RESUMEN
Intestinal epithelial cells are covered by the brush border, which consists of densely packed microvilli. The Intermicrovillar Adhesion Complex (IMAC) links the microvilli and is required for proper brush border organization. Whether microvillus crosslinking is involved in the intestinal barrier function or colitis is currently unknown. We investigate the role of microvillus crosslinking in colitis in mice with deletion of the IMAC component CDHR5. Electron microscopy shows pronounced brush border defects in CDHR5-deficient mice. The defects result in severe mucosal damage after exposure to the colitis-inducing agent DSS. DSS increases the permeability of the mucus layer and brings bacteria in direct contact with the disorganized brush border of CDHR5-deficient mice. This correlates with bacterial invasion into the epithelial cell layer which precedes epithelial apoptosis and inflammation. Single-cell RNA sequencing data of patients with ulcerative colitis reveals downregulation of CDHR5 in enterocytes of diseased areas. Our results provide experimental evidence that a combination of microvillus crosslinking defects with increased permeability of the mucus layer sensitizes to inflammatory bowel disease.
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Recent research into miniaturized illumination sources has prompted the development of alternative microscopy techniques. Although they are still being explored, emerging nano-light-emitting-diode (nano-LED) technologies show promise in approaching the optical resolution limit in a more feasible manner. This work presents the exploration of their capabilities with two different prototypes. In the first version, a resolution of less than 1 µm was shown thanks to a prototype based on an optically downscaled LED using an LED scanning transmission optical microscopy (STOM) technique. This research demonstrates how this technique can be used to improve STOM images by oversampling the acquisition. The second STOM-based microscope was fabricated with a 200 nm GaN LED. This demonstrates the possibilities for the miniaturization of on-chip-based microscopes.
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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.
Asunto(s)
Matriz Extracelular/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Animales , Proteínas del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Matriz Extracelular/patología , Fibrinógeno/metabolismo , Regulación de la Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/patología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Inhibidores de Proteasas/metabolismoRESUMEN
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.
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Neoplasias de la Mama/genética , Células Endoteliales/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción SOXF/genética , Proteínas Supresoras de Tumor/genética , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/genética , Ácidos Araquidónicos/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metástasis de la NeoplasiaRESUMEN
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.
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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.
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Neoplasias Colorrectales/patología , Células Endoteliales/fisiología , Endotelio Vascular/fisiología , Flavonoides/farmacología , Esferoides Celulares/fisiología , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Metástasis de la Neoplasia/fisiopatología , Preparaciones FarmacéuticasRESUMEN
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.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Fibroblastos Asociados al Cáncer/patología , Colon/patología , Neoplasias Colorrectales/patología , Recto/patología , Transducción de Señal , Calcio/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Miosinas Cardíacas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Humanos , Cadenas Ligeras de Miosina/metabolismo , Invasividad Neoplásica/patología , Recto/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Since cancer cells, when grown as spheroids, display drug sensitivity and radiation resistance patterns such as seen in vivo we recently established a threedimensional (3D) in vitro model recapitulating colorectal cancer (CRC)-triggered lymphatic endothelial cell (LEC)barrier breaching to study mechanisms of intra/extravasation. CRC metastasizes not only through lymphatics but also through blood vessels and here we extend the 3D model to the interaction of blood endothelial cells (BECs) with naïve and 5fluorouracil (5FU)resistant CRC CCL227 cells. The 3D model enabled quantifying effects of tumourderived microRNA200 (miR200) miR200a, miR200b, miR200c, miR141 and miR429 regarding the induction of so-called 'circular chemorepellentinduced defects' (CCIDs) within the BECbarrier, which resemble gates for tumour transmigration. For this, miR200 precursors were individually transfected and furthermore, the modulation of ZEB family expression was analysed by western blotting. miR200c, miR141 and miR429, which are contained in exosomes from naïve CCL227 cells, downregulated the expression of ZEB2, SNAI and TWIST in BECs. The exosomes of 5FUresistant CCL227RH cells, which are devoid of miR200, accelerated CCID formation in BEC monolayers as compared to exosomes from naïve CCL227 cells. This confirmed the reported role of ZEB2 and SNAI in CRC metastasis and highlighted the active contribution of the stroma in the metastatic process. CCL227 spheroids affected the integrity of BEC and LEC barriers alike, which was in agreement with the observation that CRC metastasizes via blood stream (into the liver) as well as via lymphatics (into lymph nodes and lungs). This further validated the CRC/LEC and CRC/BEC in vitro model to study mechanisms of CRC spreading through vascular systems. Treatment of CCL227RH cells with the HDAC inhibitors mocetinostat and sulforaphane reduced CCID formation to the level triggered by naïve CCL227 spheroids, however, without significantly influencing miR200 expression in CCL227-RH cells.
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Neoplasias Colorrectales/patología , Células Endoteliales/patología , Endotelio Vascular/patología , MicroARNs/genética , Benzamidas/administración & dosificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Neoplasias Colorrectales/genética , Endotelio Vascular/metabolismo , Exosomas/genética , Exosomas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isotiocianatos/administración & dosificación , Metástasis Linfática , Vasos Linfáticos/patología , FN-kappa B/metabolismo , Pirimidinas/administración & dosificación , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , SulfóxidosRESUMEN
INTRODUCTION: Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. MATERIALS AND METHODS: Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. RESULTS: Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. DISCUSSION: This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed.
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Hipocampo/fisiología , Glicoproteínas de Membrana/fisiología , Memoria/fisiología , Plasticidad Neuronal , Animales , Técnicas de Inactivación de Genes , Hipocampo/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , RatonesRESUMEN
Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Neoplasias de la Mama/patología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Movimiento Celular , Células Endoteliales/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Quelantes del Calcio/farmacología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Miosinas Cardíacas/metabolismo , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Femenino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Metástasis Linfática , Vasos Linfáticos/metabolismo , Células MCF-7 , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Permeabilidad , Fosforilación , Interferencia de ARN , Serina , Esferoides Celulares , Factores de Tiempo , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Endotelio Linfático/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Receptores Eicosanoides/metabolismo , Factor Rho/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Línea Celular Tumoral , Humanos , Permeabilidad , FosforilaciónRESUMEN
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.
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Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Neoplasias de la Mama/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Metástasis Linfática , Linfocitos/metabolismo , Células MCF-7 , Metaloproteinasa 1 de la Matriz/metabolismo , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Transducción de Señal , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited "circular chemo-repellent induced defects" (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20-30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10-15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca(2+) release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 - MMP1 (MDA-MB231) - PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.
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Neoplasias de la Mama/metabolismo , Células Endoteliales/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , FN-kappa B/metabolismo , Receptor PAR-1/metabolismo , Proteínas de Arabidopsis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Humanos , Células MCF-7 , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Comunicación Paracrina , Receptor PAR-1/genética , Esferoides Celulares , TransfecciónRESUMEN
BACKGROUND: The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. PURPOSE: Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. METHODS: ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. RESULTS: In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rß expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. CONCLUSION: Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.
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Asteraceae/química , Lactonas/farmacología , Linfoma Anaplásico de Células Grandes/patología , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sesquiterpenos de Germacrano/farmacología , Sesquiterpenos/farmacología , Apoptosis , Ciclo Celular , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Estructura Molecular , Plantas Medicinales/química , Transducción de SeñalRESUMEN
Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 = 8.9 µM after 72 h) and 10 µM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100 µM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25 µM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.
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Apoptosis/efectos de los fármacos , Asteraceae/química , Proliferación Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Sesquiterpenos de Eudesmano/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Concentración 50 Inhibidora , Masculino , Ratas , Ratas Sprague-Dawley , Saponinas/farmacología , Espirostanos/farmacologíaRESUMEN
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 â¼700 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.
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Neoplasias del Colon/metabolismo , Células Endoteliales/metabolismo , Fluorouracilo/farmacología , MicroARNs/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Células Endoteliales/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Metástasis Linfática , MicroARNs/genética , Familia de Multigenes , Invasividad Neoplásica , Factores de Transcripción de la Familia Snail , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.
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Antibióticos Antineoplásicos/farmacología , Óxido Nítrico/antagonistas & inhibidores , ARN Mensajero/genética , Receptores Depuradores de Clase B/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , HDL-Colesterol/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/antagonistas & inhibidores , Receptores Depuradores de Clase B/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
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Carbamazepina/farmacología , Técnicas de Cultivo de Célula/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Endoteliales/efectos de los fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipooxigenasa/metabolismo , Miosinas Cardíacas/metabolismo , Técnicas de Cocultivo , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliales/citología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Células MCF-7/efectos de los fármacos , Células MCF-7/patología , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Esferoides Celulares/efectos de los fármacosRESUMEN
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.
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Neoplasias de la Mama/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Metástasis Linfática/prevención & control , Terpenos/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Western Blotting , Neoplasias de la Mama/patología , Movimiento Celular , Técnicas de Cocultivo , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Células MCF-7 , FN-kappa B/metabolismo , Paxillin/metabolismoRESUMEN
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.