RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance. METHODS: We analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy. RESULTS: Spatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration. CONCLUSIONS: Finally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Mucinas , Gencitabina , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologiaRESUMO
Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.
Assuntos
Mesoderma/patologia , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Estresse do Retículo Endoplasmático/genética , Feminino , Genes myc , Genes ras , Humanos , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Mesoderma/metabolismo , Camundongos , Mosaicismo , Proteína Oncogênica p55(v-myc)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/metabolismo , Transcriptoma/genética , GencitabinaRESUMO
Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Citometria de Fluxo , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/terapia , Estudos ProspectivosRESUMO
AIM: The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization. METHOD: Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed. RESULTS: Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1ß, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients. CONCLUSION: A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients.
Assuntos
Citocinas , Fístula Retal , Humanos , Quimiocinas/metabolismo , Macrófagos/metabolismo , Fístula Retal/etiologia , Fístula Retal/cirurgiaRESUMO
Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.
Assuntos
Neoplasias Colorretais , Neoplasias , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia , Instabilidade de Microssatélites , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genética , PrognósticoRESUMO
FOXP3-expressing regulatory T-cells (Tregs), which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. It has been shown that there is an increased proportion of Tregs in several different human malignancies, although the actual mechanism remains unclear. The research aims to explore the relationship between the number of Tregs and a predict prognosis in particular hematological diseases as monoclonal gammopathies of uncertain significance (MGUS). Tregs were evaluated by means of flow cytometry (CD4+CD25high/+ CD127low/-) in whole peripheral blood of 56 patients with MGUS to predict progression to overt multiple myeloma (MM). In two groups of patients, MGUS versus MGUS evolved to MM, we found a significative difference for the number of white blood cells, but not in terms of clinical and laboratory features evaluated at diagnosis. The study demonstrated the absence of a prognostic relevance of Tregs in MGUS. Nevertheless, their role in these disorders is still to be defined.
Assuntos
Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Progressão da Doença , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/patologia , PrognósticoRESUMO
Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3ß, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.
Assuntos
Autofagia/efeitos dos fármacos , Mitose/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Adulto , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/fisiologia , Humanos , Moduladores de Mitose/farmacologia , Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidade , Análise de SobrevidaRESUMO
Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.
Assuntos
Proliferação de Células/efeitos da radiação , Neoplasias Colorretais/radioterapia , Osteogênese/efeitos da radiação , Ondas Ultrassônicas , Caderinas/genética , Diferenciação Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/efeitos da radiação , Vesículas Extracelulares/genética , Vesículas Extracelulares/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HT29 , Humanos , Células-Tronco Mesenquimais/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.
Assuntos
Moléculas de Adesão Celular/genética , Transformação Celular Neoplásica/genética , Interleucina-6/genética , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Animais , Comunicação Autócrina/genética , Moléculas de Adesão Celular/deficiência , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Citocinas/genética , Citocinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Matriz Extracelular/química , Matriz Extracelular/genética , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Wnt/ß-Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60-70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/ß-Catenin pathway and preventing ß-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/ß-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/ß-Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active ß-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/ß-Catenin signaling is modulated at multiple levels by TRAP1.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteólise , Células Tumorais Cultivadas , Ubiquitinação , beta Catenina/metabolismoRESUMO
Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.
Assuntos
Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Fluoresceínas/química , Tamanho Celular , Difusão Dinâmica da Luz , Vesículas Extracelulares/química , Células HT29 , Humanos , ImunofenotipagemRESUMO
Cholangiocarcinoma is a primary malignancy of the biliary tract characterized by late and unspecific symptoms, unfavorable prognosis, and few treatment options. The advent of next-generation sequencing has revealed potential targetable or actionable molecular alterations in biliary tumors. Among several identified genetic alterations, the IDH1 mutation is arousing interest due to its role in epigenetic and metabolic remodeling. Indeed, some IDH1 point mutations induce widespread epigenetic alterations by means of a gain-of-function of the enzyme, which becomes able to produce the oncometabolite 2-hydroxyglutarate, with inhibitory activity on α-ketoglutarate-dependent enzymes, such as DNA and histone demethylases. Thus, its accumulation produces changes in the expression of several key genes involved in cell differentiation and survival. At present, small-molecule inhibitors of IDH1 mutated enzyme are under investigation in preclinical and clinical phases as promising innovative treatments for IDH1-mutated intrahepatic cholangiocarcinomas. This review examines the molecular rationale and the results of preclinical and early-phase studies on novel pharmacological agents targeting mutant IDH1 in cholangiocarcinoma patients. Contextually, it will offer a starting point for discussion on combined therapies with metabolic and epigenetic drugs, to provide molecular support to target the interplay between metabolism and epigenetics, two hallmarks of cancer onset and progression.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Isocitrato Desidrogenase/antagonistas & inibidores , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , HumanosRESUMO
Azorella glabra Wedd. (AG) is traditionally used to treat gonorrhea or kidney's problems. The antioxidant, antidiabetic, anticholinesterase and in vitro antitumor activities of AG extracts were recently reported. The aim of this work was to investigate anti-leukemic properties of AG chloroform fraction (AG CHCl3) and of its ten sub-fractions (I-X) and to identify their possible bioactive compounds. We determined their in vitro antioxidant activity using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), nitric oxide (NO) and superoxide anion (SO) assays, and their phytochemical profile by spectrophotometric and LC-MS/MS techniques. I-X action on two acute myeloid leukemia (AML) cell lines viability, apoptosis and cell cycle were evaluated by MTS, western blotting and cytofluorimetric assays. Different polyphenol, flavonoid and terpenoid amount, and antioxidant activity were found among all samples. Most of I-X induced a dose/time dependent reduction of cell viability higher than parent extract. IV and VI sub-fractions showed highest cytotoxic activity and, of note, a negligible reduction of healthy cell viability. They activated intrinsic apoptotic pathway, induced a G0/G1 block in leukemic cells and, interestingly, led to apoptosis in patient AML cells. These activities could be due to mulinic acid or azorellane terpenoids and their derivatives, tentatively identified in both IV and VI. In conclusion, our data suggest AG plant as a source of potential anti-AML agents.
Assuntos
Antineoplásicos Fitogênicos/química , Antioxidantes/química , Apiaceae/química , Flavonoides/química , Leucemia Mieloide Aguda/tratamento farmacológico , Extratos Vegetais/isolamento & purificação , Polifenóis/química , Terpenos/química , Idoso , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clorofórmio/química , Descoberta de Drogas , Feminino , Humanos , Pessoa de Meia-Idade , Extratos Vegetais/farmacologia , Solventes/química , Espectrometria de Massas em TandemRESUMO
Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E2 significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 waf1 and p27 Kip1 , associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients.
RESUMO
Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.
Assuntos
Plaquetas/metabolismo , Colite/induzido quimicamente , Colite/enzimologia , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/genética , Sulfato de Dextrana/farmacologia , Deleção de Genes , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Colite/sangue , Colite/genética , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Prostaglandinas/biossínteseRESUMO
The technologically interesting properties of palladium nanoparticles (Pd-NPs) allowed their widespread industrial application, although concerns emerged on increasing general and occupational levels of exposure. In this context, to assess the toxicological behavior of Pd-NPs, and particularly their endocrine disruptive potential, has become a public health priority. Therefore, we evaluated Pd-NP impact on the female endocrine reproductive system of Wistar rats sub-chronically treated for 90 days with increasing doses of this xenobiotic (0.12, 1.2, and 12 µg/kg, administered at days 1, 30, and 60 for cumulative doses of 0.36, 3.6, and 36 µg/kg) via the intravenous route. In this regard, we investigated potential alterations in different sex hormone, for example, estradiol, follicle-stimulating hormone (FSH), luteinizing hormone, progesterone, and testosterone, serum concentrations. All treated groups showed significantly greater levels of FSH compared to controls, suggesting a possible impact of Pd-NPs on the regulatory system that controls the normal physiology of female reproductive function. Although relevant, since obtained under sub-chronic, low-dose conditions of exposure resembling those encountered in real-world scenarios, the present results are preliminary and require confirmation as well as identification of the possible underlining molecular mechanisms. From a public and occupational health perspective, implications for the reproductive health of exposed subjects and the next generations of women exposed during their childbearing age or pregnancy should be elucidated. This information is essential to elaborate adequate preventive strategies for assessing and controlling possible Pd-NPs adverse effects on the endocrine system.
Assuntos
Hormônio Foliculoestimulante/sangue , Genitália/efeitos dos fármacos , Hormônio Luteinizante/sangue , Nanopartículas Metálicas/análise , Paládio/sangue , Animais , Disruptores Endócrinos/farmacologia , Feminino , Sistema Hipotálamo-Hipofisário , Nanopartículas Metálicas/toxicidade , Paládio/toxicidade , Dados Preliminares , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
Despite the significant recent advances in clinical practice, gastric cancer (GC) represents a leading cause of cancer-related deaths in the world. In fact, occurrence of chemo-resistance still remains a daunting hindrance to effectiveness of the current approach to GC therapy. There is accumulating evidence that a plethora of cellular and molecular factors is implicated in drug-induced phenotypical switching of GC cells. Among them, epithelial-mesenchymal transition (EMT), autophagy, drug detoxification, DNA damage response and drug target alterations, have been reported as major determinants. Intriguingly, resistant GC phenotype may be the result of GC cell-induced tumor microenvironment (TME) remodeling, which is currently emerging as a key player in promoting drug resistance and overcoming cytotoxic effects of drugs. In this review, we discuss the possible mechanisms of drug resistance and their involvement in determining current GC therapies failure.
Assuntos
Autofagia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica/genética , Neoplasias Gástricas/genética , Microambiente Tumoral/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sobrevivência Celular/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/patogenicidade , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Inativação Metabólica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development; nevertheless, the precise space-temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium-mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Organogênese , Pâncreas/embriologia , Pâncreas/metabolismo , Transdução de Sinais , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , CamundongosRESUMO
Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells.
Assuntos
Antígeno AC133/metabolismo , Neoplasias do Colo/metabolismo , Exossomos/metabolismo , Antígeno AC133/genética , Ácido Butírico/efeitos adversos , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Fosforilação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-ß-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20-500 µmol l-1) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC50 value was 200 µmol l-1 when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.