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1.
Biomed Res Int ; 2021: 5845554, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34513994

RESUMO

Objective: Neuroblastoma (NB) is a highly metastatic tumor in children that develops in the sympathetic nervous system and has a low curative rate. Saikosaponin A (SSA), an active ingredient isolated from the root of Radix Bupleuri, is a natural compound with various pharmacological activities and shows good application prospects in antitumors. This study investigated the antihuman NB activity of SSA and underlying mechanisms associated with its actions. Materials and Methods: The MTT method was used to detect the activity of SSA in inhibiting human NB cell SK-N-AS proliferation. Cell morphology was observed. The flow cytometry technology was used in analyzing the cell apoptosis rate. The Transwell assay evaluated cell migration and invasion following SSA treatment, apoptosis-related protein expression, and angiogenesis-related protein expression, and EMT-related proteins were detected by western blot analysis. Results: SSA showed an inhibitory effect on SK-N-AS cells with the IC50 values of 14.14 µM at 24 h and 12.41 µM at 48 h. Results indicated that SSA has proapoptotic activity, and its proapoptotic activity is positively correlated with the Bax/Bcl-2/caspase-9/caspase-7/PARP pathway. Furthermore, SSA inhibited the invasion and migration of SK-N-AS cells via regulating the angiogenesis-related VEGFR2/Src/Akt pathway and the epithelial-mesenchymal transition- (EMT-) related protein expression. Conclusion: SSA exerts an antihuman NB effect and thus provides foundations for NB treatment.


Assuntos
Neuroblastoma/metabolismo , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Bupleurum/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , China , Humanos , Concentração Inibidora 50 , Neuroblastoma/tratamento farmacológico , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , Extratos Vegetais/farmacologia , Saponinas/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
BMC Cancer ; 21(1): 1061, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34565342

RESUMO

BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.


Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 12(1): 5309, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493726

RESUMO

Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. Here we study single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compare tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles a subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations reveal different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Glândulas Suprarrenais/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Receptor trkB/genética , Transcriptoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/mortalidade , Neoplasias das Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Pré-Escolar , Células Cromafins/metabolismo , Células Cromafins/patologia , Diagnóstico Precoce , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Receptor trkB/metabolismo , Medição de Risco , Análise de Célula Única , Especificidade da Espécie , Análise de Sobrevida
4.
PLoS One ; 16(9): e0257718, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34591871

RESUMO

Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.


Assuntos
Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Antineoplásicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/administração & dosagem , Neuroblastoma/tratamento farmacológico , Pirróis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Nylons/síntese química , Nylons/química , Fosforilação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Biochim Biophys Acta Gen Subj ; 1865(11): 129998, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34474117

RESUMO

BACKGROUND: Dementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. METHODS: We used SH-SY5Y cells cultured in high-glucose media. RESULTS: S-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. CONCLUSION: Glucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. GENERAL SIGNIFICANCE: The results may be useful for designing curative treatment strategies for dementia.


Assuntos
Glucose/metabolismo , Neuroblastoma/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Humanos , Estresse Oxidativo , Células Tumorais Cultivadas
6.
Front Immunol ; 12: 668307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489927

RESUMO

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Citocinas/farmacologia , Gangliosídeos/antagonistas & inibidores , Efeito Enxerto vs Tumor , Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/transplante , Ativação Linfocitária/efeitos dos fármacos , Neuroblastoma/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia
7.
Cells ; 10(9)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34572073

RESUMO

Neuronal miRNA dysregulation may have a role in the pathophysiology of Alzheimer's disease (AD). miRNA(miR)-124 is largely abundant and a critical player in many neuronal functions. However, the lack of models reliably recapitulating AD pathophysiology hampers our understanding of miR-124's role in the disease. Using the classical human SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and the PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), we observed a sustained upregulation of miR-124/miR-125b/miR-21, but only miR-124 was consistently shuttled into their exosomes. The miR-124 mimic reduced APP gene expression in both AD models. While miR-124 mimic in SH-SWE neurons led to neurite outgrowth, mitochondria activation and small Aß oligomer reduction, in iNEU-PSEN cells it diminished Tau phosphorylation, whereas miR-124 inhibitor decreased dendritic spine density. In exosomes, cellular transfection with the mimic predominantly downregulated miR-125b/miR-21/miR-146a/miR-155. The miR-124 inhibitor upregulated miR-146a in the two experimental cell models, while it led to distinct miRNA signatures in cells and exosomes. In sum, though miR-124 function may be dependent on the neuronal AD model, data indicate that keeping miR-124 level strictly controlled is crucial for proper neuronal function. Moreover, the iNEU-PSEN cellular model stands out as a useful tool for AD mechanistic studies and perhaps for the development of personalized therapeutic strategies.


Assuntos
Doença de Alzheimer/patologia , Células-Tronco Pluripotentes Induzidas/patologia , MicroRNAs/administração & dosagem , MicroRNAs/genética , Neuroblastoma/patologia , Neurônios/patologia , Presenilina-1/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Exossomos/genética , Exossomos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neurônios/metabolismo , Transdução de Sinais
8.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576232

RESUMO

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.


Assuntos
Citocinese/fisiologia , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Neuroblastoma/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Interferência de RNA , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Centrossomo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fuso Acromático
9.
Molecules ; 26(17)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34500749

RESUMO

Imidazo[1,2-b]pyridazine compounds are a new class of promising lead molecules to which we have incorporated polar nitro and amino moieties to increase the scope of their biological activity. Two of these substituted 3-nitro-6-amino-imidazo[1,2-b]pyridazine compounds (5c and 5h) showed potent acetylcholinesterase (AChE) inhibitory activity (IC50 40-50 nM), which we have previously reported. In this study, we wanted to test the biological efficacy of these compounds. Cytotoxicity assays showed that compound 5h mediated greater cell death with over 43% of cells dead at 100 µM and activation of caspase 3-mediated apoptosis. On the other hand, compound 5c mediated a dose-dependent decrease in cell proliferation. Both compounds showed cell cycle arrest in the G0/G1 phase and reduced cellular ATP levels leading to activation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitochondrial oxidative stress. It has to be noted that all these effects were observed at doses beyond 10 µM, 200-fold above the IC50 for AChE inhibition. Both compounds also inhibited bacterial lipopolysaccharide-mediated cyclooxygenase-2 and nitric oxide release in primary rat microglial cells. These results suggested that the substituted imidazo (1,2-b) pyridazine compounds, which have potent AChE inhibitory activity, were also capable of antiproliferative, anti-migratory, and anti-inflammatory effects at higher doses.


Assuntos
Antineoplásicos/farmacologia , Inibidores da Colinesterase/farmacologia , Neuroblastoma/tratamento farmacológico , Piridazinas/farmacologia , Acetilcolinesterase/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estresse Oxidativo/efeitos dos fármacos , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
10.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360553

RESUMO

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.


Assuntos
Anticonvulsivantes/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia de Alvo Molecular , Neuroblastoma/tratamento farmacológico , Receptor trkC/metabolismo , Ácido Valproico/farmacologia , Apoptose , Proliferação de Células , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptor trkC/genética , Células Tumorais Cultivadas
11.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445601

RESUMO

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe2+ to redox-inactive Fe3+, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.


Assuntos
Ferritinas/metabolismo , Ferroptose , Células-Tronco Neurais/patologia , Neuroblastoma/patologia , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Feminino , Ferritinas/genética , Ferro/metabolismo , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Oxirredução , Oxirredutases/genética , Piperazinas/metabolismo , Proteínas ras/genética
12.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361099

RESUMO

(1) Background: Ellagitannins are natural products occurring in pomegranate and walnuts. They are hydrolyzed in the gut to release ellagic acid, which is further metabolized by the microflora into urolithins, such as urolithin A (UA). Accumulation of damaged mitochondria is a hallmark of aging and age-related neurodegenerative diseases. In this study, we investigated the neuroprotective activity of the metabolite UA against mitochondrial dysfunction in a cellular model of early Alzheimer disease (AD). (2) Methods: In the present study we used SH-SY5Y-APP695 cells and its corresponding controls (SH-SY5Ymock) to assess UA's effect on mitochondrial function. Using these cells we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, autophagy and levels of reactive oxygen species (ROS) in cells treated with UA. Furthermore, we assessed UA's effect on the expression of genes related to mitochondrial bioenergetics, mitochondrial biogenesis, and autophagy via quantitative real-time PCR (qRT-PCR). (3) Results: Treatment of SH-SY5Y-APP695 cells suggests changes to autophagy corresponding with qRT-PCR results. However, LC3B-I, LC3B-II, and p62 levels were unchanged. UA (10 µM) reduced MMP, and ATP-levels. Treatment of cells with UA (1 µM) for 24 h did not affect ROS production or levels of Aß, but significantly increased expression of genes for mitochondrial biogenesis and OXPHOS. Mitochondrial Transcription Factor A (TFAM) expression was specifically increased in SH-SY5Y-APP695. Both cell lines showed unaltered levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which is commonly associated with mitochondrial biogenesis. Results imply that biogenesis might be facilitated by estrogen-related receptor (ESRR) genes. (4) Conclusion: Urolithin A shows no effect on autophagy in SH-SY5Y-APP695 cells and its effect on mitochondrial function is limited. Instead, data suggests that UA treatment induces hormetic effects as it induces transcription of several genes related to mitochondrial biogenesis.


Assuntos
Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Cumarínicos/farmacologia , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Biogênese de Organelas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Autofagia , Metabolismo Energético , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas
13.
BMC Cancer ; 21(1): 950, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433438

RESUMO

BACKGROUND: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB. METHODS: BALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines. RESULTS: High 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32. CONCLUSION: T/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.


Assuntos
Lutécio/uso terapêutico , Neuroblastoma/radioterapia , Octreotida/análogos & derivados , Radioisótopos/uso terapêutico , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445656

RESUMO

Neuroblastoma (Nb), the most common extracranial tumor in children, exhibited remarkable phenotypic diversity and heterogeneous clinical behavior. Tumors with MYCN overexpression have a worse prognosis. MYCN promotes tumor progression by inducing cell proliferation, de-differentiation, and dysregulated mitochondrial metabolism. Cyclophosphamide (CFF) at minimum effective oral doses (metronomic therapy) exerts beneficial actions on chemoresistant cancers. Molecular iodine (I2) in coadministration with all-trans retinoic acid synergizes apoptosis and cell differentiation in Nb cells. This work analyzes the impact of I2 and CFF on the viability (culture) and tumor progression (xenografts) of Nb chemoresistant SK-N-BE(2) cells. Results showed that both molecules induce dose-response antiproliferative effects, and I2 increases the sensibility of Nb cells to CFF, triggering PPARγ expression and acting as a mitocan in mitochondrial metabolism. In vivo oral I2/metronomic CFF treatments showed significant inhibition in xenograft growth, decreasing proliferation (Survivin) and activating apoptosis signaling (P53, Bax/Bcl-2). In addition, I2 decreased the expression of master markers of malignancy (MYCN, TrkB), vasculature remodeling, and increased differentiation signaling (PPARγ and TrkA). Furthermore, I2 supplementation prevented loss of body weight and hemorrhagic cystitis secondary to CFF in nude mice. These results allow us to propose the I2 supplement in metronomic CFF treatments to increase the effectiveness of chemotherapy and reduce side effects.


Assuntos
Biomarcadores Tumorais/metabolismo , Ciclofosfamida/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Iodo/farmacologia , Neuroblastoma/tratamento farmacológico , Animais , Anti-Infecciosos Locais/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Diferenciação Celular , Proliferação de Células , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Sci ; 112(11): 4617-4626, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34464480

RESUMO

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest-derived immature cells. The prognosis of patients with high-risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti-tumor effects of CD146-targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA-mediated knockdown of CD146, or treatment with an anti-CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor-kappa B signaling pathway. Furthermore, the anti-CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma.


Assuntos
Anticorpos/uso terapêutico , Antígeno CD146/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Neuroblastoma/terapia , RNA Interferente Pequeno/uso terapêutico , Animais , Apoptose , Antígeno CD146/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , NF-kappa B/metabolismo , Recidiva Local de Neoplasia , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Prognóstico , Transdução de Sinais , Esferoides Celulares , Transdução Genética/métodos
16.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299209

RESUMO

Misfolded amyloid beta (Aß) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aß cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aß mainly through the ability to stimulate allosterically the α7 nAChRs and to regulate the calcium cytosolic concentration. Here, we examined the cytoprotective effects of two GAL derivatives, namely compounds 4b and 8, against Aß cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aß. GAL and curcumin (CU) were used in the study as reference compounds. It was found that 4b protects cells in a similar mode as GAL, while compound 8 and CU potentiate the toxic effects of Aß. Allosteric stimulation of α7 nAChRs is suggested as a possible mechanism of the cytoprotectivity of 4b. These and previous findings characterize 4b as a prospective non-toxic multi-target agent against neurodegenerative disorders with inhibitory activity on acetylcholinesterase, antioxidant, and cytoprotective properties.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Antioxidantes/farmacologia , Inibidores da Colinesterase/farmacologia , Curcumina/química , Galantamina/química , Neuroblastoma/tratamento farmacológico , Substâncias Protetoras/farmacologia , Acetilcolinesterase/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Inibidores da Colinesterase/química , Curcumina/farmacologia , Citoproteção , Galantamina/farmacologia , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Substâncias Protetoras/química , Células Tumorais Cultivadas
17.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299239

RESUMO

Estrogens are steroid hormones that play a crucial role in the regulation of the reproductive and non-reproductive system physiology. Among non-reproductive systems, the nervous system is mainly affected by estrogens due to their antioxidant, anti-apoptotic, and anti-inflammatory activities, which are mediated by membranous and nuclear estrogen receptors, and also by non-estrogen receptor-associated estrogen actions. Neuronal viability and functionality are also associated with the maintenance of mitochondrial functions. Recently, the localization of estrogen receptors, especially estrogen receptor beta, in the mitochondria of many types of neuronal cells is documented, indicating the direct involvement of the mitochondrial estrogen receptor beta (mtERß) in the maintenance of neuronal physiology. In this study, cell lines of N2A cells stably overexpressing a mitochondrial-targeted estrogen receptor beta were generated and further analyzed to study the direct involvement of mtERß in estrogen neuroprotective antioxidant and anti-apoptotic actions. Results from this study revealed that the presence of estrogen receptor beta in mitochondria render N2A cells more resistant to staurosporine- and H2O2-induced apoptotic stimuli, as indicated by the reduced activation of caspase-9 and -3, the increased cell viability, the increased ATP production, and the increased resistance to mitochondrial impairment in the presence or absence of 17-ß estradiol (E2). Thus, the direct involvement of mtERß in antioxidant and anti-apoptotic activities is documented, rendering mtERß a promising therapeutic target for mitochondrial dysfunction-associated degenerative diseases.


Assuntos
Receptor beta de Estrogênio/metabolismo , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor beta de Estrogênio/genética , Estrogênios/metabolismo , Estrogênios/farmacologia , Peróxido de Hidrogênio/metabolismo , Camundongos , Mitocôndrias/fisiologia , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Neurônios/metabolismo , Neurônios/fisiologia , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptores de Estrogênio/metabolismo
18.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199157

RESUMO

The influence of p-terphenyl polyketides 1-3 from Aspergillus candidus KMM 4676 and cerebroside flavuside B (4) from Penicillium islandicum (=Talaromyces islandicus) against the effect of neurotoxins, rotenone and paraquat, on Neuro-2a cell viability by MTT and LDH release assays and intracellular ROS level, as well as DPPH radical scavenging activity, was investigated. Pre-incubation with compounds significantly diminished the ROS level in rotenone- and paraquat-treated cells. It was shown that the investigated polyketides 1-3 significantly increased the viability of rotenone- and paraquat-treated cells in two of the used assays but they affected only the viability of paraquat-treated cells in the LDH release assay. Flavuside B statistically increased the viability of paraquat-treated cells in both MTT and LDH release assays, however, it increased the viability of rotenone-treated cells in the LDH release assay. Structure-activity relationships for p-terphenyl derivatives, as well as possible mechanisms of cytoprotective action of all studied compounds, were discussed.


Assuntos
Aspergillus/química , Citoproteção/efeitos dos fármacos , Glicoesfingolipídeos/farmacologia , Neuroblastoma/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Policetídeos/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Herbicidas/toxicidade , Inseticidas/toxicidade , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fármacos Neuroprotetores/química , Paraquat/toxicidade , Policetídeos/química , Espécies Reativas de Oxigênio , Rotenona/toxicidade
19.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200415

RESUMO

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.


Assuntos
Ácido Homovanílico/química , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Ácido Vanilmandélico/química , Bioensaio/métodos , Biomarcadores/metabolismo , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Masculino , República da Coreia , Espectrometria de Massas em Tandem/métodos
20.
Nucleic Acids Res ; 49(14): 7856-7869, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34289065

RESUMO

The MYCN gene encodes the transcription factor N-Myc, a driver of neuroblastoma (NB). Targeting G-quadruplexes (G4s) with small molecules is attractive strategy to control the expression of undruggable proteins such as N-Myc. However, selective binders to G4s are challenging to identify due to the structural similarity of many G4s. Here, we report the discovery of a small molecule ligand (4) that targets the noncanonical, hairpin containing G4 structure found in the MYCN gene using small molecule microarrays (SMMs). Unlike many G4 binders, the compound was found to bind to a pocket at the base of the hairpin region of the MYCN G4. This compound stabilizes the G4 and has affinity of 3.5 ± 1.6 µM. Moreover, an improved analog, MY-8, suppressed levels of both MYCN and MYCNOS (a lncRNA embedded within the MYCN gene) in NBEB neuroblastoma cells. This work indicates that the approach of targeting complex, hybrid G4 structures that exist throughout the human genome may be an applicable strategy to achieve selectivity for targeting disease-relevant genes including protein coding (MYCN) as well as non-coding (MYCNOS) gene products.


Assuntos
DNA/química , Quadruplex G , Proteína Proto-Oncogênica N-Myc/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Estrutura Molecular , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade
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