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1.
Methods Mol Biol ; 2206: 89-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754813

RESUMO

The rabbit corneal micropocket assay uses the avascular cornea as a substrate to study angiogenesis in vivo. The continuous monitoring of neovascular growth in the same animal allows for the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Through the use of standardized slow-release pellets, a predictable angiogenic response can be quantified over the course of 1-2 weeks. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) and a synthetic polymer to allow for their slow release. A micropocket is surgically created in the cornea thickness under anesthesia and in sterile conditions. The angiogenesis stimulus (growth factor but also tissue fragment or cell suspension) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries where vessels are preexisting. On the following days, the neovascular development and progression are measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay allow to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter the experimental details of the avascular rabbit cornea assay, the technical challenges, advantages, and limitations are discussed.

2.
Oxid Med Cell Longev ; 2020: 8363245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32832006

RESUMO

Neurodegenerative disease is an umbrella term for different conditions which primarily affect the neurons in the human brain. In the last century, significant research has been focused on mechanisms and risk factors relevant to the multifaceted etiopathogenesis of neurodegenerative diseases. Currently, neurodegenerative diseases are incurable, and the treatments available only control the symptoms or delay the progression of the disease. This review is aimed at characterizing the complex network of molecular mechanisms underpinning acute and chronic neurodegeneration, focusing on the disturbance in redox homeostasis, as a common mechanism behind five pivotal risk factors: aging, oxidative stress, inflammation, glycation, and vascular injury. Considering the complex multifactorial nature of neurodegenerative diseases, a preventive strategy able to simultaneously target multiple risk factors and disease mechanisms at an early stage is most likely to be effective to slow/halt the progression of neurodegenerative diseases.

3.
PLoS One ; 15(3): e0229973, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163477

RESUMO

PURPOSE: To describe patterns of utilization, survival and infectious events in patients treated with rituximab at the University Hospital of Siena (UHS) to explore the feasibility of combining routinely collected administrative and hospital-pharmacy data for examining the real-world use of intravenous antineoplastic drugs. METHODS: A retrospective, longitudinal cohort study was conducted using data from the Hospital Pharmacy of Siena (HPS) and the Regional Administrative Database of Tuscany (RAD). Patients aged ≥18 years with ≥1 rituximab administration recorded between January 2012 and June 2016 were identified in the HPS database. Anonymized patient-level data were linked to RAD. Rituximab utilization during the first year of treatment was described using HPS. Hospital diagnoses of adverse infectious events that occurred during the first year of follow-up and four-year survival were observed using RAD. RESULTS: A total of 311 new users of rituximab were identified: 264 patients received rituximab for non-Hodgkin's lymphoma (NHL) and 47 were treated for chronic lymphocytic leukemia (CLL). Among new users with one complete year of follow-up (n = 203) over 95% received rituximab as the first-line treatment, and approximately 70% of them received 5-8 doses. No patient in the CLL group received >8 administrations. Four-year survival was approximately 70% in both CLL and NHL patients. Sepsis was the most frequent infectious event observed (5.1%). CONCLUSION: HPS and RAD provided complementary information on rituximab utilization, demonstrating their potential for future pharmacoepidemiological studies on antineoplastic medications administered in the Italian hospital setting. Overall, this general description of the real-world utilization of rituximab in patients treated for NHL and CLL at UHS was in line with treatment guidelines and current knowledge on the rituximab safety profile.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Uso de Medicamentos/estatística & dados numéricos , Infecções/epidemiologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma não Hodgkin/tratamento farmacológico , Rituximab/administração & dosagem , Administração Intravenosa , Demandas Administrativas em Assistência à Saúde/estatística & dados numéricos , Adolescente , Adulto , Idoso , Antineoplásicos Imunológicos/efeitos adversos , Bases de Dados Factuais/estatística & dados numéricos , Feminino , Seguimentos , Humanos , Infecções/induzido quimicamente , Infecções/imunologia , Itália/epidemiologia , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Estudos Longitudinais , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/mortalidade , Masculino , Pessoa de Meia-Idade , Serviço de Farmácia Hospitalar/estatística & dados numéricos , Estudos Retrospectivos , Rituximab/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
4.
Eur J Nutr ; 59(2): 517-527, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30725211

RESUMO

PURPOSE: Endothelial-to-mesenchymal transition (EndMT) plays an important role in pathogenesis of a number of inflammatory diseases. Hydroxytyrosol (HT) and, particularly, its major plasma metabolite HT-3O sulfate (HT-3Os) are known olive oil antioxidant and anti-inflammatory polyphenols which exert benefits against vascular diseases by improving endothelial function. However, to date the HT-3Os role in EndMT is not well known. METHODS: To investigate the HT-3Os effects on EndMT in the inflamed endothelium, we used an in vitro model of endothelial dysfunction, challenging endothelial cells (EC), human umbilical EC (HUVEC) and human retinal EC (HREC) with Interleukin-1ß (IL-1ß), an inflammatory agent. HREC were used as a specific model to investigate HT-3Os effects on vascular retinal diseases. RESULTS: We found that IL-1ß treatment-induced EndMT phenotype in both cell models, also changing cell morphology. HT-3Os protected EC against IL-1ß effects, recovering cell morphology and phenotype. Mechanistically, HT-3Os targeting fibroblast growth factor receptor 1 FGFR1 expression and let-7 miRNA, controlled transforming growth factor beta (TGF-ß) signalling in EC, downregulating transcription factors expression (SNAI1 and ZEB2) and gene expression of late EndMT markers (FN1, VIM, NOTCH3, CNN1, MMP2 and MMP9). CONCLUSION: These results demonstrate that HT-3Os blunts pathological EndMT in inflamed EC, maintaining high let-7 miRNA expression and preventing activation of TGF-ß signalling.

5.
Cancers (Basel) ; 11(12)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817719

RESUMO

Melanoma and non-small-cell lung carcinoma (NSCLC) cell lines are characterized by an intrinsic population of cancer stem-like cells (CSC), and high expression of detoxifying isozymes, the aldehyde dehydrogenases (ALDHs), regulating the redox state. In this study, using melanoma and NSCLC cells, we demonstrate that ALDH3A1 isozyme overexpression and activity is closely associated with a highly aggressive mesenchymal and immunosuppressive profile. The contribution of ALDH3A1 to the stemness and immunogenic status of melanoma and NSCLC cells was evaluated by their ability to grow in 3D forming tumorspheres, and by the expression of markers for stemness, epithelial to mesenchymal transition (EMT), and inflammation. Furthermore, in specimens from melanoma and NSCLC patients, we investigated the expression of ALDH3A1, PD-L1, and cyclooxygenase-2 (COX-2) by immunohistochemistry. We show that cells engineered to overexpress the ALDH3A1 enzyme enriched the CSCs population in melanoma and NSCLC cultures, changing their transcriptome. In fact, we found increased expression of EMT markers, such as vimentin, fibronectin, and Zeb1, and of pro-inflammatory and immunosuppressive mediators, such as NFkB, prostaglandin E2, and interleukin-6 and -13. ALDH3A1 overexpression enhanced PD-L1 output in tumor cells and resulted in reduced proliferation of peripheral blood mononuclear cells when co-cultured with tumor cells. Furthermore, in tumor specimens from melanoma and NSCLC patients, ALDH3A1 expression was invariably correlated with PD-L1 and the pro-inflammatory marker COX-2. These findings link ALDH3A1 expression to tumor stemness, EMT and PD-L1 expression, and suggest that aldehyde detoxification is a redox metabolic pathway that tunes the immunological output of tumors.

6.
Prostaglandins Other Lipid Mediat ; 143: 106344, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31207300

RESUMO

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as gefitinib are standard treatment of non-small cell lung cancer (NSCLC), but resistance often occurs. This study demonstrates that NSCLC cells resistant to gefitinib (GR cells) displayed a significantly higher microsomal prostaglandin E synthase-1 (mPGES-1) expression and activity than parental cells. Overexpression of mPGES-1/prostaglandin E-2 (PGE-2) signaling in GR cells was associated with acquisition of mesenchymal and stem-like cell properties, nuclear EGFR translocation and tolerance to cisplatin. mPGES-1 inhibition reduced mesenchymal and stem-like properties, and nuclear EGFR translocation in GR cells. Consistently, inhibition of mPGES-1 activity enhanced sensitivity to cisplatin and responsiveness to gefitinib in GR cells. We propose the mPGES-1/PGE-2 signaling as a potential target for treating aggressive and resistant lung cancers.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Prostaglandina-E Sintases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Dinoprostona/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Inativação Gênica , Humanos , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Transdução de Sinais/efeitos dos fármacos
8.
J Exp Clin Cancer Res ; 37(1): 311, 2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541574

RESUMO

BACKGROUND: Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. METHODS: Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). RESULTS: In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. CONCLUSION: In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.


Assuntos
Aldeído Desidrogenase/metabolismo , Neoplasias da Mama/irrigação sanguínea , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Aldeído Desidrogenase 1 , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retinal Desidrogenase , Transdução de Sinais , Transfecção
9.
Int J Mol Sci ; 19(9)2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30200598

RESUMO

Elevated levels of bradykinin (BK) and fibroblast growth factor-2 (FGF-2) have been implicated in the pathogenesis of inflammatory and angiogenic disorders. In angiogenesis, both stimuli induce a pro-inflammatory signature in endothelial cells, activating an autocrine/paracrine amplification loop that sustains the neovascularization process. Here we investigated the contribution of the FGF-2 pathway in the BK-mediated human endothelial cell permeability and migration, and the role of the B2 receptor (B2R) of BK in this cross-talk. BK (1 µM) upregulated the FGF-2 expression and promoted the FGF-2 signaling, both in human umbilical vein endothelial cells (HUVEC) and in retinal capillary endothelial cells (HREC) by the activation of Fibroblast growth factor receptor-1 (FGFR-1) and its downstream signaling (fibroblast growth factor receptor substrate: FRSα, extracellular signal⁻regulated kinases1/2: ERK1/2, and signal transducer and activator of transcription 3: STAT3 phosphorylation). FGFR-1 phosphorylation triggered by BK was c-Src mediated and independent from FGF-2 upregulation. Either HUVEC and HREC exposed to BK showed increased permeability, disassembly of adherens and tight-junction, and increased cell migration. B2R blockade by the selective antagonist, fasitibant, significantly inhibited FGF-2/FGFR-1 signaling, and in turn, BK-mediated endothelial cell permeability and migration. Similarly, the FGFR-1 inhibitor, SU5402, and the knock-down of the receptor prevented the BK/B2R inflammatory response in endothelial cells. In conclusion, this work demonstrates the existence of a BK/B2R/FGFR-1/FGF-2 axis in endothelial cells that might be implicated in propagation of angiogenic/inflammatory responses. A B2R blockade, by abolishing the initial BK stimulus, strongly attenuated FGFR-1-driven cell permeability and migration.


Assuntos
Bradicinina/farmacologia , Células Endoteliais/citologia , Receptor B2 da Bradicinina/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
10.
Oncotarget ; 9(19): 14939-14958, 2018 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-29599917

RESUMO

Prostaglandin E2 (PGE2) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin ß1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1, PTGS2, MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE2-induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

11.
Int J Mol Sci ; 19(2)2018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29360776

RESUMO

The identification of components of the kallikrein-kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy.


Assuntos
Estresse Oxidativo , Receptor B2 da Bradicinina/genética , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Neovascularização Retiniana/genética , Animais , Bradicinina/metabolismo , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Camundongos , Modelos Biológicos , Ornitina/análogos & derivados , Ornitina/farmacologia , Coelhos , Receptor B2 da Bradicinina/metabolismo , Doenças Retinianas/patologia , Neovascularização Retiniana/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia
12.
Oncotarget ; 8(47): 83207-83224, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-29137335

RESUMO

Hydroxytyrosol (HT), a polyphenol of olive oil, downregulates epidermal growth factor (EGFR) expression and inhibits cell proliferation in colon cancer (CC) cells, with mechanisms similar to that activated by the EGFR inhibitor, cetuximab. Here, we studied whether HT treatment would enhance the cetuximab inhibitory effects on cell growth in CC cells. HT-cetuximab combination showed greater efficacy in reducing cell growth in HT-29 and WiDr cells at concentrations 10 times lower than when used as single agents. This reduction was clearly linked to cell cycle blockade, occurring at G2/M phase. The cell cycle arrest in response to combination treatment is related to cyclins B, D1, and E, and cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 down-regulation, and to the concomitant over-expression of CDK inhibitors p21 and p27. HT and cetuximab stimulated a caspase-independent cell death cascade, promotedtranslocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and activated the autophagy process. Notably, normal colon cells and keratinocytes were less susceptible to combo-induced cell death and EGFR downregulation. These results suggest a potential role of diet, containing olive oil, during cetuximab chemotherapy of colon tumor. HT may be a competent therapeutic agent in CC enhancing the effects of EGFR inhibitors.

13.
Oncotarget ; 8(19): 31270-31287, 2017 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-28415726

RESUMO

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression.


Assuntos
Adenocarcinoma/metabolismo , Dinoprostona/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Ligação Proteica , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo
14.
Nitric Oxide ; 66: 17-29, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28257996

RESUMO

Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459, originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelial-mesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus facilitating iNOS production. In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Receptores ErbB/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Prostaglandina-E Sintases/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Humanos , Fenótipo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células-Tronco , Células Tumorais Cultivadas
15.
Biochim Biophys Acta Gen Subj ; 1861(4): 860-870, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28095317

RESUMO

BACKGROUND: Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS: We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS: We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS: Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE: Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fabaceae/metabolismo , Lectinas/administração & dosagem , Lectinas/química , Lipossomos/administração & dosagem , Lipossomos/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Citoplasma/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Sistemas de Liberação de Medicamentos/métodos , Epitopos/administração & dosagem , Epitopos/química , Humanos , Lisossomos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Camundongos
16.
PLoS One ; 11(12): e0168727, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28036325

RESUMO

Iron oxide nanoparticles (NPs) have been proposed for many biomedical applications as in vivo imaging and drug delivery in cancer treatment, but their toxicity is an ongoing concern. When NPs are intravenously administered, the endothelium represents the first barrier to tissue diffusion/penetration. However, there is little information about the biological effects of NPs on endothelial cells. In this work we showed that cobalt-ferrite (CoFe2O4) NPs affect endothelial cell integrity by increasing permeability, oxidative stress, inflammatory profile and by inducing cytoskeletal modifications. To overcome these problems, NPs have be loaded into biocompatible gels to form nanocomposite hybrid material (polysaccharide hydrogels containing magnetic NPs) that can be further conjugated with anticancer drugs to allow their release close to the target. The organic part of hybrid biomaterials is a carboxymethylcellulose (CMC) polymer, while the inorganic part consists of CoFe2O4 NPs coated with (3-aminopropyl)trimethoxysilane. The biological activity of these hybrid hydrogels was evaluated in vitro and in vivo. Our findings showed that hybrid hydrogels, instead of NPs alone, were not toxic on endothelial, stromal and epithelial cells, safe and biodegradable in vivo. In conclusion, biohydrogels with paramagnetic NPs as cross-linkers can be further exploited for antitumor drug loading and delivery systems.


Assuntos
Cobalto/farmacologia , Células Endoteliais/efeitos dos fármacos , Compostos Férricos/farmacologia , Hidrogéis/farmacologia , Nanopartículas/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Carboximetilcelulose Sódica/química , Linhagem Celular , Cobalto/química , Sistemas de Liberação de Medicamentos/métodos , Compostos Férricos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/química , Inflamação/tratamento farmacológico , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos
17.
J Cell Sci ; 129(21): 4091-4104, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27656109

RESUMO

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFA-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.


Assuntos
Neovascularização Fisiológica , Pinocitose , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Modelos Biológicos , Proteína cdc42 de Ligação ao GTP/metabolismo
18.
Pharmacol Res ; 113(Pt A): 426-437, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27650753

RESUMO

Cardiovascular diseases as atherosclerosis are associated to an inflammatory state of the vessel wall which is accompanied by endothelial dysfunction, and adherence and activation of circulating inflammatory cells. Hydrogen sulfide, a novel cardiovascular protective gaseous mediator, has been reported to exert anti-inflammatory activity. We have recently demonstrated that the SH containing ACE inhibitor zofenoprilat, the active metabolite of zofenopril, controls the angiogenic features of vascular endothelium through H2S enzymatic production by cystathionine gamma lyase (CSE). Based on H2S donor/generator property of zofenoprilat, the objective of this study was to evaluate whether zofenoprilat exerts anti-inflammatory activity in vascular cells through its ability to increase H2S availability. Here we found that zofenoprilat, in a CSE/H2S-mediated manner, abolished all the inflammatory features induced by interlukin-1beta (IL-1ß) in human umbilical vein endothelial cells (HUVEC), especially the NF-κB/cyclooxygenase-2 (COX-2)/prostanoid biochemical pathway. The pre-incubation with zofenoprilat/CSE dependent H2S prevented IL-1ß induced paracellular hyperpermeability through the control of expression and localization of cell-cell junctional markers ZO-1 and VE-cadherin. Moreover, zofenoprilat/CSE dependent H2S reduced the expression of the endothelial markers CD40 and CD31, involved in the recruitment of circulating mononuclear cells and platelets. Interestingly, this anti-inflammatory activity was also confirmed in vascular smooth muscle cells and fibroblasts as zofenoprilat reduced, in both cell lines, proliferation, migration and COX-2 expression induced by IL-1ß, but independently from the SH moiety and H2S availability. These in vitro data document the anti-inflammatory activity of zofenoprilat on vascular cells, reinforcing the cardiovascular protective effect of this multitasking drug.


Assuntos
Anti-Inflamatórios/farmacologia , Captopril/análogos & derivados , Endotélio Vascular/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antígenos CD/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Antígenos CD40/metabolismo , Caderinas/metabolismo , Captopril/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Cistationina gama-Liase/metabolismo , Endotélio Vascular/metabolismo , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Sci Rep ; 6: 31295, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27498819

RESUMO

The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.


Assuntos
Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Sítios de Ligação , Movimento Celular , Proliferação de Células , Dicroísmo Circular , Cristalografia por Raios X , Células Endoteliais da Veia Umbilical Humana , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Peptídeos/química , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de Sinais
20.
Oncotarget ; 7(28): 44350-44364, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27322147

RESUMO

Prostaglandin E-2 (PGE-2) promotes tumor angiogenesis via paracrine secretion of pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF). Since miRNAs regulate several cell processes, including angiogenesis, we sought to determine whether they would influence PGE-2-induced VEGF. We compared DU145 and PC3 prostate cancer cells bearing the mPGES-1 enzyme (mPGES-1+/+) and producing PGE-2, with those in which the enzyme was silenced or deleted (mPGES-1-/-). We demonstrated that mPGES-1/PGE-2 signaling decreased Dicer expression and miRNA biogenesis. Genome-wide sequencing of miRNAs revealed that miR-15a and miR-186, associated with expression of VEGF and hypoxia inducible factor-1α (HIF-1α), were down-regulated in mPGES-1+/+ cells. As a consequence, mPGES-1+/+ tumor cells expressed high levels of VEGF and HIF-1α, induced endothelial cells activation and formed highly vascularized tumors. Mir-186 mimic inhibited VEGF expression in mPGES-1+/+ tumor xenografts and reduced tumor growth. In human prostate cancer specimens, mPGES-1 was over-expressed in tumors with high Gleason score, elevated expression of VEGF and HIF-1α, high microvessel density and decreased expression of Dicer, miR15a and miR-186. Thus, clear evidence for regulating miRNA processing and VEGF output by intrinsic PGE-2 production provides a means to distinguish between aggressive and indolent prostate tumors and suggests a potential target for controlling tumor progression.


Assuntos
MicroRNAs/biossíntese , Prostaglandina-E Sintases/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Dinoprostona/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Microssomos/metabolismo , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ribonuclease III/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese
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