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1.
Molecules ; 26(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34641289

RESUMO

The dihydropyranoindole structures were previously identified as promising scaffolds for improving the anti-cancer activity of histone deacetylase inhibitors. This work describes the synthesis of related furoindoles and their ability to synergize with suberoylanilide hydroxamic acid (SAHA) against neuroblastoma and breast cancer cells. The nucleophilic substitution of hydroxyindole methyl esters with α-haloketones yielded the corresponding arylether ketones, which were subsequently cyclized to tricyclic and tetracyclic furoindoles. The furoindoles showed promising individual cytotoxic efficiency against breast cancer cells, as well as decent SAHA enhancement against cancer cells in select cases. Interestingly, the best IC50 value was obtained with the non-cyclized intermediate.


Assuntos
Neoplasias da Mama/enzimologia , Inibidores de Histona Desacetilases/farmacologia , Cetonas/síntese química , Neuroblastoma/enzimologia , Vorinostat/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Cetonas/química , Cetonas/farmacologia , Células MCF-7 , Neuroblastoma/tratamento farmacológico
2.
Cells ; 10(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440719

RESUMO

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.


Assuntos
Amina Oxidase (contendo Cobre)/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Espermina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Ratos Wistar , Transdução de Sinais , Espermina/metabolismo , Proteína Supressora de Tumor p53/genética
3.
J Mol Model ; 27(5): 134, 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33899124

RESUMO

Neuroblastoma (NB), as a metastatic form of solid tumor, has a high fatality rate found in early childhood. The two anaplastic lymphoma kinase (ALK) neoepitopes nonamer and decamer used in cancer immunotherapy against NB cancer can selectively bind to the human leukocyte antigen (HLA-B*15:01) groove with high affinities, whereas the native self-peptide is unable to interact with the HLA-B*15:01. Here, we performed molecular dynamics (MD) simulations and subsequent molecular mechanics-generalized born surface area (MM-GBSA) binding free energy calculations to explore the selective binding mechanisms of nonamer and decamer to the HLA-B*15:01 against the self-peptide. MD simulations revealed the significant conformational dynamics of the self-peptide in the HLA-B*15:01 groove against the nonamer and decamer. Binding free energy calculations showed that the binding affinities of HLA-B*15:01-neoepitope complexes were followed in the order decamer > nonamer > self-peptide. Detailed analysis of HLA-B*15:01-neoepitope structural complexes showed that compared to the nonamer, the self-peptide tended to move outward to the solvent, whereas the decamer moved deep to the HLA-B*15:01 groove. These different dynamic observations of the ALK neoepitopes can explain the distinct binding affinities of self-peptide, nonamer, and decamer to the HLA-B*15:01. The results may be useful for the design of more selective ALK neoepitopes.


Assuntos
Quinase do Linfoma Anaplásico/imunologia , Epitopos/metabolismo , Antígeno HLA-B15/metabolismo , Neuroblastoma/enzimologia , Epitopos/química , Antígeno HLA-B15/química , Humanos , Imunoterapia , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Termodinâmica
4.
Am J Physiol Cell Physiol ; 320(4): C635-C651, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33356946

RESUMO

Disruption of copper homeostasis is closely involved in neurodegenerative disorders. This study examined whether a hybrid copper-binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), is able to protect NG108-15 cells against oxidative stress. We found that treatment of cells with rotenone or hydrogen peroxide increased cellular oxidative stress and resulted in mitochondrial dysfunction and apoptosis. The cellular levels of Nrf2 and the Cu2+ chaperone DJ-1 were also decreased. These oxidative detrimental effects were all inhibited when cells were cotreated with DPMQ. DPMQ increased cellular Cu2+ content, DJ-1 protein level, superoxide dismutase (SOD) activity, and Nrf2 nuclear translocation under basal state. The activity of SOD decreased under redox imbalance and this decrease was blocked by DPMQ treatment, while the protein level of SOD1 remained unaltered regardless of the oxidative stress and DPMQ treatment. Using endogenous proteins, coimmunoprecipitation showed that DJ-1 bound with SOD1 and Nrf2 individually. The amount of Nrf2, bound to DJ-1, consistently reflected its cellular level, while the amount of SOD1, bound to DJ-1, was potentiated by DPMQ, being greater in the basal state than under redox imbalance. Simultaneous inclusion of nonpermeable Cu2+ chelator tetrathiomolybdate or triethylenetetramine during DPMQ treatment blocked all aforementioned effects of DPMQ, showing that the dependency of the effect of DPMQ on extracellular Cu2+. In addition, silencing of DJ-1 blocked the protection of DPMQ against oxidative stress. Taken all together, our results suggest that DPMQ stabilizes DJ-1 in a Cu2+-dependent manner, which then brings about SOD1 activation and Nrf2 nuclear translocation; these together alleviate cellular oxidative stress.


Assuntos
Antioxidantes/farmacologia , Quelantes/farmacologia , Cobre/metabolismo , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteína Desglicase DJ-1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Glioma/enzimologia , Glioma/patologia , Humanos , Hibridomas , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Neurônios/enzimologia , Neurônios/patologia , Proteína Desglicase DJ-1/genética , Ratos , Rotenona/toxicidade , Superóxido Dismutase-1/metabolismo
5.
Int J Mol Sci ; 21(21)2020 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-33138280

RESUMO

We evaluated the potential of nine vitamin B3 scaffold-based derivatives as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors, as a starting point for the development of novel drugs for treating disorders with cholinergic neurotransmission-linked pathology. As the results indicate, all compounds reversibly inhibited both enzymes in the micromolar range pointing to the preference of AChE over BChE for binding the tested derivatives. Molecular docking studies revealed the importance of interactions with AChE active site residues Tyr337 and Tyr124, which dictated most of the observed differences. The most potent inhibitor of both enzymes with Ki of 4 µM for AChE and 8 µM for BChE was the nicotinamide derivative 1-(4'-phenylphenacyl)-3-carbamoylpyridinium bromide. Such a result places it within the range of several currently studied novel cholinesterase inhibitors. Cytotoxicity profiling did not classify this compound as highly toxic, but the induced effects on cells should not be neglected in any future detailed studies and when considering this scaffold for drug development.


Assuntos
Butirilcolinesterase/química , Proliferação de Células , Inibidores da Colinesterase/farmacologia , Neuroblastoma/patologia , Niacinamida/química , Acetilcolinesterase , Domínio Catalítico , Inibidores da Colinesterase/química , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Simulação de Acoplamento Molecular , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas
6.
Biochem J ; 477(20): 4053-4070, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33043964

RESUMO

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is ∼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.


Assuntos
Neuroblastoma/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Domínio Catalítico , Diferenciação Celular/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Cinética , Mutação , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/genética , Células PC12 , Fosforilação , Domínios Proteicos , RNA Interferente Pequeno , Ratos , Receptor trkA/química , Receptor trkA/genética , Receptor trkB/química , Receptor trkB/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos
7.
Sci Rep ; 10(1): 11997, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686724

RESUMO

Neuroblastoma is the most common paediatric cancer type. Patients diagnosed with high-risk neuroblastoma have poor prognosis and occasionally tumours relapse. As a result, novel treatment strategies are needed for relapse and refractory neuroblastoma patients. Here, we found that high expression of Mps1 kinase (mitotic kinase Monopolar Spindle 1) was associated with relapse-free neuroblastoma patient outcomes and poor overall survival. Silencing and inhibition of Mps1 in neuroblastoma or PDX-derived cells promoted cell apoptosis via the caspase-dependent mitochondrial apoptotic pathway. The mechanism of cell death upon Mps1 inhibition was dependent on the polyploidization/aneuploidization of the cells before undergoing mitotic catastrophe. Furthermore, tumour growth retardation was confirmed in a xenograft mouse model after Mps1-inhibitor treatment. Altogether, these results suggest that Mps1 expression and inhibition can be considered as a novel prognostic marker as well as a therapeutic strategy for the treatment of high-risk neuroblastoma patients.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Mitose , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Mitocôndrias/metabolismo , Poliploidia , Proteínas Tirosina Quinases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cell Oncol (Dordr) ; 43(6): 1067-1084, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32705581

RESUMO

PURPOSE: Neuroblastoma, a common childhood tumor, remains one of the most elusive diseases to treat. To date, high-risk neuroblastoma is associated with low survival rates. To address this, novel and more effective therapeutic strategies must continue to be explored. METHODS: We employed a bioinformatics approach corroborated with in vitro and in vivo data. Samples from neuroblastoma patients were retrieved and immuno-stained for Bruton's tyrosine kinase (BTK). To evaluate its effect on cellular functions, BTK expression in SK-N-BE(2) and SH-SY5Y neuroblastoma cells was downregulated using gene silencing or inhibition with ibrutinib or acalabrutinib. Xenograft mouse models were used to investigate the in vivo role of BTK in neuroblastoma tumorigenesis. RESULTS: We found that BTK was highly expressed in primary neuroblastoma samples, preferentially in MYCN-amplified neuroblastoma cases, and was associated with a poor prognosis. Immunohistochemical staining of tissues from our neuroblastoma cohort revealed a strong BTK immunoreactivity. We also found that neuroblastoma SK-N-BE(2) and SH-SY5Y cells were sensitive to treatment with ibrutinib and acalabrutinib. Pharmacologic or molecular inhibition of BTK elicited a reduction in the migratory and invasive abilities of neuroblastoma cells, and ibrutinib considerably attenuated the neurosphere-forming ability of neuroblastoma cells. Both inhibitors showed synergism with cisplatin. In vivo assays showed that acalabrutinib effectively inhibited neuroblastoma tumorigenesis. CONCLUSIONS: From our data we conclude that BTK is a therapeutically targetable driver of neuroblastoma.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Carcinogênese/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
9.
Biomed Pharmacother ; 129: 110268, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32563146

RESUMO

The dysregulation of non-coding RNAs (ncRNAs) often caused aberrant cell behaviors. In the present study, we focused on the role of long noncoding RNA (lncRNA) non-coding RNA activated by DNA damage (NORAD) in the development of neuroblastoma (NB). The enrichment of NORAD, miRNA-144-3p (miR-144-3p) and histone deacetylase 8 (HDAC8) was measured by quantitative real time polymerase chain reaction (qRT-PCR). The proliferation, chemoresistance, apoptosis, metastasis and autophagy of NB cells were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, transwell migration and invasion assays and Western blot assay, respectively. The target relationship between miR-144-3p and NORAD or HDAC8 was predicted by Starbase software and validated through dual-luciferase reporter assay, RIP and RNA-pull down assays. The protein expression of HDAC8 was measured by Western blot assay. Murine xenograft model was used to verify the function of NORAD in vivo. We found that the level of NORAD was up-regulated in NB tissues and cells, and the level of NORAD was negatively correlated with the prognosis of NB patients. NORAD promoted the proliferation, metastasis and doxorubicin (DOX) resistance while inhibited the apoptosis and autophagy of NB cells. MiR-144-3p was a target of NORAD in NB cells, and NORAD accelerated the progression and DOX resistance of NB through sponging miR-144-3p. HDAC8 was a direct target of miR-144-3p in NB cells, and miR-144-3p suppressed the progression of NB through down-regulating HDAC8. NORAD up-regulated the expression of HDAC8 through sponging miR-144-3p in NB cells. NORAD accelerated the growth of NB tumors at least partly through miR-144-3p/HDAC8 signaling in vivo. In conclusion, NORAD promoted the progression and DOX resistance of NB through miR-144-3p/HDAC8 axis in vivo and in vitro.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Histona Desacetilases/metabolismo , MicroRNAs/metabolismo , Neuroblastoma/tratamento farmacológico , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/genética , Humanos , Masculino , Camundongos , MicroRNAs/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/secundário , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Biosci Rep ; 40(5)2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32412051

RESUMO

Neuroblastoma (NB) is an extracranial solid tumor in children with complex mechanism. Increasing reports indicated that long non-coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) account for the pathogenesis of NB. Nevertheless, the precise functions of SNHG16 needed to be further exposed in NB progression. Our data revealed that SNHG16 and hepatocyte nuclear factor 4 α (HNF4α) were up-regulated, but miR-542-3p was down-regulated in NB. Knockdown of SNHG16 or HNF4α could impede cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Interestingly, the role of SNHG16 detetion in cell behaviors was rescued by HNF4α overexpression in NB cells. Mechanically, SNHG16 modulated the progression of tumor growth via miR-542-3p/HNF4α axis in NB. Also, SNHG16 knockdown inactivated rat sarcoma/effector of RAS/mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases (RAS/RAF/MEK/ERK) signaling pathway through HNF4α. Therefore, SNHG16/miR-542-3p/HNF4α axis modified NB progression via RAS/RAF/MEK/ERK signaling pathway, might highlight a novel therapeutic approach for NB.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neuroblastoma/enzimologia , RNA Longo não Codificante/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo , Fatores Etários , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Criança , Pré-Escolar , Transição Epitelial-Mesenquimal , Fator 4 Nuclear de Hepatócito/genética , Humanos , Lactente , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Longo não Codificante/genética , Transdução de Sinais , Carga Tumoral
11.
Int J Mol Sci ; 21(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085516

RESUMO

Transglutaminase 2 (TG2) is a multifunctional enzyme and two isoforms, TG2-L and TG2-S, exerting opposite effects in the regulation of cell death and survival, have been revealed in cancer tissues. Notably, in cancer cells a hypoxic environment may stimulate tumor growth, invasion and metastasis. Here we aimed to characterize the role of TG2 isoforms in neuroblastoma cell fate under hypoxic conditions. The mRNA levels of TG2 isoforms, hypoxia-inducible factor (HIF)-1α, p16, cyclin D1 and B1, as well as markers of cell proliferation/death, DNA damage, and cell cycle were examined in SH-SY5Y (non-MYCN-amplified) and IMR-32 (MYCN-amplified) neuroblastoma cells in hypoxia/reoxygenation conditions. The exposure to hypoxia induced the up-regulation of HIF-1α in both cell lines. Hypoxic conditions caused the up-regulation of TG2-S and the reduction of cell viability/proliferation associated with DNA damage in SH-SY5Y cells, while in IMR-32 did not produce DNA damage, and increased the levels of both TG2 isoforms and proliferation markers. Different cell response to hypoxia can be mediated by TG2 isoforms in function of MYCN amplification status. A better understanding of the role of TG2 isoforms in neuroblastoma may open new venues in a diagnostic and therapeutic perspective.


Assuntos
Proteínas de Ligação ao GTP/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Transglutaminases/genética , Ciclo Celular/genética , Morte Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Fragmentação do DNA , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
J Biochem Mol Toxicol ; 34(3): e22439, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31909875

RESUMO

Nicotinamide N-methyltransferase (NNMT) plays a central role in cellular metabolism, regulating pathways including epigenetic regulation, cell signalling, and energy production. Our previous studies have shown that the expression of NNMT in the human neuroblastoma cell line SH-SY5Y increased complex I activity and subsequent ATP synthesis. This increase in ATP synthesis was lower than the increase in complex I activity, suggesting uncoupling of the mitochondrial respiratory chain. We, therefore, hypothesised that pathways that reduce oxidative stress are also increased in NNMT-expressing SH-Y5Y cells. The expression of uncoupling protein-2 messenger RNA and protein were significantly increased in NNMT-expressing cells (57% ± 5.2% and 20.1% ± 1.5%, respectively; P = .001 for both). Total GSH (22 ± 0.3 vs 35.6 ± 1.1 nmol/mg protein), free GSH (21.9 ± 0.2 vs 33.5 ± 1 nmol/mg protein), and GSSG (0.6 ± 0.02 vs 1 ± 0.05 nmol/mg protein; P = .001 for all) concentrations were significantly increased in NNMT-expressing cells, whereas the GSH:GSSG ratio was decreased (39.4 ± 1.8 vs 32.3 ± 2.5; P = .02). Finally, reactive oxygen species (ROS) content was decreased in NNMT-expressing cells (0.3 ± 0.08 vs 0.12 ± 0.03; P = .039), as was the concentration of 8-isoprostane F2α (200 ± 11.5 vs 45 ± 2.6 pg/mg protein; P = .0012). Taken together, these results suggest that NNMT expression reduced ROS generation and subsequent lipid peroxidation by uncoupling the mitochondrial membrane potential and increasing GSH buffering capacity, most likely to compensate for increased complex I activity and ATP production.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neuroblastoma/enzimologia , Nicotinamida N-Metiltransferase/biossíntese , Estresse Oxidativo , Linhagem Celular Tumoral , Humanos , Neuroblastoma/patologia
13.
Eur Arch Psychiatry Clin Neurosci ; 270(1): 119-126, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30560291

RESUMO

Repetitive transcranial magnetic stimulation (rTMS) is a neuromodulation technique that stimulates cortical regions via time-varying electromagnetic fields; in several countries this technique has been approved as a treatment for major depressive disorder. One empirically established target in antidepressant pharmacotherapy is the flavin-containing monoamine oxidoreductase (MAO). The function of MAO enzymes is based on oxidation processes that may be sensitive towards strong electromagnetic fields. Therefore, we hypothesized that rTMS-induced electromagnetic fields impact the activity of this enzyme. Using crude synaptosomal cell preparations from human SH-SY5Y neuroblastoma cells and rat cortex as well as viable cells, we assessed the effects of rTMS on MAO-A and -B activity in a well-controlled in vitro set up. In short, samples were stimulated at maximal intensity with an equal number of total stimuli at frequencies of 5, 20, and 100 Hz. Sham stimulation was performed in parallel. Treatment at frequencies of 5 and 20 Hz significantly decreased mainly MAO-B activity in all tissue preparations and species, whereas 100 Hz stimulation remained without effect on any MAO activity. Our results support the hypothesis, that rTMS-induced electromagnetic fields affect MAO activity and provide further evidence for intracellular effects possibly contributing to therapeutic effects of this neuromodulatory method. On a cautionary note, however, our findings are solely based on in vitro evidence.


Assuntos
Córtex Cerebral/enzimologia , Monoaminoxidase/metabolismo , Sinaptossomos/enzimologia , Estimulação Magnética Transcraniana , Células Tumorais Cultivadas/enzimologia , Animais , Linhagem Celular Tumoral , Humanos , Neuroblastoma/enzimologia , Ratos
14.
Cell Signal ; 65: 109425, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31689507

RESUMO

Sildenafil, a phosphodiesterase-5 inhibitor is FDA approved drug against erectile dysfunction. It is currently undergoing many clinical trials, alone or in combinations against different diseases. Treatment of neural progenitor cells with sildenafil is known to regulate their basal cGMP levels and enhance neurogenesis and differentiation. cGMP as well as cAMP are known to play a central role in the maintenance, repair and remodelling of the nervous system. In the present study, we report the neurodifferentiation property of sildenafil in neuroblastoma cancer cell line IMR-32. Sildenafil was found to induce the formation of neurite outgrowths that were found expressing neuronal markers, such as NeuN, NF-H and ßIII tubulin. IS00384, a recently discovered PDE5 inhibitor by our laboratory, was also found to induce neurodifferentiation of IMR-32 cells. The effect of IS00384 on differentiation was even more profound than sildenafil. Both the compounds were found to elevate and activate the Guanine nucleotide exchange factor C3G, which is a regulator of differentiation in IMR-32 cells. They were also found to elevate the levels of cGMP and activate the AMPK-ACC and PI3K-Akt signalling pathways. These pathways are known to play important role in cytoskeletal rearrangements necessary for differentiation. This study highlights the role of phosphodiesterases-5 in neurodifferentiation and use of sildenafil and IS00384 as small molecule tools to study the process of cellular differentiation.


Assuntos
Neuroblastoma/metabolismo , Neurogênese/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Citrato de Sildenafila/farmacologia , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/enzimologia , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Citrato de Sildenafila/química , Tubulina (Proteína)/metabolismo
15.
J Clin Pathol ; 73(3): 154-161, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31542727

RESUMO

AIMS: To investigate the relations between anaplastic lymphoma kinase (ALK) and v-myc myelocytomatosis viral related oncogene neuroblastoma derived homolog (MYCN) protein expression and their prognostic roles in neuroblastoma tumours. METHODS: Sixty-one neuroblastoma tumours obtained at diagnosis were stained with anti-MYCN and anti-ALK antibodies by immunohistochemical staining. The correlations between protein expression of MYCN, ALK and clinicopathological and biological variables of neuroblastoma tumours were analysed. RESULTS: High expression of ALK protein could be detected in 25 (41%) and high expression of MYCN protein could be detected in 24 (39.3%) of the 61 neuroblastoma tumours, respectively. The majority of neuroblastoma tumours with evident of ALK or MYCN protein high expression exhibited undifferentiated or poorly differentiated histology (30/35, 85.7%). ALK or MYCN protein high expression in neuroblastoma tumours was associated with adverse clinical prognostic factors and ALK protein high expression was significantly associated with MYCN protein high expression. In addition, either ALK or MYCN protein high expression in neuroblastoma tumours was the independent adverse prognostic factor and also predicted worse survival outcomes for neuroblastoma patients with MYCN non-amplified status or non-high-risk Children's Oncology Group grouping. CONCLUSIONS: Our study showed a novel coordinately prognostic role of ALK and MYCN protein expression in neuroblastoma and is the first report to demonstrate the correlation between ALK and MYCN protein expression in primary neuroblastoma tumours.


Assuntos
Quinase do Linfoma Anaplásico/análise , Biomarcadores Tumorais/análise , Proteína Proto-Oncogênica N-Myc/análise , Neuroblastoma/enzimologia , Adolescente , Diferenciação Celular , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neuroblastoma/terapia , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Regulação para Cima
16.
Int J Biochem Cell Biol ; 119: 105680, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866508

RESUMO

It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPSc-neurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106-126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Calcineurina/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Calcineurina/farmacologia , Linhagem Celular Tumoral , Humanos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Tacrolimo/farmacologia
17.
Sci Rep ; 9(1): 19353, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852910

RESUMO

Neuroblastoma is the most commonly diagnosed extracranial tumor in the first year of life. Approximately 9% of neuroblastoma patients present germline or somatic aberrations in the gene encoding for anaplastic lymphoma kinase (ALK). This increases in high-risk neuroblastomas, which have a 14% frequency of ALK aberrations at the time of diagnosis and show increasing numbers at relapse. Abrogating ALK activity with kinase inhibitors is employed as clinical therapy in malignancies such as non-small cell lung cancer and has shown good results in pediatric inflammatory myofibroblastic tumors and anaplastic large cell lymphomas. A phase I clinical trial of the first generation ALK inhibitor, crizotinib, in neuroblastoma patients showed modest results and suggested that further investigation was needed. Continuous development of ALK inhibitors has resulted in the third generation inhibitor repotrectinib (TPX-0005), which targets the active kinase conformations of ALK, ROS1 and TRK receptors. In the present study we investigated the effects of repotrectinib in a neuroblastoma setting in vitro and in vivo. Neuroblastoma cell lines were treated with repotrectinib to investigate inhibition of ALK and to determine its effect on proliferation. PC12 cells transfected with different ALK mutant variants were used to study the efficacy of repotrectinib to block ALK activation/signaling. The in vivo effect of repotrectinib was also analyzed in a neuroblastoma xenograft model. Our results show that repotrectinib is capable of inhibiting signaling activity of a range of ALK mutant variants found in neuroblastoma patients and importantly it exhibits strong antitumor effects in a xenograft model of neuroblastoma.


Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Compostos Macrocíclicos/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Pirazóis/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Concentração Inibidora 50 , Compostos Macrocíclicos/farmacologia , Camundongos Endogâmicos BALB C , Mutação/genética , Neovascularização Patológica/tratamento farmacológico , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuroblastoma/irrigação sanguínea , Neuroblastoma/enzimologia , Células PC12 , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Pirazóis/farmacologia , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Adv Exp Med Biol ; 1161: 89-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31562624

RESUMO

The importance of prostaglandin E2 in cancer progression is well established, but research on its role in cancer has so far mostly been focused on epithelial cancer in adults while the knowledge about the contribution of prostaglandin E2 to childhood malignancies is limited. Neuroblastoma, an extracranial solid tumor of the sympathetic nervous system, mainly affects young children. Patients with tumors classified as high-risk have poor survival despite receiving intensive treatment, illustrating a need for new treatments complimenting existing ones. The basis of neuroblastoma treatment e.g. chemotherapy and radiation therapy, target the proliferating genetically unstable tumor cells leading to treatment resistance and relapses. The tumor microenvironment is an avenue, still to a great extent, unexplored and lacking effective targeted therapies. Cancer-associated fibroblasts is the main source of prostaglandin E2 in neuroblastoma contributing to angiogenesis, immunosuppression and tumor growth. Prostaglandin E2 is formed from its precursor arachidonic acid in a two-step enzymatic reaction. Arachidonic acid is first converted by cyclooxygenases into prostaglandin H2 and then further converted by microsomal prostaglandin E synthase-1 into prostaglandin E2. We believe targeting of microsomal prostaglandin E synthase-1 in cancer-associated fibroblasts will be an effective future therapeutic strategy in fighting neuroblastoma.


Assuntos
Dinoprostona , Neuroblastoma , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases , Antineoplásicos/uso terapêutico , Ácido Araquidônico/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Neuroblastoma/fisiopatologia , Prostaglandina-E Sintases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Microambiente Tumoral
19.
Sci Rep ; 9(1): 13259, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519958

RESUMO

Patient-derived xenografts (PDXs) provide an opportunity to evaluate the effects of therapies in an environment that more closely resembles the human condition than that seen with long-term passage cell lines. In the current studies, we investigated the effects of FAK inhibition on two neuroblastoma PDXs in vitro. Cells were treated with two small molecule inhibitors of FAK, PF-573,228 (PF) and 1,2,4,5-benzentetraamine tetrahydrochloride (Y15). Following FAK inhibition, cell survival and proliferation decreased significantly and cell cycle arrest was seen in both cell lines. Migration and invasion assays were used to determine the effect of FAK inhibition on cell motility, which decreased significantly in both cell lines in the presence of either inhibitor. Finally, tumor cell stemness following FAK inhibition was evaluated with extreme limiting dilution assays as well as with immunoblotting and quantitative real-time PCR for the expression of stem cell markers. FAK inhibition decreased formation of tumorspheres and resulted in a corresponding decrease in established stem cell markers. FAK inhibition decreased many characteristics of the malignant phenotype, including cancer stem cell like features in neuroblastoma PDXs, making FAK a candidate for further investigation as a potential target for neuroblastoma therapy.


Assuntos
Movimento Celular , Proliferação de Células , Quinase 1 de Adesão Focal/antagonistas & inibidores , Neuroblastoma/patologia , Quinolonas/farmacologia , Sulfonas/farmacologia , Animais , Apoptose , Ciclo Celular , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nature ; 572(7771): 676-680, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391581

RESUMO

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)6, is overexpressed in several cancers7-9, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma10 cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Terapia de Alvo Molecular , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Fenótipo , Ligação Proteica
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