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Cellular and molecular-level interactions of nanoparticles with biological systems are a rapidly evolving field requiring an improved understanding of endocytic trafficking as the principal driver and regulator of signaling events and cellular responses. An understanding of these processes is vital to nanomedicine applications. Studies investigating the complex interplay of these processes and their relationship to targeted nanoparticles exploiting endocytic pathways are notably lacking. It is known that integrins traffic through the endosomal pathway and participate in diverse roles controlling signal transduction, cell migration, and proliferation. Here, it is shown that ultrasmall, nontoxic, core-shell silica nanoparticles (C-dots), surface-functionalized with cRGDY peptides, modestly activate integrin-signaling pathways, in turn, promoting the enhancement of cellular functions. First, nanomolar concentrations, two orders of magnitude higher than clinical trial doses, internalize within αvß3 integrin-expressing melanoma and endothelial cells, predominantly through an integrin receptor-dependent endocytic route. Second, integrin-mediated activation of focal adhesion kinase (FAK) and downstream signaling pathways occurs, in turn, upregulating phosphorylated protein expression levels and promoting concentration-dependent cellular migration and proliferative activity. Inhibiting FAK catalytic activity leads to decreased phosphorylation levels and cellular migration rates. These findings may inform the design of more effectively-targeted nanomedicines and provide insights into endocytic regulation of signal transduction.
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Nanopartículas , Transducción de Señal , Dióxido de Silicio/química , Ciclo Celular , Endocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrinas/metabolismoRESUMEN
BACKGROUND: The purpose of our study was to find a novel targeted imaging and drug delivery vehicle for inflammatory bowel disease (IBD). IBD is a common and troublesome disease that still lacks effective therapy and imaging options. As an attempt to improve the disease treatment, we tested αMSH for the targeting of nanoliposomes to IBD sites. αMSH, an endogenous tridecapeptide, binds to the melanocortin-1 receptor (MC1-R) and has anti-inflammatory and immunomodulating effects. MC1-R is found on macrophages, neutrophils and the renal tubule system. We formulated and tested a liposomal nanoparticle involving αMSH in order to achieve a specific targeting to the inflamed intestines. METHODS: NDP-αMSH peptide conjugated to Alexa Fluor™ 680 was linked to the liposomal membrane via NSuccinyl PE and additionally loaded into the lumen of the liposomes. Liposomes without the αMSH-conjugate and free NDP-αMSH were used as a control. The liposomes were also loaded with ICG to track them. The liposomes were tested in DSS treated mice, which had received DSS via drinking water order to develop a model IBD. Inflammation severity was assessed by the Disease Activity Index (DAI) score and ex vivo histological CD68 staining of samples taken from different parts of the intestine. The liposome targeting was analyzed by analyzing the ICG and ALEXA 680 fluorescence in the intestine compared to the biodistribution. RESULTS: NPD-αMSH was successfully labeled with Alexa and retained its biological activity. Liposomes were identified in expected regions in the inflamed bowel regions and in the kidneys, where MC1-R is abundant. In vivo liposome targeting correlated with the macrophage concentration at the site of the inflammation supporting the active targeting of the liposomes through αMSH. The liposomal αMSH was well tolerated by animals. CONCLUSION: This study opens up the possibility to further develop an αMSH targeted theranostic delivery to different clinically relevant applications in IBD inflammation but also opens possibilities for use in other inflammations like lung inflammation in Covid 19.
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Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Liposomas , Nanopartículas , Receptor de Melanocortina Tipo 1/química , alfa-MSH/química , Animales , Colorantes Fluorescentes/química , Ratones , Distribución TisularRESUMEN
CONTEXT: Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors. OBJECTIVES: Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type. RESULTS: Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts. CONCLUSION: Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.
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Carcinoma/genética , Resistencia a Antineoplásicos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Bencimidazoles/farmacología , Carcinoma/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Mutación Missense , Especificidad por Sustrato , Neoplasias de la Tiroides/enzimología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CONTEXT: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.
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Técnicas de Cultivo de Célula/normas , ADN de Neoplasias/genética , Perfilación de la Expresión Génica , Mutación , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Neoplasias de la Tiroides/genética , Adenocarcinoma Folicular/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Femenino , Humanos , Masculino , Melanoma/genética , ARN Mensajero/genética , Reproducibilidad de los Resultados , Neoplasias de la Tiroides/clasificaciónRESUMEN
The poor prognosis associated with malignant melanoma has not changed substantially over the past 30 years. Targeted molecular therapies, such as immunotherapy, have shown promise but suffer from resistance and off-target toxicities, underscoring the need for alternative therapeutic strategies that can be used in combination with existing protocols. Moreover, peptides targeting melanoma-specific markers, like the melanocortin-1 receptor (MC1-R), for imaging and therapy exhibit high renal uptake that limits clinical translation. In the current study, the application of ultrasmall fluorescent (Cy5) silica nanoparticles (C' dots), conjugated with MC1-R targeting alpha melanocyte stimulating hormone (αMSH) peptides on the polyethylene glycol (PEG) coated surface, is examined for melanoma-selective imaging. αMSH peptide sequences, evaluated for conjugation to the PEG-Cy5-C' dot nanoparticles, bound to MC1-R with high affinity and targeted melanoma in syngenetic and xenografted melanoma mouse models. Results demonstrated a 10-fold improvement in MC1-R affinity over the native peptide alone following surface attachment of the optimal αMSH peptide. Systematic in vivo studies further demonstrated favorable in vivo renal clearance kinetics as well as receptor-mediated tumor cell internalization of as-developed radiolabeled particle tracers in B16F10 melanoma bearing mice. These findings highlight the ability of αMSH-PEG-Cy5-C' dots to overcome previous hurdles that prevented clinical translation of peptide and antibody-based melanoma probes and reveal the potential of αMSH-PEG-Cy5-C' dots for melanoma-selective imaging, image-guided surgery, and therapeutic applications.
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Nanopartículas , Animales , Humanos , Melanoma , Melanoma Experimental , Ratones , Receptor de Melanocortina Tipo 1 , Dióxido de Silicio , alfa-MSHRESUMEN
PURPOSE: Within the multidomain structure of ZO-1 are motifs responsible for ZO-1's localization to intercellular junctions and its newly demonstrated localization to the leading edge of lamellipodia in corneal fibroblasts. Since ZO-1 also has two nuclear localization signals, this study was undertaken to determine whether stimuli associated with wounding would induce nuclear translocation of ZO-1 METHODS: Immunocytochemistry and immunoblot analysis were used to localize endogenous and exogenous ZO-1 in nuclear and cytoplasmic sites in corneal fibroblasts and 293T fibroblasts, with and without myc-ZO-1 transfection. Cells were serum starved by growth for 48 hours in DMEM/F12 with 0.2% FBS and subsequently were either scrape wounded or treated with 10% FBS, PDGF, or FGF-2 for 6 hours. For immunoblot analysis, after lysis, the nuclear and cytosolic fractions were separated and analyzed by SDS-PAGE. Cells on companion coverslips were fixed with 3% p-formaldehyde and permeabilized with 1% Triton before immunocytochemical detection of ZO-1 and nuclear proteins. RESULTS: ZO-1 was rarely detected in the nucleus of serum-starved corneal fibroblasts. In contrast, it colocalized with nucleolin in the nucleoli of corneal fibroblasts after serum-starved cells were treated with 10% FBS, PDGF, or FGF-2. Immunoblot analysis confirmed the immunocytochemical results: Little ZO-1 was detected in the nuclear fraction of lysates of serum-starved cells, but ZO-1 was found in the nuclear fractions of rabbit corneal and 293T fibroblasts treated with 10% FBS, PDGF, or FGF-2. Furthermore in scrape-wounded corneal fibroblasts, ZO-1 was localized to nucleoli of both serum-starved and serum-treated cells. CONCLUSIONS: Localization of ZO-1 to nucleoli of corneal and 293T fibroblasts under proliferative and promigratory conditions suggests a physiologically significant interaction of ZO-1 with proteins in nucleoli during the healing process.
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Nucléolo Celular/metabolismo , Córnea/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Animales , Línea Celular , Movimiento Celular/fisiología , Lesiones de la Cornea , Citosol , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Immunoblotting , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Proteínas de Unión al ARN/metabolismo , Conejos , Transfección , Cicatrización de Heridas/fisiología , Proteína de la Zonula Occludens-1 , NucleolinaRESUMEN
Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFbeta) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGFbeta induces expression of smooth muscle alpha actin (SMalphaA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFbeta induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGFbeta-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFbeta-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK -/- MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMalphaA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK -/- [corrected]MEFs. We conclude that FAK does not contribute to TGFbeta-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SMalphaA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.
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Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Actinas/metabolismo , Diferenciación Celular , Células Cultivadas , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/genética , Eliminación de Gen , Regulación de la Expresión Génica , Interferencia de ARN , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Although a large body of literature exists on the potential use of nanoparticles for medical applications, the number of probes translated into human clinical trials is remarkably small. A major challenge of particle probe development and their translation is the elucidation of safety profiles associated with their structural complexity, not only in terms of size distribution and heterogeneities in particle composition but also their effects on biological activities and the relationship between particle structure and pharmacokinetics. Here, we report on the synthesis, characterization, and long-term stability of ultrasmall (<10 nm diameter) dual-modality (optical and positron emission tomography) and integrintargeting silica nanoparticles (cRGDY-PEG-Cy5-C' dots and 124I-(or 131I-) cRGDY-PEG-Cy5-C'dots) and the extent to which their surface ligand density differentially modulates key in vitro and in vivo biological activities in melanoma models over a range of ligand numbers (i.e., ~6-18). Gel permeation chromatography, established as an important particle characterization tool, revealed a two-year shelf life for cRGDY-PEG-Cy5-C' dots. Radiochromatography further demonstrated the necessary radiochemical stability for clinical applications. The results of subsequent ligand density-dependent studies elucidate strong modulations in biological response, including statistically significant increases in integrin-specific targeting and particle uptake, cellular migration and adhesion, renal clearance, and tumor-to-blood ratios with increasing ligand number. We anticipate that nanoprobe characteristics and a better understanding of the structure-function relationships determined in this study will help guide identification of other lead nanoparticle candidates for in vitro and in vivo biological assessments and product translation.
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PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.
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Córnea/metabolismo , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Seudópodos/metabolismo , Cicatrización de Heridas , Animales , Western Blotting , Cadherinas/metabolismo , Técnicas de Cultivo de Célula , Conexina 43/metabolismo , Lesiones de la Cornea , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Inmunoprecipitación , Uniones Intercelulares , Fosforilación , Conejos , Proteína de la Zonula Occludens-1RESUMEN
Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR) inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380), in combination with nanocarrier vehicles and metabolic imaging tools (microPET) to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB)-driven genetically engineered mouse model (GEMM) of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using (18)F-SKI249380 and (18)F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg) versus saline vehicle (P < .001) in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for (18)F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g); P = .002] compared to control values (1.6%ID/g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g), consistent with in vivo binding specificity. Using (18)F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24-60 nM) that were comparable to in vitro 50% inhibitory concentration values. (18)F-SKI249380 is a PDGFR-selective tracer, which demonstrates improved delivery to PDGFB-driven high-grade gliomas and facilitates treatment planning when coupled with nanoformulations and quantitative PET imaging approaches.
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Glioblastoma/tratamiento farmacológico , Terapia Molecular Dirigida , Nanoconjugados/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Tiazoles/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dasatinib , Modelos Animales de Enfermedad , Radioisótopos de Flúor/farmacocinética , Glioblastoma/diagnóstico por imagen , Glioblastoma/patología , Estimación de Kaplan-Meier , Liposomas , Ratones , Micelas , Células 3T3 NIH , Nanoconjugados/uso terapéutico , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-sis/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/uso terapéutico , Trazadores Radiactivos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacocinética , Tiazoles/uso terapéuticoRESUMEN
Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine-glycine-aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvß3 integrin-expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.
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Portadores de Fármacos/química , Melanoma/tratamiento farmacológico , Nanopartículas/química , Dióxido de Silicio/química , Animales , Ensayos Clínicos como Asunto , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Radioisótopos de Yodo/química , Radioisótopos de Yodo/metabolismo , Ligandos , Masculino , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Estructura Molecular , Trasplante de Neoplasias , Tamaño de la Partícula , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Tomografía de Emisión de Positrones , Coloración y Etiquetado/métodos , Distribución Tisular , Trasplante HeterólogoRESUMEN
Breast cancer 1, early onset (BRCA1) expression is often reduced in sporadic breast tumors, even in the absence of BRCA1 genetic modifications, but the molecular basis for this is unknown. In this study, we identified homeobox A9 (HOXA9) as a gene frequently downregulated in human breast cancers and tumor cell lines and noted that reduced HOXA9 transcript levels associated with tumor aggression, metastasis, and patient mortality. Experiments revealed that loss of HOXA9 promoted mammary epithelial cell growth and survival and perturbed tissue morphogenesis. Restoring HOXA9 expression repressed growth and survival and inhibited the malignant phenotype of breast cancer cells in culture and in a xenograft mouse model. Molecular studies showed that HOXA9 restricted breast tumor behavior by directly modulating the expression of BRCA1. Indeed, ectopic expression of wild-type BRCA1 phenocopied the tumor suppressor function of HOXA9, and reducing BRCA1 levels or function inhibited the antitumor activity of HOXA9. Consistently, HOXA9 expression correlated with BRCA1 in clinical specimens and with tumor aggression in patients lacking estrogen receptor/progesterone receptor expression in their breast tissue. These findings indicate that HOXA9 restricts breast tumor aggression by modulating expression of the tumor suppressor gene BRCA1, which we believe provides an explanation for the loss of BRCA1 expression in sporadic breast tumors in the absence of BRCA1 genetic modifications.
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Proteína BRCA1/biosíntesis , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/fisiología , Adulto , Animales , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Persona de Mediana Edad , Modelos Genéticos , Trasplante de Neoplasias , Fenotipo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Resultado del TratamientoRESUMEN
A series of nine synthetic polyaromatic compounds were synthesized by polymerization of aromatic ring monomers with formaldehyde, which yield substantially ordered backbones with different functional anionic groups (hydroxyl and carboxyl) on the phenol ring. These compounds were tested for their heparin-mimicking activity: (1) inhibition of heparanase activity; (2) inhibition of SMC proliferation; and (3) release of bFGF from the ECM. We demonstrate that compounds that have two hydroxyl groups para and ortho to the carboxylic group and a carboxylic group at a distance of two carbons from the phenol ring inhibit heparanase activity and SMC proliferation, as well as induced an almost complete release of bFGF from ECM. Addition of a methyl group next to the carboxylic group led to a preferential inhibition of heparanase activity. Similar results were obtained with a compound that contains one hydroxyl group para to the carboxylic group and an ether group near the carboxylic group on the phenol ring. Preferential inhibition of SMC proliferation was best achieved when the position of the hydroxyl group is para and ortho to the carboxylic group and the carboxylic group is at a distance of one carbon from the phenol ring. On the other hand, for maximal release of bFGF from ECM, the position of the carboxylic group should be three carbons away from the phenol ring. These new heparin-mimicking compounds may have a potential use in inhibition of tumor metastasis, arteriosclerosis, and inflammation.
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Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/química , Heparina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Glucuronidasa/metabolismo , Heparitina Sulfato/metabolismo , Hidrocarburos Aromáticos/química , Hidrocarburos Aromáticos/farmacología , Imitación Molecular , Músculo Liso Vascular/citología , Relación Estructura-Actividad , Trombina/farmacologíaRESUMEN
BRCA1 is a tumor suppressor gene mutated in cases of hereditary breast and ovarian cancer. BRCA1 protein is involved in apoptosis and growth/tumor suppression. In this study, we present evidence that p65/RelA, one of the two subunits of the transcription factor NF-kappaB, binds to the BRCA1 protein. Treatment of 293T cells with the cytokine tumor necrosis factor-alpha induces an interaction between endogenous p65/RelA and BRCA1. GST-protein affinity assay experiments reveal that the Rel homology domain of the p65/RelA subunit of NF-kappaB interacts with multiple sites within the N-terminal region of BRCA1. Transient transfection of BRCA1 significantly enhances the ability of the tumor necrosis factor-alpha or interleukin-1beta to activate transcription from the promoters of NF-kappaB target genes. Mutation of the NF-kappaB-binding sites in the NF-kappaB reporter blocks the effect of BRCA1 on transcription. Also the ability of BRCA1 to activate NF-kappaB target genes is inhibited by a super-stable inhibitor of NF-kappaB and by the chemical inhibitor SN-50. These data indicate that BRCA1 acts as a co-activator with NF-kappaB. In addition, we show that cells infected with an adenovirus expressing BRCA1 up-regulate the endogenous expression of NF-kappaB target genes Fas and interferon-beta. Together, this information suggests that BRCA1 may play a role in cell life-death decisions following cell stress by modulation of the activity of NF-kappaB.