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1.
Zhonghua Zhong Liu Za Zhi ; 41(7): 516-521, 2019 Jul 23.
Artigo em Chinês | MEDLINE | ID: mdl-31357838

RESUMO

Objective: To investigate the effects and mechanisms of miR-144 on proliferation, apoptosis and cisplatin (DDP) resistance of neuroblastoma cells. Methods: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of miR-144 and MYCN in neuroblastoma cell lines, including SH-SY5Y and SK-N-SH, and human umbilical vein endothelial cells HUVEC. The miR-negative control, miR-144 mimics, si-negative control, si-MYCN, miR-144 mimics and pcDNA, miR-144 mimics and pcDNA-MYCN co-transfected SH-SY5Y cells were described as miR-NC, miR-144, si-NC, si-MYCN, miR-144+ pcDNA and miR-144+ pcDNA-MYCN group, respectively. The half maximal inhibitory concentration (IC(50)) and cell proliferation were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The protein expressions of MYCN, p21, cyclin D1, Bax, Bcl-2 were analyzed by western blot. Cell apoptosis was detected by flow cytometry. The cell fluorescence activity was detected by double luciferase reporter gene assay. Results: Compared with HUVEC cells, the expressions of miR-144 in neuroblastoma cells SH-SY5Y and SK-N-SH significantly decreased, while the mRNA and protein expression of MYCN significantly increased. The IC(50) of DDP was 9.16 µg/ml in SH-SY5Y cells. The absorbance value in 490nm (A(490) value) of miR-144 group was 0.30±0.03, significantly lower than 0.46±0.03 of miR-NC group. The cell apoptotic rate of miR-144 group was 26.94%±2.01%, significantly higher than 9.68%±0.52% of miR-NC group. The IC(50) value of DDP in miR-144 group was 2.95±0.26, significantly lower than 9.23±0.61 of miR-NC group. The expressions of p21, cyclin D1, Bax, Bcl-2 in miR-NC and miR-144 group were 2.67±0.19, 0.41±0.04, 2.12±0.21, 0.18±0.01 and 1.01±0.07, 1.00±0.06, 1.00±0.05, 1.00±0.06, respectively, with statistical significance (all P<0.05). Knockdown of MYCN showed the similar effects with those of miR-144 overexpression in SH-SYSY cells. MiR-144 significantly inhibited the fluorescence activity of ectopic MYCN expressing cells and negatively regulated the expression of MYCN. Overexpression of MYCN can reverse the effects of miR-144 on proliferation inhibition, apoptosis promotion and sensitization of SH-SY5Y cells to DDP. Conclusion: MiR-144 inhibits proliferation, promotes apoptosis and enhances the sensitivity of neuroblastoma cells to DDP through targeting MYCN, which provides a potential treatment for neuroblastoma.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Antineoplásicos/farmacologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Criança , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Cell Physiol Biochem ; 53(1): 258-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31313541

RESUMO

BACKGROUND/AIMS: Although neuroblastoma is a heterogeneous cancer, a substantial portion overexpresses CD71 (transferrin receptor 1) and MYCN. This study provides a mechanistically driven rationale for a combination therapy targeting neuroblastomas that doubly overexpress or have amplified CD71 and MYCN. For this subset, CD71 was targeted by its natural ligand, gambogic acid (GA), and MYCN was targeted with an HDAC inhibitor, vorinostat. A combination of GA and vorinostat was then tested for efficacy in cancer and non-cancer cells. METHODS: Microarray analysis of cohorts of neuroblastoma patients indicated a subset of neuroblastomas overexpressing both CD71 and MYCN. The viability with proliferation changes were measured by MTT and colony formation assays in neuroblastoma cells. Transfection with CD71 or MYCN along with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect expression changes. For pathway analysis, gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of GA and vorinostat in treated cells. RESULTS: For both GA and vorinostat, their pathways were explored for specificity and dependence on their targets for efficacy. For GA-treated cells, the viability/proliferation loss due to GA was dependent on the expression of CD71 and involved activation of caspase-3 and degradation of EGFR. It relied on the JNK-IRE1-mTORC1 pathway. The drug vorinostat also reduced cell viability/proliferation in the treated cells and this was dependent on the presence of MYCN as MYCN siRNA transfection led to a blunting of vorinostat efficacy and conversely, MYCN overexpression improved the vorinostat potency in those cells. Vorinostat inhibition of MYCN led to an increase of the pro-apoptotic miR183 levels and this, in turn, reduced the viability/proliferation of these cells. The combination treatment with GA and vorinostat synergistically reduced cell survival in the MYCN and CD71 overexpressing tumor cells. The same treatment had no effect or minimal effect on HEK293 and HEF cells used as models of non-cancer cells. CONCLUSION: A combination therapy with GA and vorinostat may be suitable for MYCN and CD71 overexpressing neuroblastomas.


Assuntos
Antígenos CD , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistemas de Liberação de Medicamentos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma , Receptores da Transferrina , Antígenos CD/genética , Antígenos CD/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Células HEK293 , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores da Transferrina/antagonistas & inibidores , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Vorinostat/farmacologia , Xantonas/farmacologia
3.
Anticancer Res ; 39(7): 3579-3584, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262882

RESUMO

BACKGROUND/AIM: Neuroblastoma (NB) is the most common extracranial solid tumor in childhood; treatments with greater effectiveness are required for NB, especially in advanced cases. This study aimed at evaluating the combined effect of anaplastic lymphoma kinase (ALK) inhibitor alectinib and histone deacetylase inhibitor vorinostat on NB cell lines harboring wild-type or mutated ALK. MATERIALS AND METHODS: Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. Protein expression was analyzed using western blotting. RESULTS: Combination treatment with alectinib and vorinostat had a synergistic effect on growth inhibition of the NB cell line with ALK R1275Q mutation. Cleavage of caspase-3 and poly-(ADP-ribose) polymerase increased, indicating enhanced caspase-dependent apoptosis. In addition, this combination reduced the protein levels of MYCN proto-oncogene and nuclear factor kappa B, both of which are important for NB tumorigenesis and progression. CONCLUSION: Combined treatment with alectinib and vorinostat might be a novel therapeutic option for NB harboring the ALK R1275Q mutation.


Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Carbazóis/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Neuroblastoma/tratamento farmacológico , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Vorinostat/farmacologia , Quinase do Linfoma Anaplásico/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , NF-kappa B/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo
4.
Int J Oncol ; 55(1): 93-102, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180520

RESUMO

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children, which has complex molecular mechanisms. Increasing evidence has suggested that long noncoding RNAs (lncRNAs) account for NB pathogenesis. However, the function of small nucleolar RNA host gene 16 (SNHG16) in NB is currently unclear. In the present study, publically available data and clinical specimens were employed to verify the expression of SNHG16 in NB. Colony formation, real­time cell proliferation and migration assays were performed to demonstrate the status of cellular proliferation and migration. Flow cytometry was used to examine cell cycle progression in SH­SY5Y cells, and acridine orange/ethidium bromide staining and caspase­3/7 activity measurements were applied to study cell apoptosis. To explore the underlying mechanism of SNHG16 function, an online database was used to identify potential RNA­binding proteins that bind SNHG16. The expression of SNHG16 was revealed to be in line with the clinical staging of NB, and high SNHG16 expression was positively associated with poor clinical outcome. Furthermore, SNHG16 silencing inhibited cell proliferation, repressed migration, and induced cell cycle arrest at the G0/G1 phase in SH­SY5Y cells. Additionally, apoptosis was undetectable in SH­SY5Y cells following SNHG16 silencing. Bioinformatics analysis revealed that SNHG16 regulated cell proliferation in NB through transcriptional and translational pathways. These results suggested that SNHG16 may serve important roles in the development and progression of NB, and could represent a potential target for NB therapy.


Assuntos
Neuroblastoma/genética , RNA Longo não Codificante/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Inativação Gênica , Humanos , Lactente , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oncogenes , RNA Longo não Codificante/biossíntese , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção
5.
Med Oncol ; 36(8): 66, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183633

RESUMO

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima
6.
Am J Chin Med ; 47(4): 895-912, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31091975

RESUMO

In children, neuroblastomas are the most common and deadly solid tumor. Our previous studies showed that honokiol can cross the blood-brain barrier and kill neuroblastoma cells. In this study, we further evaluated if exposure to honokiol for short periods could induce autophagy and subsequent apoptosis of neuroblastoma cells and possible mechanisms. Exposure of neuroblastoma neuro-2a cells to honokiol for 24 h induced morphological shrinkage and cell death. As to the mechanisms, honokiol consecutively induced cytochrome c release from mitochondria, caspase-3 activation, DNA fragmentation and cell apoptosis. Separately, honokiol time-dependently augmented the proportion of autophagic cells and the ratio of light chain 3 (LC3)-II/LC3-I. Pretreatment of neuro-2a cells with 3-methyladenine, an inhibitor of autophagy, attenuated honokiol-induced cell autophagy, caspase-3 activation, DNA damage and cell apoptosis. In contrast, stimulation of autophagy by rapamycin, an inducer of autophagy, significantly enhanced honokiol-induced cell apoptosis. Furthermore, honokiol-induced autophagic apoptosis was confirmed in neuroblastoma NB41A3 cells. Knocking down translation of p53 using RNA interference attenuated honokiol-induced autophagy and apoptosis in neuro-2a and NB41A3 cells. Taken together, this study showed that at early periods, honokiol can induce autophagic apoptosis of neuroblastoma cells through activating a p53-dependent mechanism. Consequently, honokiol has the potential to be a therapeutic option for neuroblastomas.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , Neuroblastoma/genética , Neuroblastoma/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Fatores de Tempo , Células Tumorais Cultivadas
8.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010127

RESUMO

In the last decades, glucosinolates (GLs), precursors of isothiocyanates (ITCs), have been studied mostly for their chemopreventive and chemotherapeutic properties. The aim of our research was to study the antiproliferative effect of 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (glucomoringin; GMG) bioactivated by myrosinase enzyme to form the corresponding isothiocyanate 4-(α-L-rhamnopyranosyloxy) benzyl C (moringin) in SH-SY5Y human neuroblastoma cells. We found that moringin significantly reduced SH-SY5Y cell growth in a time and concentration-dependent (p < 0.05, 0.01, and 0.001 vs. ctrl, after treatment with 16.4 µM moringin for 24, 48, and 72 h, respectively) manner through a mechanism involving the activation of apoptotic machinery. In addition, it altered the normal progression of cells through the cell cycle, increasing the cell population in both G2 and S phases, as well as decreasing that in the G1 phase. Studying the drug mechanism of action, we found that moringin was able to increase the expression of p53, p21, and Bax at both the protein and transcriptional level. Moreover, exposure of SH-SY5Y cells to moringin significantly increased the gene expression of both caspase 3 and 9 and enhanced their cleavage, thereby initiating an intrinsic apoptotic cascade. Finally, moringin inhibited nuclear translocation of NF-κB. Our study demonstrates the ability of moringin to reduce the growth of SH-SY5Y cells and reveals its mechanism of action, suggesting its promising role as an anticancer drug.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Isotiocianatos/farmacologia , Moringa oleifera/química , NF-kappa B/metabolismo , Neuroblastoma/patologia , Sementes/química , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neuroblastoma/genética
9.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979007

RESUMO

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.


Assuntos
Endocanabinoides/genética , Neuroblastoma/tratamento farmacológico , Receptores de Canabinoides/química , Tunicamicina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Endocanabinoides/química , Endocanabinoides/farmacologia , Citometria de Fluxo , Glicosilação/efeitos dos fármacos , Humanos , Ligantes , Microscopia Confocal , Neuroblastoma/genética , Neuroblastoma/patologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Receptores de Canabinoides/genética , Canais de Cátion TRPV/genética , Tunicamicina/química
10.
Cell Physiol Biochem ; 52(4): 893-907, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30964607

RESUMO

BACKGROUND/AIMS: Previous studies have shown that a 63-hour, intermittent exposure to a 50 Hz, 100 µT magnetic field (MF) induces in the NB69 line of human neuroblastoma a proliferative response that is mediated by activation of the MAPK pathways ERK1/2 and p38. The present study aims to investigate the potential involvement of the epidermal growth factor receptor (EGFR) in the field-induced cell proliferation and activation of MAPK pathways. METHODS: NB69 cultures were MF- or sham-exposed for 5 to 30 minute intervals and 63 hours. Cell proliferation and activation of MAPK-ERK1/2, -p38 and -JNK was analyzed in the presence or absence of erlotinib, an effective inhibitor of EGFR tyrosine kinase. The expression of p-EGFR and MMP-9 in the presence or absence of MF was also studied. Between 3 and 7 replicates of each experiment were performed, using between 3 and 4 samples per experimental condition and replicate. At the end of each replicate, the samples were analyzed at short times (5-30 min) through immunofluorescence and Western blotting, and the growth response was assessed (63 hours interval) through dye exclusion with Trypan blue. RESULTS: The results confirmed that field exposure induces cell proliferation and activation of ERK1/2, p38 and JNK, and revealed that these effects were blocked with erlotinib. The data also showed that, compared to shamexposed controls, the MF exposure induces early and transient increases in the expression of p-EGFR and MMP-9 at 15 and 5 min from the exposure onset, respectively. CONCLUSION: The obtained results reveal that the activation of the MAPK-ERK1/2 and -p38 pathways by the MF is mediated by the EGF receptor. Taken together with our previously published results, this dataset suggests that the proliferative response induced in NB69 by a 63-hour exposure to a weak, power frequency MF, is mediated by early transient activation of EGFR in which MMP-9 would be involved.


Assuntos
Proliferação de Células , Sistema de Sinalização das MAP Quinases , Campos Magnéticos , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patologia
11.
RNA ; 25(7): 840-856, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988100

RESUMO

ZC3H12B is the most enigmatic member of the ZC3H12 protein family. The founding member of this family, Regnase-1/MCPIP1/ZC3H12A, is a well-known modulator of inflammation and is involved in the degradation of inflammatory mRNAs. In this study, for the first time, we characterized the properties of the ZC3H12B protein. We show that the biological role of ZC3H12B depends on an intact NYN/PIN RNase domain. Using RNA immunoprecipitation, experiments utilizing actinomycin D and ELISA, we show that ZC3H12B binds interleukin-6 (IL-6) mRNA in vivo, regulates its turnover, and results in reduced production of IL-6 protein upon stimulation with IL-1ß. We verified that regulation of IL-6 mRNA stability occurs via interaction of ZC3H12B with the stem-loop structure present in the IL-6 3'UTR. The IL-6 transcript is not the only target of ZC3H12B. ZC3H12B also interacts with other known substrates of Regnase-1 and ZC3H12D, such as the 3'UTRs of IER3 and Regnase-1, and binds IER3 mRNA in vivo. Using immunofluorescence, we examined the localization of ZC3H12B within the cell. ZC3H12B forms small, granule-like structures in the cytoplasm that are characteristic of proteins involved in mRNA turnover. The overexpression of ZC3H12B inhibits proliferation by stalling the cell cycle in the G2 phase. This effect of ZC3H12B is also NYN/PIN dependent. The analysis of the ZC3H12B mRNA level reveals its highest expression in the human brain and the neuroblastoma cell line SH-SY5Y, although the factors regulating its expression remain elusive. Down-regulation of ZC3H12B in SH-SY5Y cells by specific shRNAs results in up-regulation of ZC3H12B-target mRNAs.


Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Interleucina-6/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Encéfalo/metabolismo , Células HeLa , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Domínios Proteicos , Estabilidade de RNA , RNA Mensageiro/genética , Ribonucleases/genética , Homologia de Sequência , Fatores de Transcrição/genética
12.
Cancer Res ; 79(7): 1293-1294, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936073

RESUMO

Both natural killer (NK) cells and exosomes released from these cells induce tumor cell cytotoxicity by way of the cell killing proteins perforin and granzyme. TGFß1 protein in the tumor microenvironment generates an immune escape mechanism rendering NK cells inactive. The tumor-suppressive miR-186 that is downregulated in neuroblastoma and in TGFß-treated NK cells represses oncogenic proteins in neuroblastoma (MYCN and AURKA) and components of the TGFß pathway. Restoration of miR-186 levels in neuroblastoma through NK cell-derived exosomes or by nanoparticle delivery reduces tumor burden, promotes survival, and restores the cell-killing abilities of NK cells, demonstrating the therapeutic potential of tumor-suppressive miRNAs in neuroblastoma.See related article by Neviani and colleagues; Cancer Res 79(6):1151-64.


Assuntos
Exossomos/genética , MicroRNAs/genética , Neuroblastoma/genética , Linhagem Celular Tumoral , Humanos , Células Matadoras Naturais/imunologia , Microambiente Tumoral
13.
Nature ; 567(7749): 545-549, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894746

RESUMO

MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.


Assuntos
Proteína BRCA1/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Estabilidade Proteica , Tioléster Hidrolases/metabolismo
14.
Int J Biol Macromol ; 130: 878-891, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30836187

RESUMO

Myocardial Infarction Associated Transcript (MIAT) is a subnuclear lncRNA that interferes with alternative splicing and is associated with increased risk of various heart conditions and nervous system tumours. The current study aims to elucidate the role of MIAT in cell survival, apoptosis and migration in neuroblastoma and glioblastoma multiforme. To this end, MIAT was silenced by MIAT-specific siRNAs in neuroblastoma and glioblastoma cell lines, and RNA sequencing together with a series of functional assays were performed. The RNA sequencing has revealed that the expression of an outstanding number of genes is altered, including genes involved in cancer-related processes, such as cell growth and survival, apoptosis, reactive oxygen species (ROS) production and migration. Furthermore, the functional studies have confirmed the RNA sequencing leads, with our key findings suggesting that MIAT knockdown eliminates long-term survival and migration and increases basal apoptosis in neuroblastoma and glioblastoma cell lines. Taken together with the recent demonstration of the involvement of MIAT in glioblastoma, our observations suggest that MIAT could possess tumour-promoting properties, thereby acting as an oncogene, and has the potential to be used as a reliable biomarker for neuroblastoma and glioblastoma and be employed for prognostic, predictive and, potentially, therapeutic purposes for these cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Neuroblastoma/genética , RNA Longo não Codificante/genética , Apoptose , Biomarcadores , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neuroblastoma/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/genética , Análise de Sequência de RNA , Transdução de Sinais
15.
Cell Mol Biol Lett ; 24: 19, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30891073

RESUMO

Background: Cyclin-dependent kinase-like 1 (CDKL1) is a member of the cell division control protein 2-related serine-threonine protein kinase family. It is known to occur in various malignant tumors, but its role in neuroblastoma (NB) remains unclear. Methods: We constructed a CDKL1-silenced NB cell strain (SH-SY5Y) and used real-time PCR and western blotting to confirm the silencing. Functional analyses were performed using the MTT, colony-formation, FACS, wound-healing and transwell invasion assays. Results: The expression of CDKL1 was significantly upregulated in NB tissue as compared to the adjacent normal tissue. CDKL1 knockdown significantly suppressed cell viability and colony formation ability. It also induced cell cycle G0/G1 phase arrest and apoptosis, and suppressed the migration and invasion ability of SH-SY5Y cells. CDKL1 knockdown decreased the CDK4, cyclin D1 and vimentin expression levels, and increased the caspase-3, PARP and E-cadherin expression levels in SH-SY5Y cells. Conclusions: Our findings suggest that CDKL1 plays an important role in NB cell proliferation, migration and invasion. It might serve as a potential target for NB therapy.


Assuntos
Movimento Celular , Proliferação de Células , Quinases Ciclina-Dependentes/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuroblastoma/genética , Apoptose , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neuroblastoma/fisiopatologia
16.
Biochim Biophys Acta Bioenerg ; 1860(5): 391-401, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885735

RESUMO

Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a "signaling protein" under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1 h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies.


Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Proteínas de Neoplasias/biossíntese , Neuroblastoma/metabolismo , Proteína Desacopladora 2/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Metabolismo Energético/genética , Glucose/genética , Glucose/metabolismo , Glutamina/genética , Camundongos , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteína Desacopladora 2/genética
17.
Int J Mol Sci ; 20(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866462

RESUMO

Dual-specificity phosphatases (DUSPs) are important regulators of neuronal cell growth and differentiation by targeting proteins essential to neuronal survival in signaling pathways, among which the MAP kinases (MAPKs) stand out. DUSPs include the MAPK phosphatases (MKPs), a family of enzymes that directly dephosphorylate MAPKs, as well as the small-size atypical DUSPs, a group of low molecular-weight enzymes which display more heterogeneous substrate specificity. Neuroblastoma (NB) is a malignancy intimately associated with the course of neuronal and neuroendocrine cell differentiation, and constitutes the source of more common extracranial solid pediatric tumors. Here, we review the current knowledge on the involvement of MKPs and small-size atypical DUSPs in NB cell growth and differentiation, and discuss the potential of DUSPs as predictive biomarkers and therapeutic targets in human NB.


Assuntos
Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Neuroblastoma/enzimologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , Transdução de Sinais
18.
Int J Mol Sci ; 20(5)2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30862004

RESUMO

Neuroblastoma (NB) is the most common childhood cancer, with a very poor prognosis. More than 60% of children with NB die within five years; therefore, a more effective therapy for NB is required. Although ginsenoside has been shown to significantly inhibit the growth of various cancers, the effect of ginsenoside Rk1 on neuroblastoma has not been known yet. Hence, we examined the anticancer effects of highly pure Rk1 on neuroblastoma cell lines. The apoptotic effects of Rk1 on neuroblastoma cells were examined using cell viability assay, flow cytometry and cell staining assay, and the change in gene expression levels were analysed using RT-PCR, western blots, and immunohistochemistry. The metastatic effect of Rk1 was monitored by wound healing assay, invasion and migration with Matrigels. Rk1 inhibited neuroblastoma cell viability dose-dependently. Rk1-induced apoptosis was investigated through nuclear condensation and mitochondrial membrane potential loss, and it showed that Rk1 can induce cell cycle arrest at the G0/G1 phase but also inhibit the metastatic ability of neuroblastoma cells. Moreover, Rk1 (30 mg/kg) injections markedly inhibited xenograft tumor growth. These findings demonstrate that Rk1 might be valuable in the development of anti-cancer agents for neuroblastoma treatment.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ginsenosídeos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neuroblastoma/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/genética , Ginsenosídeos/química , Humanos , Masculino , Camundongos , Estrutura Molecular , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
BMC Cancer ; 19(1): 243, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885150

RESUMO

BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.


Assuntos
Benzamidas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Amplificação de Genes , Técnicas de Silenciamento de Genes , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cell Mol Life Sci ; 76(13): 2615-2632, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30863908

RESUMO

The Tar DNA-Binding Protein 43 (TDP-43) and its phosphorylated isoform (pTDP-43) are the major components associated with ubiquitin positive/Tau-negative inclusions found in neurons and glial cells of patients suffering of amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration-TDP-43 (FTLD-TDP). Many studies have revealed that TDP-43 is also in the protein inclusions associated with neurodegenerative conditions other than ALS and FTLD-TDP, thus suggesting that this protein may be involved in the pathogenesis of a variety of neurological disorders. In brains of Huntington-affected patients, pTDP-43 aggregates were shown to co-localize with mutant Huntingtin (mHtt) inclusions. Here, we show that expression of mHtt carrying 80-97 polyglutamines repeats in human cell cultures induces the aggregation and the phosphorylation of endogenous TDP-43, whereas non-pathological Htt with 25 polyglutamines repeats has no effect. Mutant Htt aggregation precedes accumulation of pTDP-43 and pTDP-43 co-localizes with mHtt inclusions reminding what it was previously described in brains of Huntington-affected patients. Detergent-insoluble fractions from cells expressing mHtt and containing mHtt-pTDP-43 co-aggregates can function as seeds for further TDP-43 aggregation in human cell culture. The human cellular prion protein PrPC was previously identified as a negative modulator of mHtt aggregation; here, we show that PrPC-mediated reduction of mHtt aggregation is tightly correlated with a decrease of TDP-43 aggregation and phosphorylation, thus confirming the close relationships between TDP-43 and mHtt.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Huntingtina/metabolismo , Mutação , Neuroblastoma/patologia , Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Agregados Proteicos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Proteína Huntingtina/genética , Corpos de Inclusão , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fosforilação , Proteínas Priônicas/genética , Células Tumorais Cultivadas
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