RESUMO
Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteína Quinase CDC2/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Perfilação da Expressão Gênica , Humanos , Análise dos Mínimos Quadrados , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes , Fosforilação , Software , Transfecção , UbiquitinaçãoRESUMO
The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent-chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 µg/ml) and MK-5108 inhibitor (0.1 µM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.
Assuntos
Apoptose/genética , Aurora Quinase A/genética , Autofagia/genética , Neuroblastoma/genética , Fatores de Transcrição/genética , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Aurora Quinase A/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/farmacologia , Gangliosídeos/antagonistas & inibidores , Gangliosídeos/genética , Humanos , Camundongos , Neuroblastoma/patologia , Tiazóis/farmacologia , Fatores de Transcrição/antagonistas & inibidoresRESUMO
The main physiological function of MCPIP1 (regnase-1) is negative regulation of inflammation. Moreover, roles of regnase-1 in apoptosis and differentiation have also been described, but its involvement in cancer is yet to be fully recognized. Earlier, we showed a lack of expression of MCPIP1 in both primary tumors and several neuroblastoma cell lines. Additionally, we reported that levels of MCPIP1 and the key neuroblastoma oncoprotein-MYCN were inversely correlated in BE(2)-C clones overexpressing the MCPIP1 gene. Here, we show that exogenous expression of the MCPIP1 protein decreases MYCN mRNA and protein levels without changing the MYCN mRNA half-life. Furthermore, it was shown that MCPIP1-wt exogenous expression affects levels and phosphorylation of MYCN partners such as Aurora A (Thr288), CDC2 (Tyr15 and Thr161), GSK3ß (Ser9), and key cellular components of Akt/mTOR signaling, which regulate MYCN stability and activation. In accordance with the obtained results, we found increased phosphorylation of MYCN protein at Thr58 that causes destabilization of the oncoprotein. Moreover, it is shown that exogenous expression of MCPIP1 does not cause apoptosis. Our data extend knowledge on roles of MCPIP1 in our model and link the protein to regulation of expression and stability of MYCN through decrease of signaling via Akt/mTOR pathway. J. Cell. Biochem. 118: 1741-1755, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/genética , Apoptose/fisiologia , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Western Blotting , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Fosforilação/genética , Fosforilação/fisiologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ribonucleases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genéticaRESUMO
Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a "key and lock" interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.
Assuntos
Anticorpos Monoclonais , Gangliosídeos , Fragmentos Fab das Imunoglobulinas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Gangliosídeos/química , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Mimetismo Molecular , Conformação ProteicaRESUMO
The recently discovered MCPIP1 (monocyte chemoattractant protein-induced protein 1), a multidomain protein encoded by the MCPIP1 (ZC3H12A) gene, has been described as a new differentiation factor, a ribonuclease, and a deubiquitination-supporting factor. However, its role in cancer is poorly recognized. Our recent analysis of microarrays data showed a lack of expression of the MCPIP1 transcript in primary neuroblastoma, the most common extracranial solid tumor in children. Additionally, enforced expression of the MCPIP1 gene in BE(2)-C cells caused a significant decrease in neuroblastoma proliferation and viability. Aim of the present study was to further investigate the role of MCPIP1 in neuroblastoma, using expression DNA microarrays and microRNA microarrays. Transient transfections of BE(2)-C cells were used for overexpression of either wild type of MCPIP1 (MCPIP1-wt) or its RN-ase defective mutant (MCPIP1-ΔPIN). We have analyzed changes of transcriptome and next, we have used qRT-PCR to verify mRNA levels of selected genes responding to MCPIP1 overexpression. Additionally, protein levels were determined for some of the selected genes. The choline transporter, CTL1, encoded by the SLC44A1 gene, was significantly repressed at the specific mRNA and protein levels and most importantly this translated into a decreased choline transport in MCPIP1-overexpressing cells. Then, we have found microRNA-3613-3p as the mostly altered in the pools of cells overexpressing the wild type MCPIP1. Next, we analyzed the predicted targets of the miR-3613-3p and validated them using qRT-PCR and western blot. These results indicate that the expression of miR-3613-3p might be regulated by MCPIP1 by cleavage of its precursor form.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neuroblastoma/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Linhagem Celular Tumoral , Colina/metabolismo , Humanos , MicroRNAs/metabolismo , Neuroblastoma/genética , Interferência de RNA , Ribonucleases/genética , Fatores de Transcrição/genética , TranscriptomaRESUMO
Gangliosides are a diverse group of sialic acid containing glycosphigolipids that are abundantly present in an outer plasma membrane of some cells. Biological roles of gangliosides and other lipids in cell fate regulation are being extensively studied. Gangliosides are well known to be involved in interactions between cells and in signal transduction to regulate growth, adhesion and motility. Moreover, many gangliosides are tumor-associated antigens over-expressed on several tumor types. As a result, monoclonal antibodies binding gangliosides can be used to diagnose, monitor and to treat cancer patients. In the review, we gather and discuss data of various research groups on direct cytotoxic effects elicited by several ganglioside-specific antibodies, which bind to GM2, N-acetyl-GM2, N-glycolyl-GM2, GM3, GD3, GD2, O-acetyl-GD2, without involvement of immunological mechanisms. Thus, in cultures of numerous human and mouse cancer cell lines, the antibodies were reported to cause morphological changes, aggregation and detachment of cells, inhibition of proliferation and cell death involving necrosis, apoptosis and oncosis-like mechanisms. Additionally, data on proteome alterations were reviewed that encompass, among others, changes in kinome (P38, JNK, c-MET, ERK1/2, PI3K, AKT, FAK, aurora A, B, C), protein levels of transcription factors (P53, MYCN, HSF1) and pro-apoptotic proteins (caspase 3, BAX). Next, we collected data on application of the antibodies to enhance cytotoxicity of chemotherapeutic drugs and small molecule inhibitors. Finally, further research perspectives on the topic are discussed.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose , Gangliosídeos/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.
RESUMO
Neuroblastoma (NB) is a pediatric neuroendocrine neoplasm. It arises from the sympatho-adrenal lineage of neural-crest-derived multipotent progenitor cells that fail to differentiate. NB is the most common extracranial tumor in children, and it manifests undisputed heterogeneity. Unsatisfactory outcomes of high-risk (HR) NB patients call for more research to further inter-relate treatment and molecular features of the disease. In this regard, it is well established that in the tumor microenvironment (TME), malignant cells are engaged in complex and dynamic interactions with the extracellular matrix (ECM) and stromal cells. The ECM can be a source of both pro- and anti-tumorigenic factors to regulate tumor cell fate, such as survival, proliferation, and resistance to therapy. Moreover, the ECM composition, organization, and resulting signaling networks are vastly remodeled during tumor progression and metastasis. This review mainly focuses on the molecular mechanisms and effects of interactions of selected ECM components with their receptors on neuroblastoma cells. Additionally, it describes roles of enzymes modifying and degrading ECM in NB. Finally, the article gives examples on how the knowledge is exploited for prognosis and to yield new treatment options for NB patients.
Assuntos
Neuroblastoma , Comunicação Celular , Criança , Matriz Extracelular/patologia , Humanos , Neuroblastoma/patologia , Neuroblastoma/terapia , Transdução de Sinais , Microambiente TumoralRESUMO
In recent years, immunotherapy has been identified as an effective treatment method for high-risk neuroblastoma. A previous study demonstrated that an anti-GD2 ganglioside (GD2) mouse 14G2a monoclonal antibody (mAb) combined with a small molecule, i.e., an aurora A kinase inhibitor (MK-5108), significantly increased cytotoxicity against human neuroblastoma cells, as compared to monotherapy. This study aimed to demonstrate the mechanism of neuroblastoma cell death in vitro following the addition of an anti-GD2 human-mouse chimeric ch14.18/CHO mAb (presently used in clinics) and two aurora A inhibitors (MK-5108 and MK-8745). The effects of the aforementioned agents on neuroblastoma cells were determined by measuring the level of ATP, the level of apoptotic and necroptotic markers, and the activity of caspase 3/7. The results revealed that the ch14.18/CHO mAb decreased cellular ATP levels in the IMR-32 and CHP-134 neuroblastoma cell lines, similarly to the 14G2a mAb. Regarding ch14.18/CHO mAb treated IMR-32 cells, the observed cytotoxic effect was concomitant with induced caspase 3 cleavage, which indicated the induction of apoptosis in IMR-32 cells, but not in CHP-134 cells. Furthermore, the MK-5108 inhibitor induced apoptosis, as indicated by the increased cleavage of caspase 3 and increased activity of caspase 3/7. However, the presence of necroptosis was ruled out in MK-5108-treated IMR-32 and CHP-134 cells. In summary, the effects of the combination of ch14.18/CHO mAb and aurora A kinase inhibitors (MK-5108 and MK-8745) were shown to enhance apoptosis in IMR-32 cells compared to when used individually.
Assuntos
Antineoplásicos , Neuroblastoma , Trifosfato de Adenosina , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose , Aurora Quinase A , Caspase 3 , Linhagem Celular Tumoral , Ácidos Cicloexanocarboxílicos , Gangliosídeos/metabolismo , Humanos , Camundongos , Neuroblastoma/tratamento farmacológico , Piperazinas , TiazóisRESUMO
BACKGROUND: MCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored. RESULTS: Here we report that the proinflammatory cytokine IL-1beta rapidly induces the synthesis of MCPIP in primary monocyte-derived macrophages and HepG2 cells. This up-regulation takes place through the MAP kinase pathway and following activation of the transcription factor Elk-1. Using a ZC3H12A reporter construct we have shown that a ZC3H12A promoter region, stretching from -76 to +60, mediates activation by IL-1beta. This region contains binding sites for Elk-1 and its partner SRF. Chromatin immunoprecipitation analysis confirms in vivo binding of both transcription factors to this region of the ZC3H12A promoter. CONCLUSIONS: We conclude that the transcription factor Elk-1 plays an important role in the activation of ZC3H12A expression in response to IL-1beta stimulation.
Assuntos
Interleucina-1beta/farmacologia , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas Elk-1 do Domínio ets/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , Células Hep G2 , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Ribonucleases , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional gene regulation. Furthermore, dysregulation of miRNA expression is an important factor in the pathogenesis of neuroblastoma. Our previous study identified that overexpression of monocyte chemoattractant proteininduced protein 1 protein led to a significant downregulation of a novel miRNA molecule, miRNA36133p. In the present study, the potential involvement of miRNA36133p in the cell biology of neuroblastoma was investigated. It was identified that the expression of miRNA36133p varies among a range of human neuroblastoma cell lines. As the delineation of the functions of a miRNA requires the identification of its target genes, seven putative mRNAs that may be regulated by miRNA36133p were selected. Furthermore, it was identified that overexpression of miRNA36133p causes significant downregulation of several genes exhibiting tumor suppressive potential [encoding apoptotic proteaseactivating factor 1 (APAF1), Dicer, DNA fragmentation factor subunit ß, von HippelLindau protein and neurofibromin 1] in BE(2)C human neuroblastoma cells. APAF1 mRNA was the most significantly decreased transcript in the cells with miRNA36133p overexpression. In accordance with the aforementioned results, the downregulation of cleaved caspase-9 and lack of activation of executive caspases in BE(2)C cells following miRNA36133p overexpression was observed. The results of the present study suggest a potential underlying molecular mechanism of apoptosis inhibition via APAF1 downregulation in human neuroblastoma BE(2)C cells with miRNA36133p overexpression.
Assuntos
Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neuroblastoma/genética , Regiões 3' não Traduzidas/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.
Assuntos
Gangliosídeos/química , Mimetismo Molecular , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Linhagem Celular Tumoral , Células Clonais , Dissulfetos , Ensaio de Imunoadsorção Enzimática , Gangliosídeos/imunologia , Humanos , Immunoblotting , Dados de Sequência Molecular , Neuroblastoma/patologia , Peptídeos/química , Ligação Proteica , Análise de Sequência de ProteínaRESUMO
The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.
Assuntos
Vacinas Anticâncer/imunologia , Gangliosídeos/imunologia , Imunoglobulina G/biossíntese , Imunoterapia Ativa/métodos , Peptídeos/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Vacinas Anticâncer/genética , Vacinas Anticâncer/farmacologia , Reações Cruzadas , Feminino , Gangliosídeos/genética , Humanos , Imunoglobulina G/imunologia , Melanoma/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Dados de Sequência Molecular , Neuroblastoma/imunologia , Neuroblastoma/terapia , Biblioteca de Peptídeos , Peptídeos/genética , Vacinas de DNA/genética , Vacinas de DNA/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1ß1 heterodimer), BIO 1211 (inhibiting active α4ß1 heterodimer), cilengitide and SB273005 (inhibitors of αVß3, αVß5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Matriz Extracelular/imunologia , Gangliosídeos/imunologia , Neuroblastoma/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Caspase 3/biossíntese , Linhagem Celular Tumoral , Diterpenos , Gangliosídeos/genética , Humanos , Camundongos , Neuroblastoma/genética , Neuroblastoma/imunologia , Neuroblastoma/patologia , Bibliotecas de Moléculas Pequenas/administração & dosagem , Venenos de Serpentes/administração & dosagemRESUMO
We have recently shown that mRNA and protein of PHLDA1 (pleckstrin-homology-like domain family A, member 1) were by far the most upregulated molecules upon treatment of IMR-32 cells with the anti-GD2 ganglioside monoclonal antibody 14G2a. Hence, we decided to study functions of PHLDA1 using human neuroblastoma IMR-32 cells as a model. Here, we show that constitutive expression of mRNA and protein of the PHLDA1 gene in IMR-32 cells was inversely correlated with transcript of the AURKA gene and Aurora A oncoprotein. Next, we silenced PHLDA1 expression in IMR-32 cells using an shRNA interference method. We report that IMR-32 cells with stable downregulation of PHLDA1 showed enhanced cellular ATP levels and an increase in mitochondrial membrane potential, as compared to control and non-transduced cells. We demonstrated that downregulation of PHLDA1 leads to a significant increase in expression of Aurora A and TRKB that are markers of poor prognosis in neuroblastoma. Also, we measured an increase in Aurora A and Akt kinases phosphorylation in the cells. Most importantly, PHLDA1-silenced cells were less susceptible to apoptosis than control cells, as shown by the lower expression of cleaved caspase-3 and PARP as well as a decreased activity of caspase-3 and -7. Our study negatively correlates expression of PHLDA1 and Aurora A in IMR-32 cells and sheds new light on functions of PHLDA1 in the neuroblastoma tumor cells, suggesting its role as a pro-apoptotic protein. Additionally, our results show possible links of the protein to regulation of features of mitochondria and formation of autophagosomes.
Assuntos
Aurora Quinase A/biossíntese , Glicoproteínas de Membrana/biossíntese , Neuroblastoma/genética , Proteínas Tirosina Quinases/biossíntese , Fatores de Transcrição/genética , Trifosfato de Adenosina/biossíntese , Apoptose/genética , Aurora Quinase A/genética , Autofagia/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neuroblastoma/patologia , Proteínas Tirosina Quinases/genética , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Receptor trkB , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossínteseRESUMO
Mechanisms leading to inhibitory effects of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) and PI3K/Akt/mTOR pathway inhibitors on human neuroblastoma cell survival were studied in vitro. We have recently shown on IMR-32, CHP134, and LA-N-1 neuroblastoma cells that targeting GD2 with the mAb decreases cell viability of the cell lines. In this study we used cytotoxicity assays, proteomic arrays and immunoblotting to evaluate the response of the three cell lines to the antiGD2 14G2a mAb and specific PI3K/Akt/mTOR pathway inhibitors. We show here that the mAb modulates intracellular signal transduction through changes in several kinases and their substrates phosphorylation. More detailed analysis of the PI3K/Akt/mTOR pathway showed significant decrease in activity of Akt, mTOR, p70 S6 and 4E-BP1 proteins and transient increase in PTEN (a suppressor of the pathway), leading to inhibition of the signaling network responsible for stimulation of translation and proliferation. Additionally, combining the GD2-specific 14G2a mAb with an Akt inhibitor (perifosine), dual mTOR/PI3K inhibitors (BEZ-235 and SAR245409), and a pan-PI3K inhibitor (LY294002) was shown to enhance cytotoxic effects against IMR-32, CHP134 and LA-N-1 cells. Our study extends knowledge on mechanisms of action of the 14G2a mAb on the neuroblastoma cells. Also, it stresses the need for further delineation of molecular signal orchestration aimed at more reasonable selection of drugs to target key cellular pathways in quest for better cure for neuroblastoma patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Gangliosídeos/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/farmacologia , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables, which has been shown to exert an anti-cancer effect when tested in vitro and in vivo. The anti-cancer effects of SFN encompass induction of cytoprotective autophagy; therefore, the present study aimed to determine whether the chemopreventive activity of SFN may be potentiated by inhibition of autophagy. The present study provided detailed insight into the susceptibility of human neuroblastoma cells to treatment with synthetic SFN, in combination with an inhibitor of autophagy, 3-methyladenine (3-MA). The present study confirmed the suppression of the viability of the human neuroblastoma cell line BE(2)-C by SFN and reported the inhibition of DNA synthesis, as determined by a decrease in tritiated thymidine incorporation. Furthermore, the results verified the effectiveness of SFN in inducing apoptosis in the BE(2)-C cell line as demonstrated by caspase activation, increased protein expression levels of B-cell lymphoma 2-associated X protein and loss of mitochondrial membrane potential. Combined treatment of the cells with SFN with 3-MA proved to be effective in decreasing cell viability, through a mechanism that may proceed via the early induction of autophagy by SFN, followed by induction of apoptosis, as well as inhibition of autophagy by 3-MA.
Assuntos
Adenina/análogos & derivados , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Adenina/administração & dosagem , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Isotiocianatos/administração & dosagem , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neuroblastoma/genética , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , SulfóxidosRESUMO
Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.
Assuntos
Doxorrubicina/química , Gangliosídeos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neuroblastoma/patologia , Animais , Antibióticos Antineoplásicos/química , Anticorpos Monoclonais/química , Linhagem Celular Tumoral/efeitos dos fármacos , Análise por Conglomerados , Relação Dose-Resposta a Droga , Humanos , Camundongos , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Software , Topotecan/química , TranscriptomaRESUMO
Aberrant glycosylation is a universal feature of a cancer cell. The examples of such differences are the observed quantitative and qualitative changes in the expression of gangliosides of the tumors of the neuroectodermal origin. The role of gangliosides in cancer progression has been described, as well as their abilities to act as immunosuppressors. The presence of large amounts of these tumor associated carbohydrate antigens (TACA) on cancer cells, as compared to normal cells, opens the possibilities to use them in diagnosis and immunotherapeutic approaches which engage the immune system to fight with a tumor disease. The passive immunotherapy of neuroblastoma with anti-gangliosides monoclonal antibodies and their derivatives has been reviewed. The advantages and the disadvantages of using carbohydrate antigens as vaccines have been summarized. The examples of use of active specific immunotherapy with gangliosides have been described, as well as the approaches to modify the immunogenic potential of these antigens with carbohydrate-protein conjugate vaccines, and antiidiotypic antibodies used with immunomodulators such as QA-21. Finally, in a separate paragraph, the application of anti-carbohydrate antibodies to screen phage display peptide libraries for mimotopes has been described. The perspectives of using carbohydrate mimicking surrogate antigens in the immunotherapy of cancer have been discussed.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Gangliosídeos/imunologia , Imunização Passiva/métodos , Neuroblastoma/imunologia , Neuroblastoma/terapia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Vacinas Anticâncer/isolamento & purificação , Vacinas Anticâncer/uso terapêutico , Glicosilação , HumanosRESUMO
The importance of monocyte chemotactic protein-1-induced protein 1 (MCPIP1) in the negative regulation of inflammatory reactions has already been extensively studied. However, its role in cancer is not yet established. We studied MCPIP1 gene expression in primary human neuroblastomas and several neuroblastoma cell lines. Our results showed a lack of MCPIP1 expression in primary neuroblastoma tumors. Moreover, it was found that the low expression of the protein measured in human neuroblastoma cell lines might be important for neuroblastoma survival, since enforced MCPIP1 gene expression in human neuroblastoma BE(2)-C cells caused a significant decrease in neuroblastoma cell viability and proliferation.