RESUMEN
Despite the importance of the tumor microenvironment in regulating tumor progression, few in vitro models have been developed to understand the effects of non-neoplastic cells and extracellular matrix (ECM) on drug resistance in glioblastoma (GBM) cells. Using CellTrace-labeled human GBM and microglial (MG) cells, we established a 2D co-culture including various ratios of the two cell types. Viability, proliferation, migration, and drug response assays were carried out to assess the role of MG. A 3D model was then established using a hyaluronic acid-gelatin hydrogel to culture a mixture of GBM and MG and evaluate drug resistance. A contact co-culture of fluorescently labeled GBM and MG demonstrated that MG cells modestly promoted tumor cell proliferation (17%-30% increase) and greater migration of GBM cells (>1.5-fold increase). Notably, the presence of MG elicited drug resistance even when in a low ratio (10%-20%) relative to co-cultured tumor cells. The protective effect of MG on GBM was greater in the 3D model (>100% survival of GBM when challenged with cytotoxics). This new 3D human model demonstrated the influence of non-neoplastic cells and matrix on chemoresistance of GBM cells to three agents with different mechanisms of action suggesting that such sophisticated in vitro approaches may facilitate improved preclinical testing.
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Neoplasias Encefálicas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citotoxinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Microglía/efectos de los fármacos , Anciano , Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/patología , Glioblastoma/patología , Humanos , Ácido Hialurónico/farmacología , Hidrogeles/farmacología , Masculino , Microglía/patología , Persona de Mediana Edad , Microambiente Tumoral/efectos de los fármacosRESUMEN
PURPOSE: Polyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers. METHODS: Anionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS). RESULTS: Dendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies. CONCLUSIONS: PAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.
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Dendrímeros/metabolismo , Portadores de Fármacos/metabolismo , Pulmón/metabolismo , Células Epiteliales Alveolares/metabolismo , Animales , Aniones/metabolismo , Transporte Biológico , Línea Celular , Células Cultivadas , Endocitosis , RatasRESUMEN
PURPOSE: To examine if pulmonary P-glycoprotein (P-gp) is functional in an intact lung; impeding the pulmonary absorption and increasing lung retention of P-gp substrates administered into the airways. Using calculated physico-chemical properties alone build a predictive Quantitative Structure-Activity Relationship (QSAR) model distinguishing whether a substrate's pulmonary absorption would be limited by P-gp or not. METHODS: A panel of 18 P-gp substrates were administered into the airways of an isolated perfused mouse lung (IPML) model derived from Mdr1a/Mdr1b knockout mice. Parallel intestinal absorption studies were performed. Substrate physico-chemical profiling was undertaken. Using multivariate analysis a QSAR model was established. RESULTS: A subset of P-gp substrates (10/18) displayed pulmonary kinetics influenced by lung P-gp. These substrates possessed distinct physico-chemical properties to those P-gp substrates unaffected by P-gp (8/18). Differential outcomes were not related to different intrinsic P-gp transporter kinetics. In the lung, in contrast to intestine, a higher degree of non-polar character is required of a P-gp substrate before the net effects of efflux become evident. The QSAR predictive model was applied to 129 substrates including eight marketed inhaled drugs, all these inhaled drugs were predicted to display P-gp dependent pulmonary disposition. CONCLUSIONS: Lung P-gp can affect the pulmonary kinetics of a subset of P-gp substrates. Physico-chemical relationships determining the significance of P-gp to absorption in the lung are different to those operative in the intestine. Our QSAR framework may assist profiling of inhaled drug discovery candidates that are also P-gp substrates. The potential for P-gp mediated pulmonary disposition exists in the clinic.
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Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Pulmón/metabolismo , Preparaciones Farmacéuticas/metabolismo , Absorción a través del Sistema Respiratorio , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Masculino , Ratones , Ratones Noqueados , Preparaciones Farmacéuticas/química , Especificidad por Sustrato , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
We have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. We show here that the formulation of peptides LL37 and magainin-2B amide in polysorbate 20 (Tween 20) results in greater reductions in virus titer than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high-ionic-strength buffer. In contrast, formulation with polysorbate 20 or high-ionic-strength buffer has the opposite effect on bactericidal activity of both peptides, resulting in lesser reductions in titer for both Gram-positive and Gram-negative bacteria. Circular dichroism spectroscopy shows that the differential action of polysorbate 20 and salt on the virucidal and bactericidal activities correlates with the α-helical content of peptide secondary structure in solution, suggesting that the virucidal and bactericidal activities are mediated through distinct mechanisms. The correlation of a defined structural feature with differential activity against a host-derived viral membrane and the membranes of both Gram-positive and Gram-negative bacteria suggests that the overall helical content in solution under physiological conditions is an important feature for consideration in the design and development of candidate peptide-based antimicrobial compounds.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Catelicidinas/farmacología , Escherichia coli/efectos de los fármacos , Polisorbatos/farmacología , Staphylococcus aureus/efectos de los fármacos , Virus Vaccinia/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Antivirales/química , Catelicidinas/química , Línea Celular , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Escherichia coli/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Concentración Osmolar , Polisorbatos/química , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conejos , Especificidad de la Especie , Staphylococcus aureus/crecimiento & desarrollo , Relación Estructura-Actividad , Virus Vaccinia/crecimiento & desarrollo , Virión/efectos de los fármacos , Virión/crecimiento & desarrolloRESUMEN
PURPOSE: We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. MATERIALS AND METHODS: Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg ml(-1) mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. RESULTS: Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 µg cm(-2), respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. CONCLUSIONS: To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C.
Asunto(s)
Mitomicina/farmacocinética , Uréter/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Animales , Técnicas In Vitro , Permeabilidad , PorcinosRESUMEN
Transurothelial drug delivery continues to be an attractive treatment option for a range of urological conditions; however, dosing regimens remain largely empirical. Recently, intravesical delivery of the nonsteroidal anti-inflammatory ketorolac has been shown to significantly reduce ureteral stent-related pain. While this latest development provides an opportunity for advancing the management of stent-related pain, clinical translation will undoubtedly require an understanding of the rate and extent of delivery of ketorolac into the bladder wall. Using an ex vivo porcine model, we evaluate the urothelial permeability and bladder wall distribution of ketorolac. The subsequent application of a pharmacokinetic (PK) model enables prediction of concentrations achieved in vivo. Ketorolac was applied to the urothelium and a transurothelial permeability coefficient (Kp) calculated. Relative drug distribution into the bladder wall after 90 min was determined. Ketorolac was able to permeate the urothelium (Kp = 2.63 × 10(-6) cm s(-1)), and after 90 min average concentrations of 400, 141 and 21 µg g(-1) were achieved in the urothelium, lamina propria and detrusor respectively. An average concentration of 87 µg g(-1) was achieved across the whole bladder wall. PK simulations (STELLA) were then carried out, using ex vivo values for Kp and muscle/saline partition coefficient (providing an estimation of vascular clearance), to predict 90 min in vivo ketorolac tissue concentrations. When dilution of the drug solution with urine and vascular clearance were taken into account, a reduced ketorolac concentration of 37 µg g(-1) across the whole bladder wall was predicted. These studies reveal crucial information about the urothelium's permeability to agents such as ketorolac and the concentrations achievable in the bladder wall. It would appear that levels of ketorolac delivered to the bladder wall intravesically would be sufficient to provide an anti-inflammatory effect. The combination of such ex vivo data and PK modeling provides an insight into the likelihood of achieving clinically relevant concentrations of drug following intravesical administration.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ketorolaco/farmacocinética , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Administración Intravesical , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Simulación por Computador , Sistemas de Liberación de Medicamentos , Ketorolaco/administración & dosificación , Cinética , Porcinos , Distribución TisularRESUMEN
BACKGROUND: Up to 40% of patients initially diagnosed with clinically-confined renal cell carcinoma (RCC) and who undergo curative surgery will nevertheless relapse with metastatic disease (mRCC) associated with poor long term survival. The discovery of novel prognostic/predictive biomarkers and drug targets is needed and in this context the aim of the current study was to investigate a putative caveolin-1/ERK signalling axis in clinically confined RCC, and to examine in a panel of RCC cell lines the effects of caveolin-1 (Cav-1) on pathological processes (invasion and growth) and select signalling pathways. METHODS: Using immunohistochemistry we assessed the expression of both Cav-1 and phosphorylated-ERK (pERK) in 176 patients with clinically confined RCC, their correlation with histological parameters and their impact upon disease-free survival. Using a panel of RCC cell lines we explored the functional effects of Cav-1 knockdown upon cell growth, cell invasion and VEGF-A secretion, as well Cav-1 regulation by cognate cell signalling pathways. RESULTS: We found a significant correlation (P = 0.03) between Cav-1 and pERK in a cohort of patients with clinically confined disease which represented a prognostic biomarker combination (HR = 4.2) that effectively stratified patients into low, intermediate and high risk groups with respect to relapse, even if the patients' tumours displayed low grade and/or low stage disease. In RCC cell lines Cav-1 knockdown unequivocally reduced cell invasive capacity while also displaying both pro-and anti-proliferative effects; targeted knockdown of Cav-1 also partially suppressed VEGF-A secretion in VHL-negative RCC cells. The actions of Cav-1 in the RCC cell lines appeared independent of both ERK and AKT/mTOR signalling pathways. CONCLUSION: The combined expression of Cav-1 and pERK serves as an independent biomarker signature with potential merit in RCC surveillance strategies able to predict those patients with clinically confined disease who will eventually relapse. In a panel of in-vitro RCC cells Cav-1 promotes cell invasion with variable effects on cell growth and VEGF-A secretion. Cav-1 has potential as a therapeutic target for the prevention and treatment of mRCC.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Caveolina 1/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Carcinoma de Células Renales/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Renales/enzimología , Análisis Multivariante , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación/efectos de los fármacos , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando RANK/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Antimicrobial peptides (AMPs) have therapeutic potential, particularly for localized infections such as those of the lung. Here we show that airway administration of a pegylated AMP minimizes lung tissue toxicity while nevertheless maintaining antimicrobial activity. CaLL, a potent synthetic AMP (KWKLFKKIFKRIVQRIKDFLR) comprising fragments of LL-37 and cecropin A peptides, was N-terminally pegylated (PEG-CaLL). PEG-CaLL derivatives retained significant antimicrobial activity (50% inhibitory concentrations [IC(50)s] 2- to 3-fold higher than those of CaLL) against bacterial lung pathogens even in the presence of lung lining fluid. Circular dichroism and fluorescence spectroscopy confirmed that conformational changes associated with the binding of CaLL to model microbial membranes were not disrupted by pegylation. Pegylation of CaLL reduced AMP-elicited cell toxicity as measured using in vitro lung epithelial primary cell cultures. Further, in a fully intact ex vivo isolated perfused rat lung (IPRL) model, airway-administered PEG-CaLL did not result in disruption of the pulmonary epithelial barrier, whereas CaLL caused an immediate loss of membrane integrity leading to pulmonary edema. All AMPs (CaLL, PEG-CaLL, LL-37, cecropin A) delivered to the lung by airway administration showed limited (<3%) pulmonary absorption in the IPRL with extensive AMP accumulation in lung tissue itself, a characteristic anticipated to be beneficial for the treatment of pulmonary infections. We conclude that pegylation may present a means of improving the lung biocompatibility of AMPs designed for the treatment of pulmonary infections.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Péptidos/química , Péptidos/farmacología , Polietilenglicoles/química , Animales , Antiinfecciosos/síntesis química , Línea Celular , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Masculino , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Ratas , Ratas Sprague-Dawley , Espectrometría de FluorescenciaRESUMEN
The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree (Fontainea picrosperma), and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.
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Forboles , Animales , Antibacterianos/farmacología , Biopelículas , Bovinos , Humanos , Queratinocitos , Ratones , Cicatrización de HeridasRESUMEN
Glioblastoma (GBM) is the most aggressive malignant brain tumor and is associated with a very poor prognosis. The standard treatment for newly diagnosed patients involves total tumor surgical resection (if possible), plus irradiation and adjuvant chemotherapy. Despite treatment, the prognosis is still poor, and the tumor often recurs within two centimeters of the original tumor. A promising approach to improving the efficacy of GBM therapeutics is to utilize biomaterials to deliver them locally at the tumor site. Local delivery to GBM offers several advantages over systemic administration, such as bypassing the blood-brain barrier and increasing the bioavailability of the therapeutic at the tumor site without causing systemic toxicity. Local delivery may also combat tumor recurrence by maintaining sufficient drug concentrations at and surrounding the original tumor area. Herein, we critically appraised the literature on local delivery systems based within the following categories: polymer-based implantable devices, polymeric injectable systems, and hydrogel drug delivery systems. We also discussed the negative effect of hypoxia on treatment strategies and how one might utilize local implantation of oxygen-generating biomaterials as an adjuvant to enhance current therapeutic strategies.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Materiales Biocompatibles/uso terapéutico , Barrera Hematoencefálica , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Glioblastoma/tratamiento farmacológico , HumanosRESUMEN
Glioblastoma (GB) remains an aggressive malignancy with an extremely poor prognosis. Discovering new candidate drug targets for GB remains an unmet medical need. Caveolin-1 (Cav-1) has been shown to act variously as both a tumour suppressor and tumour promoter in many cancers. The implications of Cav-1 expression in GB remains poorly understood. Using clinical and genomic databases we examined the relationship between tumour Cav-1 gene expression (including its spatial distribution) and clinical pathological parameters of the GB tumour and survival probability in a TCGA cohort (n=155) and CGGA cohort (n=220) of GB patients. High expression of Cav-1 represented a significant independent predictor of shortened survival (HR = 2.985, 5.1 vs 14.9 months) with a greater statistically significant impact in female patients and in the Proneural and Mesenchymal GB subtypes. High Cav-1 expression correlated with other factors associated with poor prognosis: IDH w/t status, high histological tumour grade and low KPS score. A total of 4879 differentially expressed genes (DEGs) in the GB tumour were found to correlate with Cav-1 expression (either positively or negatively). Pathway enrichment analysis highlighted an over-representation of these DEGs to certain biological pathways. Focusing on those that lie within a framework of epithelial to mesenchymal transition and tumour cell migration and invasion we identified 27 of these DEGs. We then examined the prognostic value of Cav-1 when used in combination with any of these 27 genes and identified a subset of combinations (with Cav-1) indicative of co-operative synergistic mechanisms of action. Overall, the work has confirmed Cav-1 can serve as an independent prognostic marker in GB, but also augment prognosis when used in combination with a panel of biomarkers or clinicopathologic parameters. Moreover, Cav-1 appears to be linked to many signalling entities within the GB tumour and as such this work begins to substantiate Cav-1 or its associated signalling partners as candidate target for GB new drug discovery.
RESUMEN
Novel therapeutics designed to target the polymeric matrix of biofilms requires innovative techniques to accurately assess their efficacy. Here, multiple particle tracking (MPT) was developed to characterize the physical and mechanical properties of antimicrobial resistant (AMR) bacterial biofilms and to quantify the effects of antibiotic treatment. Studies employed nanoparticles (NPs) of varying charge and size (40-500 nm) in Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA) biofilms and also in polymyxin B (PMB) treated Escherichia coli biofilms of PMB-sensitive (PMBSens) IR57 and PMB-resistant (PMBR) PN47 strains. NP size-dependent and strain-related differences in the diffusion coefficient values of biofilms were evident between PAO1 and MRSA. Dose-dependent treatment effects induced by PMB in PMBSens E. coli biofilms included increases in diffusion and creep compliance (P < 0.05), not evident in PMB treatment of PMBR E. coli biofilms. Our results highlight the ability of MPT to quantify the diffusion and mechanical effects of antibiotic therapies within the AMR biofilm matrix, offering a valuable tool for the pre-clinical screening of anti-biofilm therapies.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Staphylococcus aureus Resistente a Meticilina/fisiología , Polimixina B/farmacología , Pseudomonas aeruginosa/fisiología , Imagen Individual de Molécula/métodos , Biopelículas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Confocal , Nanopartículas , Tamaño de la Partícula , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
The current decline in antimalarial drug efficacy due to the evolution of resistant Plasmodium strains calls for new strategies capable of improving the bioavailability of antimalarials, especially of those whose lipophilic character imparts them a low solubility in biological fluids. Here we have designed, synthesized and characterized amphiphilic zwitterionic block copolymers forming nanoparticles capable of penetrating the intestinal epithelium that can be used for oral administration. Poly(butyl methacrylate-co-morpholinoethyl sulfobetaine methacrylate) (PBMA-MESBMA)-based nanoparticles exhibited a specific targeting to Plasmodium falciparum-infected vs. parasite-free red blood cells (74.8%/0.8% respectively), which was maintained upon encapsulation of the lipophilic antimalarial drug curcumin (82.6%/0.3%). The in vitro efficacy of curcumin upon encapsulation was maintained relative to the free compound, with an IC50 around 5 µM. In vivo assays indicated a significantly increased curcumin concentration in the blood of mice one hour after being orally fed PBMA-MESBMA-curcumin in comparison to the administration of free drug (18.7 vs. 2.1 ng/ml, respectively). At longer times, however, plasma curcumin concentration equaled between free and encapsulated drug, which was reflected in similar in vivo antimalarial activities in Plasmodium yoelii yoelii-infected mice. Microscopic analysis in blood samples of fluorescently labeled PBMA-MESBMA revealed the presence of the polymer inside P. yoelii yoelii-parasitized erythrocytes one hour after oral administration to infected animals.
Asunto(s)
Antimaláricos , Malaria , Nanopartículas , Plasmodium yoelii , Administración Oral , Animales , Malaria/tratamiento farmacológico , Ratones , Plasmodium falciparumRESUMEN
Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifen-resistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERalpha) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.
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Neoplasias de la Mama/metabolismo , Caveolina 1/biosíntesis , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Western Blotting , Neoplasias de la Mama/genética , Caveolina 1/genética , Línea Celular Tumoral , Receptores ErbB/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , TransfecciónRESUMEN
The viscous mucus coating that adheres to the epithelial surfaces of mammalian organs provides protection for the underlying tissues and is an efficient barrier to drug delivery. Pulsed-gradient spin-echo NMR and small-angle neutron scattering have been used to study the aqueous solution interaction of various model polymer therapeutics with mucin, the principle organic component within mucus. Nonionic polymers such as linear and star-branched poly(ethylene oxide) (PEO) and dextrin showed no appreciable interaction with mucin but suffered a moderate retardation in their rate of diffusion through the mucin solution. A strong interaction with mucin was observed for a series of polyamidoamine (PAMAM) dendrimers and hyperbranched poly(ethylene imine) (PEI), which displayed a characteristic pH-dependent profile and led to significant reductions in their rates of diffusion. These observations have implications for the design of optimized polymer therapeutic structures being adopted for the delivery of therapeutic moieties through mucin-rich environments.
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Sistemas de Liberación de Medicamentos , Espectroscopía de Resonancia Magnética , Mucinas/metabolismo , Polímeros/metabolismo , Dispersión del Ángulo Pequeño , Agua/química , Animales , Difusión , Modelos Moleculares , Mucinas/química , Difracción de Neutrones , Polímeros/química , Soluciones , PorcinosRESUMEN
BACKGROUND: The lack of a brain-derived endothelial cell-based model has led researchers to exploit non-brain endothelial/epithelial cells as in vitro models for blood-brain barrier (BBB) investigations. Of these, the endothelial-like human ECV304 cell line was initially characterized as endothelial cells and has been widely used as an in vitro BBB model. It was later shown to be T24 bladder carcinoma epithelial cells. To pursue its potential as a cell-based model for drug screening and transport machineries, ECV304 cells were evaluated for their barrier and plasma membrane characteristics. MATERIAL/METHODS: ECV304 cells treated with some tight-junction modulators (e.g. cAMP elevators, butyric acid, dexamethasone, gamma-linolenic acid, and astrocytic factors) were examined for bioelectrical resistance, transcellular/paracellular permeability, and functionality expression of some key transporters. RESULTS: A significant (p<0.05) increase in trans-endothelial electrical resistance (TEER) was observed in ECV304 cells treated with astrocytic factors and cAMP elevators (i.e. to approximately 110Omega*cm2 vs. approximately 60 Omega*cm2 in the control). Such treatment also yielded high discrimination in the permeability coefficients of the transcellular marker propranolol (25x10-6 cm/sec) and the paracellular marker sucrose (11.3 x 10-6 cm/sec). Carrier-mediated transporters such as GLUT-1, system L, and P-glycoprotein (P-gp) as well as endocytic transport machineries (e.g. clathrin and caveolin) were found to be functionally expressed. CONCLUSIONS: ECV304 cells failed to generate a discriminative tight barrier even in the presence of tight-junction modulators. Thus their implementation in drug permeability screening is not recommended. They display some important key carrier-mediated transport systems and can be considered as a useful cell-based in vitro model.
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Barrera Hematoencefálica/citología , Células Epiteliales/citología , Regulación de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Uniones Estrechas/metabolismo , Ácido Butírico , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/fisiología , Dexametasona , Impedancia Eléctrica , Humanos , Ácido gammalinolénicoRESUMEN
The secondary structures of Scherer commonly known as perineuronal and perivascular satellitosis have been identified as a histopathological hallmark of diffuse, invasive, high-grade gliomas. They are recognised as perineuronal satellitosis when clusters of neoplastic glial cells surround neurons cell bodies and perivascular satellitosis when such tumour cells surround blood vessels infiltrating Virchow-Robin spaces. In this review, we provide an overview of emerging knowledge regarding how interactions between neurons and glioma cells can modulate tumour evolution and how neurons play a key role in glioma growth and progression, as well as the role of perivascular satellitosis into mechanisms of glioma cells spread. At the same time, we review the current knowledge about the role of perineuronal satellitosis and perivascular satellitosis within the tumour microenvironment (TME), in order to highlight critical knowledge gaps in research space.
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The coronavirus disease (COVID-19) outbreak has presented enormous challenges for healthcare, societal, and economic systems worldwide. There is an urgent global need for a universal vaccine to cover all SARS-CoV-2 mutant strains to stop the current COVID-19 pandemic and the threat of an inevitable second wave of coronavirus. Carbon dioxide is safe and superior antimicrobial, which suggests it should be effective against coronaviruses and mutants thereof. Depending on the therapeutic regime, CO2 could also ameliorate other COVID-19 symptoms as it has also been reported to have antioxidant, anti-inflammation, anti-cytokine effects, and to stimulate the human immune system. Moreover, CO2 has beneficial effects on respiratory physiology, cardiovascular health, and human nervous systems. This article reviews the rationale of early treatment by inhaling safe doses of warmed humidified CO2 gas, either alone or as a carrier gas to deliver other inhaled drugs may help save lives by suppressing SARS-CoV-2 infections and excessive inflammatory responses. We suggest testing this somewhat counter-intuitive, but low tech and safe intervention for its suitability as a preventive measure and treatment against COVID-19. Overall, development and evaluation of this therapy now may provide a safe and economical tool for use not only during the current pandemic but also for any future outbreaks of respiratory diseases and related conditions.
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The epithelial permeation of water-soluble fluorescent PAMAM dendrons based on 7H-benz[de] benzimidazo [2,1-a] isoquinoline-7-one as a fluorescent core across epithelial cell models MDCK I and MDCK II has been quantified. Hydrodynamic radii have been derived from self-diffusion coefficients obtained via pulsed-gradient spin-echo Nuclear Magnetic Resonance (PGSE-NMR). Results indicate that these dendritic molecules are molecularly disperse, non-aggregating, and only slightly larger than their parent homologues. MDCK I permeability studies across epithelial barriers show that these dendritic molecules are biocompatible with the chosen epithelial in-vitro model and can permeate across MDCK cell monolayers. Permeability is demonstrated to be a property of dendritic size and cell barrier restrictiveness indicating that paracellular mechanisms play the predominant role in the transport of these molecules.
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Dendrímeros/síntesis química , Células Epiteliales/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Permeabilidad , Animales , Células Cultivadas , Perros , Espectroscopía de Resonancia MagnéticaRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive type of malignant brain tumour, which is associated with a poor two-year survival rate and a high rate of fatal recurrence near the original tumour. Focal/local drug delivery devices hold promise for improving therapeutic outcomes for GBM by increasing drug concentrations locally at the tumour site, or by facilitating the use of potent anti-cancer drugs that are poorly permeable across the blood brain barrier (BBB). For inoperable tumours, stereotactic delivery to the tumour necessitates the development of nanoscale/microscale injectable drug delivery devices. Herein we assess the ability of a novel class of polymer nanotube (based on poly(ethylene glycol) (PEG)) to load doxorubicin (a mainstay breast cancer therapeutic with poor BBB permeability) and release it slowly. The drug loading properties of the PEG nanotubes could be tuned by varying the degree of carboxylic acid functionalisation and hence the capacity of the nanotubes to electrostatically bind and load doxorubicin. 70% of the drug was released over the first seven days followed by sustained drug release for the remaining two weeks tested. Unloaded PEG nanotubes showed no toxicity to any of the cell types analysed, whereas doxorubicin loaded nanotubes decreased GBM cell viability (C6, U-87 and U-251) in a dose dependent manner in 2D in vitro culture. Finally, doxorubicin loaded PEG nanotubes significantly reduced the viability of in vitro 3D GBM models whilst unloaded nanotubes showed no cytotoxicity. Taken together, these findings show that polymer nanotubes could be used to deliver alternative anti-cancer drugs for local therapeutic strategies against brain cancers.