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1.
Int J Mol Sci ; 21(6)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197499

RESUMO

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.


Assuntos
Matriz Extracelular/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Animais , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Inibidores de Proteases/metabolismo
2.
Molecules ; 25(9)2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32365473

RESUMO

The health effects of plant phenolics in vegetables and other food and the increasing evidence of the preventive potential of flavonoids in "Western Diseases" such as cancer, neurodegenerative diseases and others, have gained enormous interest. This prompted us to investigate the effects of 20 different flavonoids of the groups of flavones, flavonols and flavanones in 3D in vitro systems to determine their ability to inhibit the formation of circular chemorepellent induced defects (CCIDs) in monolayers of lymph- or blood-endothelial cells (LECs, BECs; respectively) by 12(S)-HETE, which is secreted by SW620 colon cancer spheroids. Several compounds reduced the spheroid-induced defects of the endothelial barriers. In the SW620/LEC model, apigenin and luteolin were most active and acacetin, nepetin, wogonin, pinocembrin, chrysin and hispidulin showed weak effects. In the SW620/BEC model acacetin, apigenin, luteolin, wogonin, hispidulin and chrysin exhibited weak activity.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Flavonoides/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Técnicas de Cocultura , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Flavonoides/química , Humanos , Neovascularização Patológica/metabolismo , Esferoides Celulares
3.
Hum Mol Genet ; 25(22): 5006-5016, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28171546

RESUMO

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3'-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática , Linfócitos/metabolismo , Células MCF-7 , Metaloproteinase 1 da Matriz/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Transdução de Sinais , Esferoides Celulares , Células Tumorais Cultivadas
4.
Cell Mol Life Sci ; 74(10): 1907-1921, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28013338

RESUMO

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Fibroblastos Associados a Câncer/patologia , Colo/patologia , Neoplasias Colorretais/patologia , Reto/patologia , Transdução de Sinais , Cálcio/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Miosinas Cardíacas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Humanos , Cadeias Leves de Miosina/metabolismo , Invasividade Neoplásica/patologia , Reto/metabolismo , Quinases Associadas a rho/metabolismo
5.
Hum Mol Genet ; 24(13): 3689-98, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25832648

RESUMO

Invasive colorectal cancer is associated with poor prognosis requiring treatment with systemic chemotherapies usually including 5-fluorouracil. A consequence of prolonged treatment is the acquisition of resistance eventually resulting in the recurrence of highly metastatic cancer cells. To address the relationship between drug resistance and increased lymphatic metastatic potential, we used a 3D co-culture model of colon tumour cell spheroids of parent CCL227 cells and subclones with gradually increasing resistance against 5-fluorouracil. From each investigated cell line, homogeneous tumour spheroids were generated in the presence of methylcellulose yielding emboli of ∼700 µm diameter. When invasive, tumour spheroids disrupt the continuous lymphendothelial cell (LEC) layer and generate a 'circular chemorepellent-induced defect' (CCID), reminiscent of the entry gates through which tumour emboli intravasate lymphatic vasculature. Here we provide evidence that increasingly chemoresistant colon cancer spheroids were strongly associated with enhanced intravasative properties. In naïve CCL227 spheroids, miR-200 family members were released into exosomes thereby repressing the epithelial to mesenchymal transition-regulating transcription factors ZEB1 and SLUG in LEC. As a consequence of attenuated plasticity and migration of LEC, CCID formation was impaired. Loss of exosomal transferred miR-200c in resistant colon cells rendered LEC more susceptible to pro-migratory signals that were generated and directly transmitted by colon cancer spheroids. This observation indicates a common molecular axis in colon cancer and LEC where miR-200 family members act as regulators of ZEB proteins. The data support the notion that horizontal miR-200 signalling prevents the permeation of cells into adjacent epithelia and contributes to organ integrity.


Assuntos
Neoplasias do Colo/metabolismo , Células Endoteliais/metabolismo , Fluoruracila/farmacologia , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metástase Linfática , MicroRNAs/genética , Família Multigênica , Invasividade Neoplásica , Fatores de Transcrição da Família Snail , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de Zinco
6.
Br J Cancer ; 115(3): 364-70, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27362730

RESUMO

BACKGROUND: The arachidonic acid metabolite 12(S)-HETE is suspected to enhance metastatic spread by inducing cancer cell- and lymph endothelial cell (LEC) motility. However, the molecular mechanisms leading to 12(S)-HETE-triggered cell migration are still elusive. METHODS: To delineate the signalling pathways involved in 12(S)-HETE-mediated migration, inhibitors against RHO and ROCK, and specific siRNAs downregulating 12(S)-HETE receptor (12-HETER) and myosin light chain 2 (MLC2) were used. The breaching of the endothelial barrier was investigated by an assay measuring tumour spheroid-triggered 'circular chemorepellent-induced defects' (CCIDs), and respective signal transduction was elucidated by western blotting. RESULTS: We provide evidence that 12(S)-HETE phosphorylated (and activated) MLC2, which regulates actin/myosin-based contraction. MLC2 activation was found to be essential for LEC retraction and CCID formation. Furthermore, we show that 12(S)-HETE activated a 12-HETER-RHO-ROCK-MYPT signalling cascade to induce MLC2 function. CONCLUSIONS: Signalling via this pathway is described for this metabolite for the first time. This may provide potential targets for the intervention of metastatic colonisation.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Endotélio Linfático/metabolismo , Cadeias Leves de Miosina/metabolismo , Receptores Eicosanoides/metabolismo , Fator Rho/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Linhagem Celular Tumoral , Humanos , Permeabilidade , Fosforilação
7.
Hum Mol Genet ; 21(21): 4615-27, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22843495

RESUMO

Pancreas cancer cells escape most treatment options. Heat shock protein (Hsp)90 is frequently over-expressed in pancreas carcinomas and protects a number of cell-cycle regulators such as the proto-oncogene Cdc25A. We show that inhibition of Hsp90 with geldanamycin (GD) destabilizes Cdc25A independent of Chk1/2, whereas the standard drug for pancreas carcinoma treatment, gemcitabine (GEM), causes Cdc25A degradation through the activation of Chk2. Both agents applied together additively inhibit the expression of Cdc25A and the proliferation of pancreas carcinoma cells thereby demonstrating that both Cdc25A-destabilizing/degrading pathways are separated. The role of Hsp90 as stabilizer of Cdc25A in pancreas carcinoma cells is further supported by two novel synthetic inhibitors 4-tosylcyclonovobiocic acid and 7-tosylcyclonovobiocic acid and specific Hsp90AB1 (Hsp90ß) shRNA. Our data show that targeting Hsp90 reduced the resistance of pancreas carcinoma cells to treatment with GEM.


Assuntos
Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90 , Neoplasias Pancreáticas , Fosfatases cdc25 , Benzoquinonas/farmacologia , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Novobiocina/análogos & derivados , Novobiocina/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Proto-Oncogene Mas , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Gencitabina , Neoplasias Pancreáticas
8.
Arch Toxicol ; 88(3): 691-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24352538

RESUMO

Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 µM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.


Assuntos
Carbamazepina/farmacologia , Técnicas de Cultura de Células/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células Endoteliais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Miosinas Cardíacas/metabolismo , Técnicas de Cocultura , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliais/citologia , Quinase 1 de Adesão Focal/metabolismo , Humanos , Células MCF-7/efeitos dos fármacos , Células MCF-7/patologia , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos
9.
Nat Med ; 12(2): 230-4, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16415878

RESUMO

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.


Assuntos
Células Endoteliais/patologia , Transplante de Rim/patologia , Linfangiogênese/fisiologia , Sequência de Bases , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/patologia , Cromossomos Humanos Y , Feminino , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Humanos , Hibridização in Situ Fluorescente , Transplante de Rim/efeitos adversos , Linfangiogênese/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/patologia , Doadores de Tecidos , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
10.
Arch Toxicol ; 87(7): 1301-12, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23503627

RESUMO

Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Flavonoides/farmacologia , Propiofenonas/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/metabolismo , Adesão Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocromo P-450 CYP1A1/metabolismo , Relação Dose-Resposta a Droga , Selectina E/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células MCF-7 , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Esferoides Celulares , Transfecção
11.
Arch Toxicol ; 87(10): 1851-61, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23543012

RESUMO

Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Metástase Linfática/prevenção & controle , Terpenos/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Western Blotting , Neoplasias da Mama/patologia , Movimento Celular , Técnicas de Cocultura , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Paxilina/metabolismo
12.
Am J Pathol ; 174(4): 1191-202, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19264907

RESUMO

Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.


Assuntos
Antioxidantes/metabolismo , Ácidos Graxos/metabolismo , Nefrose/fisiopatologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Animais , Antimetabólitos Antineoplásicos/toxicidade , Western Blotting , Antígenos CD36/biossíntese , Antígenos CD36/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Imunofluorescência , Masculino , Microscopia Eletrônica de Transmissão , Nefrose/induzido quimicamente , Proteoma , Puromicina Aminonucleosídeo/toxicidade , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxido Dismutase/metabolismo
13.
Int J Oncol ; 56(4): 1034-1044, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32319559

RESUMO

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic acid [12(S)­HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription­quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced defect' assay. 12(S)­HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)­HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.


Assuntos
Movimento Celular , Endotélio Linfático/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Neoplasias/patologia , Fatores de Transcrição SOXF/metabolismo , Fator de Transcrição RelA/metabolismo , Linhagem Celular Tumoral , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/patologia , Retroalimentação Fisiológica , Quinase 1 de Adesão Focal/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Fator de Transcrição RelA/genética
14.
Nephrol Dial Transplant ; 24(6): 1979-86, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19223275

RESUMO

BACKGROUND: Several studies indicate that interstitial and intracapillary monocytes/macrophages (MO) represent a significant proportion of graft-infiltrating cells in renal allografts and that their presence may unfavourably affect clinical outcome. Much less is known about the role of MO in vascular rejection of transplanted kidneys. The aim of our study was to determine the cellular composition of immune cell infiltrates in intimal arteritis and to analyse whether it is associated with features of humoral immunity and impaired graft survival. METHODS: In 34 recipients with vascular rejection, we determined the proportion of intimal and interstitial MO and T-cells (expressed as ratio of CD68- and CD3-positive cells) in immunohistochemically double-labelled slides. RESULTS: Intimal arteritis is always composed of T-cells and MO with a median CD68/CD3 ratio of 1.03. In 47% of cases, however, T-cells predominate (CD68/CD3 ratio <1). The median interstitial CD68/CD3 ratio is 0.61, with T-cells dominating in 64% of cases. There is no correlation between the cellular composition of arterial and interstitial infiltrates. The proportion of interstitial and arterial MO has no impact on graft survival, and is, in contrast to previous reports on MO in allograft glomerulitis and capillaritis, not associated with C4d staining. CONCLUSIONS: Intimal arteritis in kidney allograft rejection is composed of a mixed infiltrate of MO and T-lymphocytes. In contrast to MO in PTCitis and glomerulitis, the MO in intimal arteritis are not associated with markers of humoral immune response and there are no different allograft outcomes between MO and T-lymphocyte-dominated groups.


Assuntos
Arterite/etiologia , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Macrófagos/imunologia , Monócitos/imunologia , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Arterite/imunologia , Arterite/patologia , Complexo CD3/metabolismo , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Estimativa de Kaplan-Meier , Transplante de Rim/imunologia , Transplante de Rim/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Linfócitos T/patologia , Transplante Homólogo , Resultado do Tratamento , Túnica Íntima/imunologia , Túnica Íntima/patologia
16.
PLoS One ; 14(3): e0214332, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921378

RESUMO

Focal and Segmental Glomerulosclerosis (FSGS) is a severe glomerulopathy that frequently leads to end stage renal disease. Only a subset of patients responds to current therapies, making it important to identify alternative therapeutic options. The interleukin (IL)-1 receptor antagonist anakinra is beneficial in several diseases with renal involvement. Here, we evaluated the potential of anakinra for FSGS treatment. Molecular process models obtained from scientific literature data were used to build FSGS pathology and anakinra mechanism of action models by exploiting information on protein interactions. These molecular models were compared by statistical interference analysis and expert based molecular signature matching. Experimental validation was performed in Adriamycin- and lipopolysaccharide (LPS)-induced nephropathy mouse models. Interference analysis (containing 225 protein coding genes and 8 molecular process segments) of the FSGS molecular pathophysiology model with the drug mechanism of action of anakinra identified a statistically significant overlap with 43 shared molecular features that were enriched in pathways relevant in FSGS, such as plasminogen activating cascade, inflammation and apoptosis. Expert adjudication of molecular signature matching, focusing on molecular process segments did not suggest a high therapeutic potential of anakinra in FSGS. In line with this, experimental validation did not result in altered proteinuria or significant changes in expression of the FSGS-relevant genes COL1A1 and NPHS1. In summary, an integrated bioinformatic and experimental workflow showed that FSGS relevant molecular processes can be significantly affected by anakinra beyond the direct drug target IL-1 receptor type 1 (IL1R1) context but might not counteract central pathophysiology processes in FSGS. Anakinra is therefore not suggested for extended preclinical trials.


Assuntos
Glomerulosclerose Segmentar e Focal/patologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Animais , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Reposicionamento de Medicamentos , Expressão Gênica/efeitos dos fármacos , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
17.
Int J Oncol ; 53(1): 307-316, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29749465

RESUMO

Metastasising breast cancer cells communicate with adjacent lymph endothelia, intravasate and disseminate through lymphatic routes, colonise lymph nodes and finally metastasize to distant organs. Thus, understanding and blocking intravasation may attenuate the metastatic cascade at an early step. As a trigger factor, which causes the retraction of lymph endothelial cells (LECs) and opens entry ports for tumour cell intravasation, MDA-MB231 breast cancer cells secrete the pro-metastatic arachidonic acid metabolite, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE]. In the current study, treatment of LECs with 12(S)-HETE upregulated the expression of the transcription factors SRY-related HMG-box 18 (SOX18) and prospero homeobox protein 1 (PROX1), which determine endothelial development. Thus, whether they have a role in LEC retraction was determined using a validated intravasation assay, small interfering RNA mediated knockdown of gene expression, and mRNA and protein expression analyses. Specific inhibition of SOX18 or PROX1 significantly attenuated in vitro intravasation of MDA-MB231 spheroids through the LEC barrier and 12(S)-HETE-triggered signals were transduced by the high and low affinity receptors, 12(S)-HETE receptor and leukotriene B4 receptor 2. In addition, the current findings indicate that there is crosstalk between SOX18 and nuclear factor κ-light-chain-enhancer of activated B cells, which was demonstrated to contribute to MDA-MB231/lymph endothelial intravasation. The present data demonstrate that the endothelial-specific and lymph endothelial-specific transcription factors SOX18 and PROX1 contribute to LEC retraction.


Assuntos
Neoplasias da Mama/genética , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição SOXF/genética , Proteínas Supressoras de Tumor/genética , Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/genética , Ácidos Araquidônicos/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Endoteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Neoplásica
18.
Front Pharmacol ; 9: 220, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29593542

RESUMO

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.

19.
Food Chem Toxicol ; 111: 114-124, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29129665

RESUMO

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.


Assuntos
Neoplasias Colorretais/patologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Flavonoides/farmacologia , Esferoides Celulares/fisiologia , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Metástase Neoplásica/fisiopatologia , Preparações Farmacêuticas
20.
Int J Oncol ; 50(5): 1879-1888, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28393180

RESUMO

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Técnicas de Cocultura , Fenofibrato/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citocromo P-450 CYP1A1/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Metástase Linfática , Células MCF-7 , NF-kappa B/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos
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