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1.
Trends Cancer ; 7(11): 974-977, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34456156

RESUMO

Lysosomal ß-galactosylceramidase (GALC) removes ß-galactose from ß-galactosylceramide, thus generating the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth and differentiation, questioning its contribution to the sphingolipid metabolism in cancer cells and its role in tumor progression.

2.
Blood ; 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34077955

RESUMO

The multiple myeloma (MM) mutational landscape has identified alterations in KRAS as the most recurring somatic variant. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying that KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We have used AZD4785, a potent and selective antisense oligonucleotide (ASO) which selectively targets and down-regulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche; and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, confirming KRAS as a driver and a therapeutic target in MM.

3.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063734

RESUMO

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.


Assuntos
Cafeína/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Xenoenxertos , Humanos , Hibridização In Situ , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Fisiológica/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166181, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34082068

RESUMO

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Macroglial Müller cells appear to play a pivotal role in the pathogenesis of iERM where they may undergo glial-to-mesenchymal transition (GMT), a transdifferentiation process characterized by the downregulation of Müller cell markers, paralleled by the upregulation of pro-fibrotic myofibroblast markers. Previous observations from our laboratory allowed the molecular identification of two major clusters of iERM patients (named iERM-A and iERM-B), iERM-A patients being characterized by less severe clinical features and a more "quiescent" iERM gene expression profile when compared to iERM-B patients. In the present work, Müller MIO-M1 cells were exposed to vitreous samples obtained before membrane peeling from the same cohort of iERM-A and iERM-B patients. The results demonstrate that iERM vitreous induces proliferation, migration, and GMT in MIO-M1 cells, a phenotype consistent with Müller cell behavior during iERM progression. However, even though the vitreous samples obtained from iERM-A patients were able to induce a complete GMT in MIO-M1 cells, iERM-B samples caused only a partial GMT, characterized by the downregulation of Müller cell markers in the absence of upregulation of pro-fibrotic myofibroblast markers. Together, the results indicate that a relationship may exist among the ability of iERM vitreous to modulate GMT in Müller cells, the molecular profile of the corresponding iERMs, and the clinical features of iERM patients.

5.
Cancers (Basel) ; 13(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066669

RESUMO

During multiple myeloma (MM) progression the activation of the angiogenic process represents a key step for the formation of the vascular niche, where different stromal components and neoplastic cells collaborate and foster tumor growth. Among the different pro-angiogenic players, Fibroblast Growth Factor 2 (FGF2) plays a pivotal role in BM vascularization occurring during MM progression. Long Pentraxin 3 (PTX3), a natural FGF antagonist, is able to reduce the activation of stromal components promoted by FGF2 in various in vitro models. An increased FGF/PTX3 ratio has also been found to occur during MM evolution, suggesting that restoring the "physiological" FGF/PTX3 ratio in plasma cells and BM stromal cells (BMSCs) might impact MM. In this work, taking advantage of PTX3-inducible human MM models, we show that PTX3 produced by tumor cells is able to restore a balanced FGF/PTX3 ratio sufficient to prevent the activation of the FGF/FGFR system in endothelial cells and to reduce the angiogenic capacity of MM cells in different in vivo models. As a result of this anti-angiogenic activity, PTX3 overexpression causes a significant reduction of the tumor burden in both subcutaneously grafted and systemic MM models. These data pave the way for the exploitation of PTX3-derived anti-angiogenic approaches in MM.

6.
Eur J Med Chem ; 221: 113529, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34004471

RESUMO

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity in vivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells in vitro by inhibiting FGF-dependent receptor activation and slowing MM growth in vivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.


Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Animais , Antineoplásicos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/análogos & derivados , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas
7.
Cancer Lett ; 507: 80-88, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33744390

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2WT. Furthermore, activated VEGFR2R1051Q augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.


Assuntos
Metabolismo Energético , Mutação com Ganho de Função , Glutamina/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
8.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671690

RESUMO

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.


Assuntos
Retinopatia Diabética/patologia , Células Ependimogliais/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Proliferação de Células , Células Cultivadas , Colesterol/análogos & derivados , Colesterol/farmacologia , Retinopatia Diabética/cirurgia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/fisiologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Humanos , Hidrocortisona/farmacologia , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Ranibizumab/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Vitrectomia
9.
Front Immunol ; 12: 617671, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679758

RESUMO

Pulmonary fibrosis is a progressive scarring disease of the lungs, characterized by inflammation, fibroblast activation, and deposition of extracellular matrix. The long pentraxin 3 (PTX3) is a member of the pentraxin family with non-redundant functions in innate immune responses, tissue repair, and haemostasis. The role played in the lungs by PTX3 during the fibrotic process has not been elucidated. In this study, the impact of PTX3 expression on lung fibrosis was assessed in an intratracheal bleomycin (BLM)-induced murine model of the disease applied to wild type animals, transgenic mice characterized by endothelial overexpression and stromal accumulation of PTX3 (Tie2-PTX3 mice), and genetically deficient Ptx3 -/- animals. Our data demonstrate that PTX3 is produced during BLM-induced fibrosis in wild type mice, and that PTX3 accumulation in the stroma compartment of Tie2-PTX3 mice limits the formation of fibrotic tissue in the lungs, with reduced fibroblast activation and collagen deposition, and a decrease in the recruitment of the immune infiltrate. Conversely, Ptx3-null mice showed an exacerbated fibrotic response and decreased survival in response to BLM treatment. These results underline the protective role of endogenous PTX3 during lung fibrosis and pave the way for the study of novel PTX3-derived therapeutic approaches to the disease.


Assuntos
Biomarcadores , Proteína C-Reativa/genética , Suscetibilidade a Doenças , Expressão Gênica , Fibrose Pulmonar/etiologia , Componente Amiloide P Sérico/genética , Animais , Bleomicina/efeitos adversos , Proteína C-Reativa/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Prognóstico , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/mortalidade , Fibrose Pulmonar/patologia , Componente Amiloide P Sérico/metabolismo
10.
Cells ; 10(2)2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525607

RESUMO

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Neovascularização Patológica/metabolismo , Orthoreovirus Aviário/metabolismo , Proteínas não Estruturais Virais/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Galinhas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Microvasos/citologia , Morfogênese/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução Genética , Regulação para Cima/efeitos dos fármacos
11.
Exp Cell Res ; 400(2): 112490, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33484747

RESUMO

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/patologia , Neovascularização Patológica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Teratoma/irrigação sanguínea , Teratoma/patologia , Animais , Embrião de Galinha , Membrana Corioalantoide , Células-Tronco Embrionárias/metabolismo , Camundongos , Camundongos Knockout , Teratoma/metabolismo
12.
Physiol Rev ; 101(2): 569-610, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32730114

RESUMO

Fibroblast growth factors (FGFs) are a family of proteins possessing paracrine, autocrine, or endocrine functions in a variety of biological processes, including embryonic development, angiogenesis, tissue homeostasis, wound repair, and cancer. Canonical FGFs bind and activate tyrosine kinase FGF receptors (FGFRs), triggering intracellular signaling cascades that mediate their biological activity. Experimental evidence indicates that FGFs play a complex role in the physiopathology of the prostate gland that ranges from essential functions during embryonic development to modulation of neoplastic transformation. The use of ligand- and receptor-deleted mouse models has highlighted the requirement for FGF signaling in the normal development of the prostate gland. In adult prostate, the maintenance of a functional FGF/FGFR signaling axis is critical for organ homeostasis and function, as its disruption leads to prostate hyperplasia and may contribute to cancer progression and metastatic dissemination. Dissection of the molecular landscape modulated by the FGF family will facilitate ongoing translational efforts directed toward prostate cancer therapy.

13.
Blood ; 137(18): 2495-2508, 2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-33197938

RESUMO

The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.

14.
Methods Mol Biol ; 2206: 151-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754817

RESUMO

This protocol focuses on the quantitative description of the angioarchitecture of experimental tumor xenografts. This semiautomatic analysis is carried out on functional vessels and microvessels acquired by confocal imaging and processed into progressively reconstructed angioarchitectures following a caliber-classification step. The protocol can be applied also to the quantification of pathological angioarchitectures other than tumor grafts as well as to the microvasculature of physiological tissue samples.


Assuntos
Microscopia Confocal/métodos , Microvasos/patologia , Neoplasias/patologia , Neovascularização Patológica/patologia , Animais , Xenoenxertos/patologia , Humanos , Camundongos
15.
Cancer Lett ; 496: 84-92, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33035615

RESUMO

In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2R1051Q potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2R1051Q, may help to predict patient prognosis and drug response, with significant clinical implications.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Melanoma/patologia , Mutação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Prognóstico , Inibidores de Proteínas Quinases/uso terapêutico , Taxa de Sobrevida , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 21(24)2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33317057

RESUMO

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Neoplasias Pulmonares/metabolismo , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Colesterol/análogos & derivados , Colesterol/farmacologia , Colesterol/uso terapêutico , Regulação para Baixo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo
17.
Methods Protoc ; 3(4)2020 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-33233345

RESUMO

The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins.

18.
Cancer Res ; 80(22): 5011-5023, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32998995

RESUMO

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. ß-Galactosylceramidase (GALC) removes ß-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that ß-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.


Assuntos
Ceramidas/metabolismo , Galactosilceramidase/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/patologia , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Galactosilceramidase/genética , Inativação Gênica , Humanos , Neoplasias Pulmonares/secundário , Melanócitos/citologia , Melanócitos/enzimologia , Melanoma/metabolismo , Melanoma/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Cutâneas/metabolismo , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima , Peixe-Zebra
19.
Front Immunol ; 11: 581288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33117388

RESUMO

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy (PDR). The pathogenesis of PDR is complex and involves vascular, inflammatory, and neuronal mechanisms. Several structural and molecular alterations associated to PDR are related to the presence of inflammation that appears to play a non-redundant role in the neovascular response that characterizes the retina of PDR patients. Vascular endothelial growth factor (VEGF) blockers have evolved over time for the treatment of retinal neovascularization. However, several limitations to anti-VEGF interventions exist. Indeed, the production of other angiogenic factors and pro-inflammatory mediators may nullify and/or cause resistance to anti-VEGF therapies. Thus, appropriate experimental models are crucial for dissecting the mechanisms leading to retinal neovascularization and for the discovery of more efficacious anti-angiogenic/anti-inflammatory therapies for PDR patients. This review focuses on the tight cross talk between angiogenesis and inflammation during PDR and describe how the chick embryo chorioallantoic membrane (CAM) assay may represent a cost-effective and rapid in vivo tool for the study of the relationship between neovascular and inflammatory responses elicited by the vitreous humor of PDR patients and for the screening of novel therapeutic agents.


Assuntos
Retinopatia Diabética/etiologia , Neovascularização Retiniana/etiologia , Inibidores da Angiogênese/uso terapêutico , Animais , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/imunologia , Membrana Corioalantoide/patologia , Retinopatia Diabética/imunologia , Retinopatia Diabética/patologia , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Modelos Animais , Modelos Biológicos , Neovascularização Retiniana/imunologia , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/fisiologia , Corpo Vítreo/imunologia , Corpo Vítreo/patologia
20.
Biochim Biophys Acta Mol Basis Dis ; 1866(12): 165938, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827649

RESUMO

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Aim of this study was to provide a molecular characterization of iERMs by gene expression analysis. To this purpose, 56 iERMs obtained by pars plana vitrectomy were analyzed for the expression levels of genes encoding biomarkers of the cellular and molecular events occurring in iERMs. RT-qPCR analysis showed significant differences in the levels of cell population, extracellular matrix and cytokine/growth factor biomarkers among the iERMs investigated. Hierarchical clustering of RT-qPCR data identified two distinct iERM clusters, Cluster B samples representing transcriptionally "activated" iERMs when compared to transcriptionally "quiescent" Cluster A specimens. Further, Cluster B could be subdivided in two subgroups, Cluster B1 iERMs, characterized by a marked glial cell activation, and Cluster B2 samples characterized by a more pro-fibrotic phenotype. Preoperative decimal best-corrected visual acuity and post-surgery inner segment/outer grading values were higher in Cluster A patients, that showed a prevalence of fovea-attached type iERMs with near-normal inner retina, than in Cluster B patients, that presented more severe clinical and spectral domain optical coherence tomography (SD-OCT) features. In conclusion, this molecular characterization has identified two major clusters of iERM specimens with distinct transcriptional activities that reflect different clinical and SD-OCT features of iERM patients. This retrospective work paves the way to prospective whole-genome transcriptomic studies to allow a molecular classification of iERMs and for the identification of molecular signature(s) of prognostic and therapeutic significance.


Assuntos
Membrana Epirretiniana/genética , Idoso , Análise por Conglomerados , Membrana Epirretiniana/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Tomografia de Coerência Óptica
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