RESUMEN
Trichomicin, a novel small-molecule compound isolated from the fungus Trichoderma harzianum and identified as new structure compound, exhibited antitumor activities in various human cancer cell lines and reversed drug resistance activity in the multidrug-resistant cancer cell line KBV. The underlying cellular and molecular mechanism was illuminated. Trichomicin can significantly induce cancer cell apoptosis and reduced IL-6 expression and phosphorylation of STAT3 were found in response to Trichomicin treatment. The blockade of IL-6 mediated JAK-STAT3 signaling pathway by Trichomicin was confirmed using reporter gene system. As a promising antitumor-activity compound, Trichomicin is presented in this study.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Hypocreales/química , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Interleucina-6/antagonistas & inhibidores , Ratones , Fosforilación , Factor de Transcripción STAT3/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
The primary purpose of this research was to explore the potential chiral recognition functions induced by two kinds of novel concealed body mesoporous silica nanoparticles (Cb-D-MSN and Cb-L-MSN) with the entrapment of indometacin (IMC). The as-synthesized Cb-D-MSN and Cb-L-MSN were successfully fabricated through grafting molecular functional groups. The TEM result showed that Cb-D-MSN and Cb-L-MSN were regular spheres with concealed pore channels. Nitrogen adsorption/desorption analysis confirmed mesoporous structure of Cb-D-MSN and Cb-L-MSN. In vitro drug release study indicated that both Cb-D-MSN and Cb-L-MSN significantly improved IMC dissolution compared with Naked-MSN (N-MSN). Interestingly, the results of anti-inflammation pharmacodynamics and pharmacokinetics indicated that Cb-D-MSN and Cb-L-MSN exerted different chiral recognition functions, which Cb-D-MSN can deliver more IMC evidenced by molecular simulations. It is expected that the herein favorable carriers with unique advantages in triggering chiral biological environment can be promising for constructing drug delivery system.
Asunto(s)
Indometacina/química , Nanopartículas/química , Dióxido de Silicio/química , Animales , Sistemas de Liberación de Medicamentos , Concentración de Iones de Hidrógeno , Masculino , Porosidad , Ratas , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification. Confirmation for the presence of the analytes was achieved by comparing ion intensity ratio. The ranges for limits of detection, quantification and confirmation were 0.003-1 ng/mL (S/N ≥ 3), 0.01-2.5 ng/mL and 0.02-5 ng/mL, respectively. The linear dynamic range of the method was 0.1-100 ng/mL. The precision and accuracy at 0.5, 5 and 50 ng/mL of all 17 barbiturates during intra-day assay were 1.6-8.6% and 96-106%, respectively. For inter-day assay, precision and accuracy at the same three concentrations were 2.6-8.9% and 96-106%, respectively. Analysis of all 17 analytes was completed within 7 min. Thus, the present method is fast, simple, sensitive and reproducibly reliable.
Asunto(s)
Barbitúricos/sangre , Doping en los Deportes , Detección de Abuso de Sustancias/métodos , Animales , Caballos , Extracción Líquido-Líquido , Éteres Metílicos , PlasmaRESUMEN
Treatment of nonunion fractures is a significant problem. Common therapeutics, including autologous bone grafts and bone morphogenetic proteins, show well-established limitations. Therefore, a need persists for the identification of novel clinical therapies to promote healing. The Notch signaling pathway regulates bone development. Clinically, loss-of-function mutations to the Notch ligand Jagged1 decrease bone mass and increase fracture risk. Jagged1 is also the most highly upregulated ligand during fracture repair, identifying it as a potential target to promote bone formation. Therefore, the objective of this study was to develop a clinically translatable construct comprised of Jagged1 and an osteoconductive scaffold, and characterize its activity in human mesenchymal stem cells (hMSC). We first evaluated the effects of Jagged1 directly immobilized to a novel poly(ß-amino ester) relative to indirect coupling via antibody. Direct was more effective at activating hMSC Notch target gene expression and osteogenic activity. We then found that directly immobilized Jagged1 constructs induced osteoblast differentiation. This is the first study to demonstrate that Jagged1 delivery transiently activates Notch signaling and increases osteogenesis. A positive correlation was found between Jagged1-induced Notch and osteogenic expression. Collectively, these results indicate that Jagged1 coupled to an osteogenic biomaterial could promote bone tissue formation during fracture healing.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Oseointegración/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Polímeros/farmacología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Materiales Biocompatibles/farmacología , Calcificación Fisiológica/efectos de los fármacos , Recuento de Células , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inmovilizadas/metabolismo , Proteína Jagged-1 , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Ratas , Proteínas Serrate-Jagged , Andamios del Tejido/químicaRESUMEN
Genome-wide association studies (GWAS) have demonstrated that genetic variation at the MADS box transcription enhancer factor 2, polypeptide C (MEF2C) locus is robustly associated with bone mineral density, primarily at the femoral neck. MEF2C is a transcription factor known to operate via the Wnt signaling pathway. Our hypothesis was that MEF2C regulates the expression of a set of molecular pathways critical to skeletal function. Drawing on our laboratory and bioinformatic experience with ChIP-seq, we analyzed ChIP-seq data for MEF2C available via the ENCODE project to gain insight in to its global genomic binding pattern. We aligned the ChIP-seq data generated for GM12878 (an established lymphoblastoid cell line) and, using the analysis package HOMER, a total of 17,611 binding sites corresponding to 8,118 known genes were observed. We then performed a pathway analysis of the gene list using Ingenuity. At 5 kb, the gene list yielded 'EIF2 Signaling' as the most significant annotation, with a P value of 5.01 × 10(-26). Moving further out, this category remained the top pathway at 50 and 100 kb, then dropped to just second place at 500 kb and beyond by 'Molecular Mechanisms of Cancer'. In addition, at 50 kb and beyond 'RANK Signaling in Osteoclasts' was a consistent feature and resonates with the main general finding from GWAS of bone density. We also observed that MEF2C binding sites were significantly enriched primarily near inflammation associated genes identified from GWAS; indeed, a similar enrichment for inflammation genes has been reported previously using a similar approach for the vitamin D receptor, an established key regulator of bone turnover. Our analyses point to known connective tissue and skeletal processes but also provide novel insights in to networks involved in skeletal regulation. The fact that a specific GWAS category is enriched points to a possible role of inflammation through which it impacts bone mineral density.
Asunto(s)
Densidad Ósea/genética , Inmunoprecipitación de Cromatina , Estudio de Asociación del Genoma Completo , Algoritmos , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Biología Computacional , Bases de Datos Genéticas , Genómica , Humanos , Inflamación , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a γ-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C δ (PKCδ) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCδ nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCδ with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCδ signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Proteína Quinasa C-delta/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacosRESUMEN
The transcription factor osterix (Sp7) is essential for osteoblastogenesis and bone formation in mice. Genome wide association studies have demonstrated that osterix is associated with bone mineral density in humans; however, the molecular significance of osterix in human osteoblast differentiation is poorly described. In this study we have characterized the role of osterix in human mesenchymal progenitor cell (hMSC) differentiation. We first analyzed temporal microarray data of primary hMSC treated with bone morphogenetic protein-6 (BMP6) using clustering to identify genes that are associated with osterix expression. Osterix clusters with a set of osteoblast-associated extracellular matrix (ECM) genes, including bone sialoprotein (BSP) and a novel set of proteoglycans, osteomodulin (OMD), osteoglycin, and asporin. Maximum expression of these genes is dependent upon both the concentration and duration of BMP6 exposure. Next we overexpressed and repressed osterix in primary hMSC using retrovirus. The enforced expression of osterix had relatively minor effects on osteoblastic gene expression independent of exogenous BMP6. However, in the presence of BMP6, osterix overexpression enhanced expression of the aforementioned ECM genes. Additionally, osterix overexpression enhanced BMP6 induced osteoblast mineralization, while inhibiting hMSC proliferation. Conversely, osterix knockdown maintained hMSC in an immature state by decreasing expression of these ECM genes and decreasing mineralization and hMSC proliferation. Overexpression of the osterix regulated gene OMD with retrovirus promoted mineralization of hMSC. These results suggest that osterix is necessary, but not sufficient for hMSC osteoblast differentiation. Osterix regulates the expression of a set of ECM proteins which are involved in terminal osteoblast differentiation.
Asunto(s)
Proteína Morfogenética Ósea 6/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo , Calcificación Fisiológica , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Análisis por Conglomerados , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Factor de Transcripción Sp7 , Factores de Tiempo , Factores de Transcripción/genética , TransfecciónRESUMEN
With IL-6R as target, a new compound 2460A was identified from fungus using HTS screening model. The taxonomics of the produced strain was confirmed to be Trichoderma hazianum rifai after sequencing analysis of rDNA-ITS (internal transcribed spacer). Results showed that this compound has a binding activity on IL-6R competed with IL-6, thus it is a new ligand of IL-6R originating from microbe. With MTT assay, the anti-tumor activities of 2460A were demonstrated on CM126 and HT-29 cell lines separately, the IC50 are 2.17 x 10(-5) mol x L(-1) and 1.8 x 10(-5) mol x L(-1) respectively. The compound affected lightly the HT-29 cell cycle at S phase. Studies for the anti-tumor activity of 2460A in vivo are in progress in our lab.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Médula Ósea/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Trichoderma/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Unión Competitiva , Línea Celular Tumoral , Células HT29 , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-6/metabolismo , Ligandos , Receptores de Interleucina-6/metabolismoRESUMEN
Cellular signal transduction pathways transduce input signals to produce corresponding output effects, ensuring correct response to extracellular signals. Manipulation of components in signaling pathways will alter correlation of input signals to output effects. Here we report that by reconstructing the components in mitogenic and apoptotic signaling pathways, Ras, Raf, and caspase-3, we manipulated the cells to couple mitogenic signal input to apoptotic output. The reconstructed biomolecules that couple mitogenesis to apoptosis are designated as "mitogenesis coupled-apoptosis molecular device" (MCAMD). As mitogenesis in cancer cells is constitutively active, MCAMD may have potential applications for cancer gene therapy.
Asunto(s)
Apoptosis , Sistema de Señalización de MAP Quinasas , Mitógenos/farmacología , Caspasa 3/metabolismo , Línea Celular , Humanos , Ingeniería de Proteínas , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
AIM: To isolate IL-6R antagonists from the cultured broth of the strain Torulomyces ovatus. METHODS: Various column chromatographyes were used to separate and purify the compounds with IL-6R antagonist activity. The spectral data and physic-chemical properties were measured for structure identification. RESULTS: One compound namely 2520 was isolated from the cultured broth of Torulomyces ovatus. CONCLUSION: 2520A is a known compound (ferrichrome). It is first reported about its antagonistic activity of IL-6R and identification of iron atom in its structure.