/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clausena/química , Clausena/metabolismo , Dano ao DNA/efeitos dos fármacos , Química Verde , Humanos , Nanopartículas Metálicas/toxicidade , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Pediatr Blood Cancer ; 67(5): e28098, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31975571

RESUMO

INTRODUCTION: WEE1 is a serine kinase central to the G2 checkpoint. Inhibition of WEE1 can lead to cell death by permitting cell-cycle progression despite unrepaired DNA damage. AZD1775 is a WEE1 inhibitor that is in clinical development for children and adults with cancer. METHODS: AZD1775 was tested using a dose of 120 mg/kg administered orally for days 1 to 5. Irinotecan was administered intraperitoneally at a dose of 2.5 mg/kg for days 1 to 5 (one hour after AZD1775 when used in combination). AZD1775 and irinotecan were studied alone and in combination in neuroblastoma (n = 3), osteosarcoma (n = 4), and Wilms tumor (n = 3) xenografts. RESULTS: AZD1775 as a single agent showed little activity. Irinotecan induced objective responses in two neuroblastoma lines (PRs), and two Wilms tumor models (CR and PR). The combination of AZD1775 + irinotecan-induced objective responses in two neuroblastoma lines (PR and CR) and all three Wilms tumor lines (CR and 2 PRs). The objective response measure improved compared with single-agent treatment for one neuroblastoma (PR to CR), two osteosarcoma (PD1 to PD2), and one Wilms tumor (PD2 to PR) xenograft lines. Of note, the combination yielded CR (n = 1) and PR (n = 2) in all the Wilms tumor lines. The event-free survival was significantly longer for the combination compared with single-agent irinotecan in all models tested. The magnitude of the increase was greatest in osteosarcoma and Wilms tumor xenografts. CONCLUSIONS: AZD1775 potentiates the effects of irinotecan across most of the xenograft lines tested, with effect size appearing to vary across tumor panels.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Tumor de Wilms/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Irinotecano/farmacologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos SCID , Neoplasias Experimentais/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Tumor de Wilms/metabolismo , Tumor de Wilms/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA