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1.
Compr Rev Food Sci Food Saf ; : e13426, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39169551

RESUMO

Bioactive compounds in fruit and vegetables have a positive impact on human health by reducing oxidative stress, inflammation, and the risk of chronic diseases such as cancer, cardiovascular (CV) diseases, and metabolic disorders. However, some fruit and vegetables must be heated before consumption and thermal processes can modify the amount of nutraceuticals, that is, polyphenols, carotenoids, glucosinolates, and ascorbic acid, that can increase or decrease in relation to different factors such as type of processing, temperature, and time but also the plant part (e.g., flower, leaf, tuber, and root) utilized as food. Another important aspect is related to the bioaccessibility and bioavailability of nutraceuticals. Indeed, the key stage of nutraceutical bioefficiency is oral bioavailability, which involves the release of nutraceuticals from fruit and vegetables in gastrointestinal fluids, the solubilization of nutraceuticals and their interaction with other components of gastrointestinal fluids, the absorption of nutraceuticals by the epithelial layer, and the chemical and biochemical transformations into epithelial cells. Several studies have shown that thermal processing can enhance the absorption of nutraceuticals from fruit and vegetable. Once absorbed, they reach the blood vessels and promote multiple biological effects (e.g., antioxidant, anti-inflammatory, antihypertensive, vasoprotective, and cardioprotective). In this review, we described the impact of different thermal processes (such as boiling, steaming and superheated steaming, blanching, and microwaving) on the retention/degradation of bioactive compounds and their health-promoting effects after the intake. We then summarized the impact of heating on the absorption of nutraceuticals and the biological effects promoted by natural compounds in the CV system to provide a comprehensive overview of the potential impact of thermal processing on the CV benefits of fruit and vegetables.

2.
Semin Cancer Biol ; 79: 18-43, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-32659257

RESUMO

Immune checkpoint inhibitors (ICIs) have shown unprecedented benefits in various adult cancers, and this success has prompted the exploration of ICI therapy even in childhood malignances. Although the use of ICIs as individual agents has achieved disappointing response rates, combinational therapies are likely to promise better results. However, only a subset of patients experienced prolonged clinical effects, thus suggesting the need to identify robust bio-markers that predict individual clinical response or resistance to ICI therapy as the main challenge. In this review, we focus on how the use of ICIs in adult cancers can be translated into pediatric malignances. We discuss the physiological mechanism of action of each IC, including PD-1, PD-L1 and CTLA-4 and the new emerging ones, LAG-3, TIM-3, TIGIT, B7-H3, BTLA and IDO-1, and evaluate their prognostic value in both adult and childhood tumors. Furthermore, we offer an overview of preclinical models and clinical trials currently under investigation to improve the effectiveness of cancer immunotherapies in these patients. Finally, we outline the main predictive factors that influence the efficacy of ICIs, in order to lay the basis for the development of a pan-cancer immunogenomic model, able to direct young patients towards more specific immunotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Adolescente , Antígenos B7/análise , Antígeno B7-H1/análise , Antígeno CTLA-4/análise , Criança , Pré-Escolar , Terapia Combinada/métodos , Humanos , Fatores Imunológicos/uso terapêutico , Lactente , Recém-Nascido , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/análise , Resultado do Tratamento
3.
Int J Mol Sci ; 24(16)2023 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-37628929

RESUMO

Nonalcoholic fatty liver disease (NAFLD, including nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH)) is a high-prevalence disorder, affecting about 1 billion people, which can evolve to more severe conditions like cirrhosis or hepatocellular carcinoma. NAFLD is often concomitant with conditions of the metabolic syndrome, such as central obesity and insulin-resistance, but a specific drug able to revert NAFL and prevent its evolution towards NASH is still lacking. With the liver being a key organ in metabolic processes, the potential therapeutic strategies are many, and range from directly targeting the lipid metabolism to the prevention of tissue inflammation. However, side effects have been reported for the drugs tested up to now. In this review, different approaches to the treatment of NAFLD are presented, including newer therapies and ongoing clinical trials. Particular focus is placed on the reverse cholesterol transport system and on the agonists for nuclear factors like PPAR and FXR, but also drugs initially developed for other conditions such as incretins and thyromimetics along with validated natural compounds that have anti-inflammatory potential. This work provides an overview of the different therapeutic strategies currently being tested for NAFLD, other than, or along with, the recommendation of weight loss.


Assuntos
Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Metabolismo dos Lipídeos , Transporte Biológico
4.
J Pathol ; 250(2): 134-147, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31518438

RESUMO

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK - a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Fatores de Transcrição E2F/metabolismo , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Genes p53 , Humanos , Camundongos Nus , Terapia de Alvo Molecular/métodos , Estadiamento de Neoplasias , Organoides/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804873

RESUMO

Different functional states determine glioblastoma (GBM) heterogeneity. Brain cancer cells coexist with the glial cells in a functional syncytium based on a continuous metabolic rewiring. However, standard glioma therapies do not account for the effects of the glial cells within the tumor microenvironment. This may be a possible reason for the lack of improvements in patients with high-grade gliomas therapies. Cell metabolism and bioenergetic fitness depend on the availability of nutrients and interactions in the microenvironment. It is strictly related to the cell location in the tumor mass, proximity to blood vessels, biochemical gradients, and tumor evolution, underlying the influence of the context and the timeline in anti-tumor therapeutic approaches. Besides the cancer metabolic strategies, here we review the modifications found in the GBM-associated glia, focusing on morphological, molecular, and metabolic features. We propose to analyze the GBM metabolic rewiring processes from a systems biology perspective. We aim at defining the crosstalk between GBM and the glial cells as modules. The complex networking may be expressed by metabolic modules corresponding to the GBM growth and spreading phases. Variation in the oxidative phosphorylation (OXPHOS) rate and regulation appears to be the most important part of the metabolic and functional heterogeneity, correlating with glycolysis and response to hypoxia. Integrated metabolic modules along with molecular and morphological features could allow the identification of key factors for controlling the GBM-stroma metabolism in multi-targeted, time-dependent therapies.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Microambiente Tumoral , Animais , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Humanos , Hipóxia Tumoral , Efeito Warburg em Oncologia
6.
Biophys J ; 110(10): 2151-61, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27224480

RESUMO

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.


Assuntos
Antineoplásicos/farmacologia , DNA/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Algoritmos , Cisplatino/farmacologia , DNA/metabolismo , Congelamento , Estrutura Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Plasmídeos/genética , Análise Espectral
7.
Tumour Biol ; 37(10): 13831-13842, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27481518

RESUMO

As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Colo/patologia , Neoplasias do Colo/genética , Variações do Número de Cópias de DNA , Mutação/genética , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Colo/metabolismo , Neoplasias do Colo/patologia , Hibridização Genômica Comparativa , Feminino , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico
8.
Plasmid ; 79: 22-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25779031

RESUMO

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.


Assuntos
5'-Nucleotidase/genética , Antígenos CD/genética , Apirase/genética , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , 5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Furina/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Heme Oxigenase-1/metabolismo , Humanos , Plasmídeos/genética , Transplante Heterólogo
9.
Nucleic Acids Res ; 41(3): 2009-19, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23248010

RESUMO

The opening of DNA double strands is extremely relevant to several biological functions, such as replication and transcription or binding of specific proteins. Such opening phenomenon is particularly sensitive to the aqueous solvent conditions in which the DNA molecule is dispersed, as it can be observed by considering the classical dependence of DNA melting temperature on pH and salt concentration. In the present work, we report a single-molecule study of the stability of DNA against denaturation when subjected to changes in solvent. We investigated the appearance of DNA instability under specific external applied force and imposed twist values, which was revealed by an increase in the temporal fluctuations in the DNA extension. These fluctuations occur in the presence of a continuous interval of equilibrium states, ranging from a plectonemic state to a state characterized by denaturation bubbles. In particular, we observe the fluctuations only around a characteristic force value. Moreover, this characteristic force is demonstrated to be notably sensitive to variations in the pH and ionic strength. Finally, an extension of a theoretical model of plectoneme formation is used to estimate the average denaturation energy, which is found to be linearly correlated to the melting temperature of the DNA double strands.


Assuntos
DNA/química , Fenômenos Biomecânicos , Concentração de Íons de Hidrogênio , Nanotecnologia , Desnaturação de Ácido Nucleico , Concentração Osmolar
10.
Insects ; 15(2)2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38392552

RESUMO

Hermetia illucens is a species of great interest for numerous industrial applications. A high-quality reference genome is already available for H. illucens. However, the worldwide maintenance of numerous captive populations of H. illucens, each with its own genotypic and phenotypic characteristics, made it of interest to perform a de novo genome assembly on one population of H. illucens to define a chromosome-scale genome assembly. By combining the PacBio and the Omni-C proximity ligation technologies, a new H. illucens chromosome-scale genome of 888.59 Mb, with a scaffold N50 value of 162.19 Mb, was assembled. The final chromosome-scale assembly obtained a BUSCO completeness of 89.1%. By exploiting the Omni-C proximity ligation technology, topologically associated domains and other topological features that play a key role in the regulation of gene expression were identified. Further, 65.62% of genomic sequences were masked as repeated sequences, and 32,516 genes were annotated using the MAKER pipeline. The H. illucens Lsp-2 genes that were annotated were further characterized, and the three-dimensional organization of the encoded proteins was predicted. A new chromosome-scale genome assembly of good quality for H. illucens was assembled, and the genomic annotation phase was initiated. The availability of this new chromosome-scale genome assembly enables the further characterization, both genotypically and phenotypically, of a species of interest for several biotechnological applications.

11.
3 Biotech ; 14(2): 45, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38261961

RESUMO

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.

12.
J Exp Clin Cancer Res ; 43(1): 292, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39438988

RESUMO

BACKGROUND: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy. METHODS: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence. RESULTS: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival. CONCLUSIONS: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.


Assuntos
Aminopeptidases , Benzamidas , Imunoterapia , Antígenos de Histocompatibilidade Menor , Neuroblastoma , Piridinas , Neuroblastoma/imunologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neuroblastoma/genética , Camundongos , Animais , Aminopeptidases/genética , Aminopeptidases/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Imunoterapia/métodos , Piridinas/farmacologia , Piridinas/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos
13.
Front Immunol ; 14: 1197053, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37359555

RESUMO

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy.


Assuntos
Células Matadoras Naturais , Neoplasias , Humanos , Ligantes , Neoplasias/genética , Neoplasias/terapia , Imunoterapia , Receptores de Antígenos/metabolismo
14.
J Neurosurg Sci ; 67(1): 55-65, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33056947

RESUMO

BACKGROUND: In the context of high-grade gliomas (HGGs), very little evidence is available concerning the optimal radiotherapy (RT) schedule to be used in radioimmunotherapy combinations. This studied was aimed at shedding new light in this field by analyzing the effects of RT dose escalation and dose fractionation on the tumor microenvironment of experimental HGGs. METHODS: Neurospheres (NS) CT-2A HGG-bearing C57BL/6 mice were treated with stereotactic RT. For dose-escalation experiments, mice received 2, 4 or 8 Gy as single administrations. For dose-fractionation experiments, mice received 4 Gy as a single fraction or multiple (1.33x3 Gy) fractions. The impact of the RT schedule on murine survival and tumor immunity was evaluated. Modifications of glioma stem cells (GSCs), tumor vasculature and tumor cell replication were also assessed. RESULTS: RT dose-escalation was associated with an improved immune profile, with higher CD8+ T cells and CD8+ T cells/regulatory T cells (Tregs) ratio (P=0.0003 and P=0.0022, respectively) and lower total tumor associated microglia/macrophages (TAMs), M2 TAMs and monocytic myeloid derived suppressor cells (mMDSCs) (P=0.0011, P=0.0024 and P<0.0001, respectively). The progressive increase of RT dosages prolonged survival (P<0.0001) and reduced tumor vasculature (P=0.069), tumor cell proliferation (P<0.0001) and the amount of GSCs (P=0.0132 or lower). Compared to the unfractionated regimen, RT dose-fractionation negatively affected tumor immunity by inducing higher total TAMs, M2 TAMs and mMDSCs (P=0.0051, P=0.0036 and P=0.0436, respectively). Fractionation also induced a shorter survival (P=0.0078), a higher amount of GSCs (P=0.0015 or lower) and a higher degree of tumor cell proliferation (P=0.0003). CONCLUSIONS: This study demonstrates that RT dosage and fractionation significantly influence survival, tumor immunity and GSCs in experimental HGGs. These findings should be taken into account when aiming at designing more synergistic and effective radio-immunotherapy combinations.


Assuntos
Glioma , Microambiente Tumoral , Animais , Camundongos , Linfócitos T CD8-Positivos/patologia , Camundongos Endogâmicos C57BL , Glioma/patologia , Células-Tronco Neoplásicas/patologia , Doses de Radiação
15.
Pharmaceuticals (Basel) ; 16(10)2023 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-37895880

RESUMO

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

17.
Mol Neurobiol ; 59(11): 6857-6873, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36048342

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a malignant prognosis. GBM is characterized by high cellular heterogeneity and its progression relies on the interaction with the central nervous system, which ensures the immune-escape and tumor promotion. This interplay induces metabolic, (epi)-genetic and molecular rewiring in both domains. In the present study, we aim to characterize the time-related changes in the GBM landscape, using a syngeneic mouse model of primary GBM. GL261 glioma cells were injected in the right striatum of immuno-competent C57Bl/6 mice and animals were sacrificed after 7, 14, and 21 days (7D, 14D, 21D). The tumor development was assessed through 3D tomographic imaging and brains were processed for immunohistochemistry, immunofluorescence, and western blotting. A human transcriptomic database was inquired to support the translational value of the experimental data. Our results showed the dynamic of the tumor progression, being established as a bulk at 14D and surrounded by a dense scar of reactive astrocytes. The GBM growth was paralleled by the impairment in the microglial/macrophagic recruitment and antigen-presenting functions, while the invasive phase was characterized by changes in the extracellular matrix, as shown by the analysis of tenascin C and metalloproteinase-9. The present study emphasizes the role of the molecular changes in the microenvironment during the GBM progression, fostering the development of novel multi-targeted, time-dependent therapies in an experimental model similar to the human disease.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Evasão Tumoral , Microambiente Tumoral , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/imunologia , Glioblastoma/patologia , Glioma/imunologia , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Tenascina/metabolismo
18.
Biomedicines ; 10(7)2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35884844

RESUMO

Chronic hypoxia drives vascular dysfunction by various mechanisms, including changes in mitochondrial respiration. Although endothelial cells (ECs) rely predominantly on glycolysis, hypoxia is known to alter oxidative phosphorylation, promote oxidative stress and induce dysfunction in ECs. Our work aimed to analyze the effects of prolonged treatment with hypoxia-mimetic agent CoCl2 on intracellular nucleotide concentration, extracellular nucleotide breakdown, mitochondrial function, and nitric oxide (NO) production in microvascular ECs. Moreover, we investigated how nucleotide precursor supplementation and adenosine deaminase inhibition protected against CoCl2-mediated disturbances. Mouse (H5V) and human (HMEC-1) microvascular ECs were exposed to CoCl2-mimicked hypoxia for 24 h in the presence of nucleotide precursors: adenine and ribose, and adenosine deaminase inhibitor, 2'deoxycoformycin. CoCl2 treatment decreased NO production by ECs, depleted intracellular ATP concentration, and increased extracellular nucleotide and adenosine catabolism in both H5V and HMEC-1 cell lines. Diminished intracellular ATP level was the effect of disturbed mitochondrial phosphorylation, while nucleotide precursors effectively restored the ATP pool via the salvage pathway and improved endothelial function under CoCl2 treatment. Endothelial protective effects of adenine and ribose were further enhanced by adenosine deaminase inhibition, that increased adenosine concentration. This work points to a novel strategy for protection of hypoxic ECs by replenishing the adenine nucleotide pool and promoting adenosine signaling.

19.
Biol Pharm Bull ; 34(8): 1204-14, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21804207

RESUMO

Heme oxygenase-1 (HO-1, encoded by the HMOX1 gene) and inducible nitric oxide synthase (iNOS) have been implicated in vascular disease; however the role of these genes remains unclear. Therefore, we studied the mechanism by which iNOS-derived nitric oxide (NO) affects the intimal hyperplasia (IH) formation in relation to HO-1. We show, in a model of balloon injury in rats, that the suppression of vascular smooth muscle cells (VSMC) proliferation by NO required HO-1, while induction of apoptosis of the VSMC by NO does not involve HO-1. To better clarify the molecular mechanism of this finding, we used Hmox1(+/+) and Hmox1(-/-) VSMC exposed to NO. In Hmox1(+/+) VSMC, NO is antiproliferative (up to 34% inhibition) and it is associated to an increase of apoptosis (up to 35%) due to a decrease of X-linked inhibitor of apoptosis protein (XIAP) expression level and to the activation of caspase-3. In the absence of HO-1 (Hmox1(-/-) VSMC) apoptosis was significantly greater (69% p<0.01 vs. Hmox1(+/+) VSMC) demonstrating that HO-1 attenuated the pro-apoptotic effect of NO on VSMC. In the context of IH, the pro-apoptotic effect of NO on VSMC is increased in the absence of HO-1 and exerts therapeutic effects with a significant reduction in IH.


Assuntos
Apoptose , Heme Oxigenase-1/antagonistas & inibidores , Músculo Liso Vascular/lesões , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Túnica Íntima/patologia , Animais , Caspase 3/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Heme Oxigenase-1/genética , Hiperplasia/prevenção & controle , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico/uso terapêutico , Ratos , Ratos Sprague-Dawley , Túnica Íntima/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
20.
Front Cell Dev Biol ; 9: 690365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34164404

RESUMO

Bruton's tyrosine kinase (BTK) is a non-receptor intracellular kinase playing a key role in the proliferation and survival of normal and malignant B-lymphocytes. Its targeting by Ibrutinib, the first specific inhibitor, represented a turning point for the therapy of certain types of B-cell leukemias/lymphomas and several more BTK inhibitors are today in the clinic or advanced clinical trials. BTK expression was successively found to occur also outside of the hematopoietic compartment. In fact, we identified p65BTK, a novel 65 kDa isoform lacking an N-term stretch of 86 amino acids (compared to the 77 kDa protein expressed in B cells) as highly expressed in colon cancer patients. We demonstrated that p65BTK is a powerful oncogene acting downstream of the RAS/MAPK pathway and necessary for RAS-mediated transformation. Notably, the kinase domain is conserved and therefore inhibited by the available BTK-targeting drugs (Ibrutinib, Spebrutinib, etc.) which we used to demonstrate that p65BTK is an actionable target in drug-resistant colorectal carcinomas. We found p65BTK expressed also in >50% non-small cell lung cancers (NSCLC) and demonstrated that it is an actionable target in KRAS-mutated/EGFR-wild type drug-resistant NSCLC models (for which no targeted therapy is available). We also reported a significant correlation between p65BTK expression and low-grade tumors and overall survival of patients with grade III gliomas and showed that its targeting induced a significant decrease in the viability of in glioma stem cells. Finally, in ovarian cancer patients, p65BTK expression levels correlate with early relapse and shorter progression-free survival, both indicators of resistance to therapy. Remarkably, Ibrutinib is more effective than standard of care (SOC) therapeutics in in vitro and ex vivo settings. On the whole, our preclinical data indicate that, depending on the tumor type, BTK inhibitors used alone can induce cytotoxicity (gliomas), be more effective than SOC chemotherapy (ovarian cancer) or can kill drug-resistant tumor cells when used in combination with SOC chemotherapy (colon cancer and NSCLC) or targeted therapy (NSCLC and ovarian cancer), thus suggesting that p65BTK may be an actionable target in different solid tumors. In addition, our data also give the proof-of-concept for starting clinical trials using BTK inhibitors, alone or in combination, to improve the therapeutic options for solid tumors treatment.

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