RESUMO
Neuroblastoma (NB) is a heterogeneous developmental tumor occurring in childhood, which arises from the embryonic sympathoadrenal cells of the neural crest. Although the recent progress that has been done on this tumor, the mechanisms involved in NB are still partially unknown. Despite some genetic aberrations having been identified, the sporadic cases represent the majority. Due to its wide heterogeneity in clinical behavior and etiology, NB represents a challenge in terms of prevention and treatment. Since a definitive therapy is lacking so far, there is an urgent necessity to unveil the molecular mechanisms behind NB onset and progression to develop new therapeutic approaches. Long non-coding RNAs (lncRNAs) are a group of RNAs longer than 200 nucleotides. Whether lncRNAs are destined to become a protein or not, they exert multiple biological functions such as regulating gene expression and functions. In recent decades, different research has highlighted the possible role of lncRNAs in the pathogenesis of many diseases, including cancer. Moreover, lncRNAs may represent potential markers or targets for diagnosis and treatment of diseases. This mini-review aimed to briefly summarize the most recent findings on the involvement of some lncRNAs in NB disease by focusing on their mechanisms of action and possible role in unveiling NB onset and progression.
Assuntos
Neuroblastoma/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , RNA Longo não Codificante/genéticaRESUMO
High-risk neuroblastoma (HR-NB) still remains the most dangerous tumor in early childhood. For this reason, the identification of new therapeutic approaches is of fundamental importance. Recently, we combined the conventional pharmacological approach to NB, represented by cisplatin, with fendiline hydrochloride, an inhibitor of several transporters involved in multidrug resistance of cancer cells, which demonstrated an enhancement of the ability of cisplatin to induce apoptosis. In this work, we co-administrated acetazolamide, a carbonic anhydrase isoform IX (CAIX) inhibitor which was reported to increase chemotherapy efficacy in various cancer types, to the cisplatin/fendiline approach in SKNBE2 xenografts in NOD-SCID mice with the aim of identifying a novel and more effective treatment. We observed that the combination of the three drugs increases more than twelvefold the differences in the cytotoxic activity of cisplatin alone, leading to a remarkable decrease of the expression of malignancy markers. Our conclusion is that this approach, based on three FDA-approved drugs, may constitute an appropriate improvement of the pharmacological approach to HR-NB.
Assuntos
Acetazolamida/farmacologia , Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Cisplatino/farmacologia , Fendilina/farmacologia , Neuroblastoma/tratamento farmacológico , Animais , Apoptose , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. METHODS: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. RESULTS: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. CONCLUSIONS: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.
Assuntos
Cateninas/metabolismo , Glioblastoma/tratamento farmacológico , Isopenteniladenosina/farmacologia , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Cateninas/genética , Linhagem Celular Tumoral , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genéticaRESUMO
The aim of this work was to deeply investigate the structure and properties of electrochemically synthesized silver nanoparticles (AgNPs) through high-resolution techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta Potential measurements, and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Strong brightness, tendency to generate nanoclusters containing an odd number of atoms, and absence of the free silver ions in solution were observed. The research also highlighted that the chemical and physical properties of the AgNPs seemed to be related to their peculiar oxidative state as suggested by X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRPD) analyses. Finally, the MTT assay tested the low cytotoxicity of the investigated AgNPs.
Assuntos
Química Verde/métodos , Nanopartículas Metálicas/química , Prata/química , Soluções/química , Microscopia Eletrônica de Transmissão/métodos , Espectrometria por Raios X/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Difração de Raios X/métodosRESUMO
CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer metastasis and inflammatory diseases. Starting from our previously-developed pharmacophoric model, we applied a combined computational and experimental approach that led to the identification of the hydantoin alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae, as novel CXCR4 antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B was identified as the most potent CXCR4 antagonist with an IC50 value of 9.3 nM, even though all the investigated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency and efficacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure-activity relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening.
Assuntos
Alcaloides/química , Antozoários/química , Receptores CXCR4/antagonistas & inibidores , Alcaloides/farmacologia , Animais , Antozoários/metabolismo , Clonagem Molecular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Descoberta de Drogas , Humanos , Hidantoínas , Simulação de Acoplamento Molecular , Ratos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Despite significant improvement of neuroblastoma (NB) patients' survival due to recent treatment advancements in recent years, NB is still associated with high mortality rate. In search of novel strategies to increase NB's susceptibility to pharmacological treatments, we investigated the in vitro and in vivo effects of fendiline hydrochloride as an enhancer of cisplatin antitumor activity. To assess the modulation of fendiline treatment on cisplatin responses, we used in vitro (evaluating NB cell proliferation by XCELLigence technology and colony formation, and gene expression by RT-PCR) and in vivo (NB cell grafts in NOD-SCID mice) models of NB. NB cell treatment with fendiline induced the expression of the ncRNA NDM29, leading to cell differentiation and to the reduction of the expression of MDRs/ABC transporters linked to multidrug resistance. These events were correlated to higher NB cell susceptibility to cisplatin and, consequently, increased its cytotoxic potency. In vivo, this drug interaction causes an enhanced ability of cisplatin to induce apoptosis in NB masses, resulting in tumor growth reduction and prolonged animal survival rate. Thus, the administration of fendiline might be a possible novel therapeutic approach to increase cisplatin efficacy in aggressive and poorly responsive NB cases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Cisplatino/administração & dosagem , Fendilina/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , RNA não Traduzido/metabolismoRESUMO
Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Metformina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cães , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/patologiaRESUMO
The aim of this review is to critically analyze promises and limitations of pharmacological inducers of autophagy against protein misfolding-associated neurodegeneration. Effective therapies against neurodegenerative disorders can be developed by regulating the "self-defense" equipment of neurons, such as autophagy. Through the degradation and recycling of the intracellular content, autophagy promotes neuron survival in conditions of trophic factor deprivation, oxidative stress, mitochondrial and lysosomal damage, or accumulation of misfolded proteins. Autophagy involves the activation of self-digestive pathways, which is different for dynamics (macro, micro and chaperone-mediated autophagy), or degraded material (mitophagy, lysophagy, aggrephagy). All neurodegenerative disorders share common pathogenic mechanisms, including the impairment of autophagic flux, which causes the inability to remove the neurotoxic oligomers of misfolded proteins. Pharmacological activation of autophagy is typically achieved by blocking the kinase activity of mammalian target of rapamycin (mTOR) enzymatic complex 1 (mTORC1), removing its autophagy suppressor activity observed under physiological conditions; acting in this way, rapamycin provided the first proof of principle that pharmacological autophagy enhancement can induce neuroprotection through the facilitation of oligomers' clearance. The demand for effective disease-modifying strategies against neurodegenerative disorders is currently stimulating the development of a wide number of novel molecules, as well as the re-evaluation of old drugs for their pro-autophagic potential.
Assuntos
Autofagia/efeitos dos fármacos , Descoberta de Drogas , Neuroproteção/efeitos dos fármacos , Animais , Autofagia/genética , Biomarcadores , Descoberta de Drogas/métodos , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Relação Estrutura-AtividadeRESUMO
Endogenous somatostatin shows anti-secretory effects in both physiological and pathological settings, as well as inhibitory activity on cell growth. Since somatostatin is not suitable for clinical practice, researchers developed synthetic somatostatin receptor ligands (SRLs) to overcome this limitation. Currently, SRLs represent pivotal tools in the treatment algorithm of neuroendocrine tumors (NETs). Octreotide and lanreotide are the first-generation SRLs developed and show a preferential binding affinity to somatostatin receptor (SST) subtype 2, while pasireotide, which is a second-generation SRL, has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). A number of studies demonstrated that first-generation and second-generation SRLs show distinct functional properties, besides the mere receptor affinity. Therefore, the aim of the present review is to critically review the current evidence on the biological effects of SRLs in pituitary adenomas and neuroendocrine tumors, by mainly focusing on the differences between first-generation and second-generation ligands.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Estudos Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Tumores Neuroendócrinos/etiologia , Tumores Neuroendócrinos/patologia , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/etiologia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Ligação Proteica , Multimerização Proteica , Receptores de Somatostatina/química , Transdução de Sinais , Resultado do TratamentoRESUMO
In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers."
Assuntos
Antineoplásicos/uso terapêutico , Canais de Cloreto/metabolismo , Regulação Neoplásica da Expressão Gênica , Moduladores de Transporte de Membrana/uso terapêutico , Neoplasias/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Progressão da Doença , Humanos , Interações Hidrofóbicas e Hidrofílicas , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Especificidade de Órgãos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.
Assuntos
Celecoxib/farmacologia , Dinoprostona/metabolismo , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Neuroglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , Proteínas Priônicas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Inflamação/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.
Assuntos
Doenças do Cão/metabolismo , Células-Tronco Neoplásicas/fisiologia , Osteossarcoma/veterinária , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Cães , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/citologiaRESUMO
BACKGROUND: Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. METHODS: Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. RESULTS: We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. CONCLUSIONS: Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias Mamárias Animais , Metformina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacocinética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Antígeno Ki-67/metabolismo , Metformina/farmacocinética , Camundongos , Fenótipo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid ß production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aß production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/genética , Ciclo Celular , Neuroblastoma/patologia , RNA Nuclear Pequeno/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloidose/metabolismo , Animais , Apoptose , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Testes para Micronúcleos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches.
Assuntos
Adiponectina/farmacologia , Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Glioblastoma/patologia , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Adiponectina/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential. METHODS: Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype. RESULTS: We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29. CONCLUSION: These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.
RESUMO
Glioblastoma (GBM) is the most malignant brain tumor, characterized by cell heterogeneity comprising stem cells (GSCs) responsible for aggressiveness. The calpain/calpastatin (calp/cast) proteolytic system is involved in critical physiological processes and cancer progression. In this work we showed the expression profile of hcast 3-25 (a Type III calpastatin variant devoid of inhibitory units) and the members of the system in several patient-derived GSCs exploring the relationship between hcast 3-25 and activation/activity of calpains. Each GSC shows a peculiar calp/cast mRNA and protein expression pattern, and hcast 3-25 is the least expressed. Differentiation promotes upregulation of all the calp/cast system components except hcast 3-25 mRNA, which increased or decreased depending on individual GSC culture. Transfection of hcast 3-25-V5 into two selected GSCs indicated that hcast 3-25 effectively associates with calpains, supporting the digestion of selected calpain targets. Hcast 3-25 possibly affects the stem state promoting a differentiated, less aggressive phenotype.
RESUMO
Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.
RESUMO
Glioblastoma (GBM), one of the deadliest brain tumors, accounts for approximately 50% of all primary malignant CNS tumors, therefore novel, highly effective remedies are urgently needed. Boron neutron capture therapy, which has recently repositioned as a promising strategy to treat high-grade gliomas, requires a conspicuous accumulation of boron atoms in the cancer cells. With the aim of selectively deliver sodium borocaptate (BSH, a 12 B atoms-including molecule already employed in the clinics) to GBM cells, we developed novel cell membrane-derived vesicles (CMVs), overcoming the limits of natural extracellular vesicles as drug carriers, while maintaining their inherent homing abilities that make them preferable to fully synthetic nanocarriers. Purified cell membrane fragments, isolated from patient-derived GBM stem-like cell cultures, were used to prepare nanosized CMVs, which retained some membrane proteins specific of the GBM parent cells and were devoid of potentially detrimental genetic material. In vitro tests evidenced the targeting ability of this novel nanosystem and ruled out any cytotoxicity. The CMVs were successfully loaded with BSH, by following two different procedures, i.e. sonication and electroporation, demonstrating their potential applicability in GBM therapy.
Assuntos
Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas , Membrana Celular , Glioblastoma , Humanos , Terapia por Captura de Nêutron de Boro/métodos , Glioblastoma/radioterapia , Glioblastoma/patologia , Glioblastoma/terapia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patologia , Membrana Celular/metabolismo , Boroidretos/química , Linhagem Celular Tumoral , Portadores de Fármacos/química , Nanopartículas/química , Compostos de SulfidrilaRESUMO
Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid ß peptide (Aß) secretion and the concomitant increment of Aß x-42/Aß x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aß formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aß peptides in the extracellular space, as it may occur in Alzheimer's Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies.