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The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.
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Quimiotaxis , Receptor Toll-Like 2 , Humanos , Adhesión Celular , Células Endoteliales/metabolismo , Integrinas , Células THP-1 , Receptor Toll-Like 2/metabolismo , Movimiento CelularRESUMEN
In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.
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Lipopolisacáridos , Receptor Toll-Like 4 , Células Endoteliales/metabolismo , Expresión Génica , Lipopolisacáridos/farmacología , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Células Endoteliales de la Vena Umbilical Humana , HumanosRESUMEN
As common industrial by-products, airborne engineered nanomaterials are considered important environmental toxins to monitor due to their potential health risks to humans and animals. The main uptake routes of airborne nanoparticles are nasal and/or oral inhalation, which are known to enable the transfer of nanomaterials into the bloodstream resulting in the rapid distribution throughout the human body. Consequently, mucosal barriers present in the nose, buccal, and lung have been identified and intensively studied as the key tissue barrier to nanoparticle translocation. Despite decades of research, surprisingly little is known about the differences among various mucosa tissue types to tolerate nanoparticle exposures. One limitation in comparing nanotoxicological data sets can be linked to a lack of harmonization and standardization of cell-based assays, where (a) different cultivation conditions such as an air-liquid interface or submerged cultures, (b) varying barrier maturity, and (c) diverse media substitutes have been used. The current comparative nanotoxicological study, therefore, aims at analyzing the toxic effects of nanomaterials on four human mucosa barrier models including nasal (RPMI2650), buccal (TR146), alveolar (A549), and bronchial (Calu-3) mucosal cell lines to better understand the modulating effects of tissue maturity, cultivation conditions, and tissue type using standard transwell cultivations at liquid-liquid and air-liquid interfaces. Overall, cell size, confluency, tight junction localization, and cell viability as well as barrier formation using 50% and 100% confluency was monitored using trans-epithelial-electrical resistance (TEER) measurements and resazurin-based Presto Blue assays of immature (e.g., 5 days) and mature (e.g., 22 days) cultures in the presence and absence of corticosteroids such as hydrocortisone. Results of our study show that cellular viability in response to increasing nanoparticle exposure scenarios is highly compound and cell-type specific (TR146 6 ± 0.7% at 2 mM ZnO (ZnO) vs. ~90% at 2 mM TiO2 (TiO2) for 24 h; Calu3 93.9 ± 4.21% at 2 mM ZnO vs. ~100% at 2 mM TiO2). Nanoparticle-induced cytotoxic effects under air-liquid cultivation conditions declined in RPMI2650, A549, TR146, and Calu-3 cells (~0.7 to ~0.2-fold), with increasing 50 to 100% barrier maturity under the influence of ZnO (2 mM). Cell viability in early and late mucosa barriers where hardly influenced by TiO2 as well as most cell types did not fall below 77% viability when added to Individual ALI cultures. Fully maturated bronchial mucosal cell barrier models cultivated under ALI conditions showed less tolerance to acute ZnO nanoparticle exposures (~50% remaining viability at 2 mM ZnO for 24 h) than the similarly treated but more robust nasal (~74%), buccal (~73%), and alveolar (~82%) cell-based models.
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Nanopartículas , Óxido de Zinc , Animales , Humanos , Óxido de Zinc/toxicidad , Nanopartículas/toxicidad , Titanio/toxicidad , Membrana MucosaRESUMEN
Malignant melanoma is the deadliest form of skin cancer and NRF2 has been proposed as a main regulator of tumor cell malignancy. Still the mechanisms how NRF2 is contributing to melanoma progression are incompletely understood. Here we analyzed the effects of either NRF2 induction or depletion, and we also quantified changes on the whole cell proteome level. Our results showed that inhibition of NRF2 leads to a loss of reactive oxygen species protection, but at the same time to an induction of an epithelial mesenchymal transition (EMT) phenotype and an up-regulation of the stem cell marker CD44. Additionally, cells devoid of NRF2 showed increased cell viability after treatment with a MYC and a BRAF inhibitor. Importantly, survival upon vemurafenib treatment was dependent on CD44 expression. Finally, analysis of archival melanoma patient samples confirmed a vice versa relationship of NRF2 and CD44 expression. In summary, we recorded changes in the proteome after NRF2 modulation in melanoma cells. Surprisingly, we identified that NRF2 inhibition lead to induction of an EMT phenotype and an increase in survival of cells after apoptosis induction. Therefore, we propose that it is important for future therapies targeting NRF2 to consider blocking EMT promoting pathways in order to achieve efficient tumor therapy.
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Melanoma , Proteoma , Apoptosis , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Regulación hacia Arriba , Vemurafenib/farmacologíaRESUMEN
Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.
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Genes Reporteros , Luz , FN-kappa B/metabolismo , Optogenética/métodos , Receptor Toll-Like 4/metabolismo , Adhesión Celular , Movimiento Celular , Células HEK293 , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oxígeno/metabolismo , Páncreas/citología , Podosomas/metabolismo , Transducción de Señal , Receptor Toll-Like 4/genéticaRESUMEN
Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Melanoma/tratamiento farmacológico , Quercetina/farmacología , Antineoplásicos/química , Hidrolasas de Éster Carboxílico/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/genética , Melanoma/patología , Factor 2 Relacionado con NF-E2/genética , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Quercetina/química , Esferoides Celulares/efectos de los fármacosRESUMEN
Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF's anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS.
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Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide. Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release. We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway. Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells. Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET. In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex. Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI. In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype. Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.
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Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Receptores Depuradores de Clase B/genética , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proto-Oncogenes Mas , Receptores Depuradores de Clase B/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
BACKGROUND: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. METHODS: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. RESULTS: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. CONCLUSION: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.
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Biblioteca de Péptidos , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Ligandos , Estreptavidina/metabolismoRESUMEN
Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell-substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.
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Anticuerpos Neutralizantes/análisis , Células Epiteliales/citología , Gliadina/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Intestinos/citología , Células CACO-2 , Comunicación Celular , Supervivencia Celular , Regulación hacia Abajo , Humanos , Inmunoglobulinas/aislamiento & purificación , Inflamación/patología , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. METHOD: Enzymol. 459, 395-433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4'-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959-968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658-24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649-662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.
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Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Staphylococcus aureus , Temperatura , Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad ProteicaRESUMEN
Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.
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Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis.
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Anticuerpos Antineoplásicos , Antígenos de Neoplasias/inmunología , Biomarcadores de Tumor/inmunología , Cromatografía de Afinidad , Melanoma/inmunología , Animales , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/aislamiento & purificación , Antígenos de Neoplasias/química , Biomarcadores de Tumor/química , Femenino , Humanos , Melanoma/mortalidad , Melanoma/patología , Ratones , Ratones SCID , Metástasis de la Neoplasia , ConejosRESUMEN
To design new compounds suitable as starting points for anticancer drug development, we have synthesized a novel series of benzoxazoles with pharmaceutically advantageous piperazine and fluorine moieties attached to them. The newly synthesized benzoxazoles and their corresponding precursors were evaluated for cytotoxicity on human A-549 lung carcinoma cells and non-cancer HepaRG hepatocyes. Some of these new benzoxazoles show potential anticancer activity, while two of the intermediates show lung cancer selective properties at low concentrations where healthy cells are unaffected, indicating a selectivity window for anticancer compounds.
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BACKGROUND: HER2 expression in breast cancer correlates with increased metastatic potential, higher tumor recurrence rates and improved response to targeted therapies. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are two methods commonly used for the analysis of HER2 in the clinic. However, lack of standardization, technical variability in laboratory protocols and subjective interpretation are major problems associated with these testing procedures. METHODS: Here we evaluated the applicability of reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for HER2 testing in breast cancer. We tested thirty formaldehyde-fixed and paraffin-embedded tumor samples by RT-qPCR, FISH and IHC and analysed and compared the data from the three methods. RESULTS: We found that laser-captured microdissection is essential for the accurate determination of HER2 expression by RT-qPCR. When isolating RNA from total tumor tissue we obtained a significant number of false negative results. However, when using RNA from purified cancer cells the RT-qPCR data were fully consistent with FISH and IHC. In addition we provide evidence that ductal carcinomas might be further classified by the differential expression of HER3 and HER4. CONCLUSIONS: Laser-captured microdissection in combination with RT-qPCR is a precise and cost-effective diagnostic approach for HER2 testing in cancer. The PCR assay is simple, accurate and robust and can easily be implemented and standardized in clinical laboratories.
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BACKGROUND: To be able to develop effective therapeutics for epidermolysis bullosa simplex (EBS), it is necessary to elucidate the molecular pathomechanisms that give rise to the disease's characteristic severe skin-blistering phenotype. RESULTS: Starting with a whole-transcriptome microarray analysis of an EBS Dowling-Meara model cell line (KEB7), we identified 207 genes showing differential expression relative to control keratinocytes. A complementary qRT-PCR study of 156 candidates confirmed 76.58 % of the selected genes to be significantly up-regulated or down-regulated (p-value <0.05) within biological replicates. Our hit list contains previously identified genes involved in epithelial cell proliferation, cell-substrate adhesion, and responses to diverse biological stimuli. In addition, we identified novel candidate genes and potential affected pathways not previously considered as relevant to EBS pathology. CONCLUSIONS: Our results broaden our understanding of the molecular processes dysregulated in EBS.
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Perfilación de la Expresión Génica/métodos , Queratinocitos/metabolismo , Transducción de Señal/genética , Transcriptoma/genética , Western Blotting , Línea Celular , Epidermólisis Ampollosa Simple/tratamiento farmacológico , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/patología , Ontología de Genes , Predisposición Genética a la Enfermedad/genética , Humanos , Queratinocitos/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.
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Resistencia a Antineoplásicos , Melanoma/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , alfa Catenina/metabolismo , Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Melanoma/genética , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , alfa Catenina/genéticaRESUMEN
Acne vulgaris is the most common skin disease, causing significant psychosocial problems such as anxiety and depression similar to a chronic illness for those afflicted. Currently, obtainable agents for acne treatment have limited use. Thus, development of novel agents to treat this disease is a high medical need. The anaerobic bacterium Propionibacterium acnes has been implicated in the inflammatory phase of acne vulgaris by activating pro-inflammatory mediators such as the interleukin-8 (IL-8) via the NF-κB and MAPK pathways. Talaromyces wortmannii is an endophytic fungus, which is known to produce high bioactive natural compounds. We hypothesize that compound C but also the crude extract from T. wortmannii may possess both antibacterial activity especially against P. acnes and also anti-inflammatory properties by inhibiting TNF-α-induced ICAM-1 expression and P. acnes-induced IL-8 release. Treatment of keratinocytes (HaCaT) with P. acnes significantly increased NF-κB and activator protein-1 (AP-1) activation, as well as IL-8 release. Compound C inhibited P. acnes-mediated activation of NF-κB and AP-1 by inhibiting IκB degradation and the phosphorylation of ERK and JNK MAP kinases, and IL-8 release in a dose-dependent manner. Based on these results, compound C has effective antimicrobial activity against P. acnes and anti-inflammatory activity, and we suggest that this substance or the crude extract are alternative treatments for antibiotic/anti-inflammatory therapy for acne vulgaris.
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Acné Vulgar/microbiología , Antibacterianos/farmacología , Antiinflamatorios/farmacología , Mezclas Complejas/farmacología , Endófitos/química , Propionibacterium acnes/fisiología , Talaromyces/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/metabolismo , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Propionibacterium acnes/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cultured human primary keratinocytes constitute suitable targets for in-depth evaluation of the proliferative or differentiative potential of compounds. There is, however, a double-edged and intrinsically inseparable transition from biological activity to cytotoxicity for any agent under investigation. For that reason, we here first of all present an established protocol for the isolation, cultivation, and analysis of primary foreskin-derived keratinocytes. Taking calcitriol as example, we then reveal how a straightforward photometric cell culture assay can be exploited to assess overall cell viability in response to increasing compound doses. With predetermined cellular cytotoxicity at hand, physiologically meaningful (sub-toxic) compound concentrations for subsequent stimulation of cells can be readily selected, and, in doing so, differentially expressed genes with biological significance can be reliably identified.
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Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica/métodos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Calcitriol/farmacología , Calcitriol/toxicidad , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Separación Celular , Supervivencia Celular/efectos de los fármacos , Criopreservación , Humanos , Queratinocitos/citología , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Aeruginosin-865 (Aer-865), isolated from terrestrial cyanobacterium Nostoc sp. Lukesová 30/93, is the first aeruginosin-type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one- and two-dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: D-Hpla, D-Leu, 5-OH-Choi, Agma, with hexanoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proinflammatory mediators IL-8 and ICAM-1 in hTNF-α-stimulated HLMVECs. Aer-865 showed significant reduction of both: with EC50 values of (3.5±1.5) µg mL(-1) ((4.0±1.7) µM) and (50.0±13.4) µg mL(-1) ((57.8±15.5) µM), respectively. Confocal laser scanning microscopy revealed that the anti-inflammatory effect of Aer-865 was directly associated with inhibition of NF-κB translocation to the nucleus. Moreover, Aer-865 did not show any cytotoxic effect.