RESUMEN
Facile assembly of intelligent DNA nanoobjects with the ability to exert in situ visualization of intracellular microRNAs (miRNAs) has long been concerned in the fields of DNA nanotechnology and basic medical study. Here, we present a driving primer (DP)-triggered polymerization-mediated metastable assembly (PMA) strategy to prepare a well-ordered metastable DNA nanoarchitecture composed of only two hairpin probes (HAPs), which has never been explored by assembly methods. Its structural features and functions are characterized by atomic force microscope (AFM) and gel electrophoresis. Even if with a metastable molecular structure, this nanoarchitecture is relatively stable at physiological temperature. The assembly strategy can be expanded to execute microRNA-21 (miRNA-21) in situ imaging inside cancer cells by labelling one of the HAPs with fluorophore and quencher. Compared with the conventional fluorescence probe-based in situ hybridization (FISH) technique, confocal images revealed that the proposed DNA nanoassembly can not only achieve greatly enhanced imaging effect within cancer cells, but also reflect the miRNA-21 expression level sensitively. We believe that the easily constructed DNA nanoarchitecture and in situ profiling strategy are significant progresses in DNA assembly and molecule imaging in cells.
Asunto(s)
ADN/ultraestructura , MicroARNs/química , MicroARNs/ultraestructura , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Nanopartículas/ultraestructura , Neoplasias Experimentales/ultraestructura , Cristalización/métodos , ADN/química , Colorantes Fluorescentes , Humanos , Células MCF-7 , Nanopartículas/química , Neoplasias Experimentales/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded â¼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos , Fraccionamiento Celular/instrumentación , Vesículas Extracelulares/fisiología , Vesículas Extracelulares/ultraestructura , Macrófagos/metabolismo , Neoplasias Experimentales/ultraestructura , Técnicas de Cultivo Celular por Lotes/métodos , Fraccionamiento Celular/métodos , Células Cultivadas , Diseño de Equipo , Células HEK293 , Humanos , Ingeniería de Tejidos/instrumentaciónRESUMEN
Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.
Asunto(s)
Compuestos de Anilina/análisis , Colorantes Fluorescentes/análisis , Genes Reporteros , Microscopía Intravital , Neoplasias Experimentales/ultraestructura , Imagen Óptica/métodos , Anticuerpos de Cadena Única/análisis , Activación Metabólica , Compuestos de Anilina/farmacocinética , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Color , Desamino Arginina Vasopresina/farmacología , Endocitosis/efectos de los fármacos , Fluorescencia , Colorantes Fluorescentes/farmacocinética , Proteínas Fluorescentes Verdes/análisis , Células HCT116/trasplante , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/química , Neoplasias Peritoneales/química , Neoplasias Peritoneales/ultraestructura , Receptores de Vasopresinas/análisis , Receptores de Vasopresinas/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Transducción GenéticaRESUMEN
OBJECTIVE: To observe the micromorphological changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis after ablation with the high-intensity focused ultrasound (HIFU), and to explore the mechanisms responsible for the thermal and non-thermal effect.â© METHODS: Forty rabbits with hepatic VX2 tumors were randomly divided into a thermal group (n=20) and a non-thermal group (n=20), and were subjected to HIFU ablation with thermal or non-thermal condition, respectively. Five animals in each group were sacrificed on the 1st, 3rd, 7th or 14th day after the ablation. The changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis were detected.â© RESULTS: The results of transmission electron microscope (TEM) revealed more severe injury on tissue and cells in the non-thermal group than that in the thermal group. The changes of apoptosis-related proteins expression and tumor cell apoptosis in transient zone were significantly different in comparison with that in the ablated area or peripheral area between the two groups. The expression of vascular endothelial growth factor (VEGF) was at low level on the 1st and 3rd day and elevated gradually on the 7th and 14th day, with no significant difference (all P>0.05). The expression of caspase-3 reached peak on the 3rd day and decreased on the 7th and 14th day. It was significantly higher in the non-thermal group than that in the thermal group on the 3rd and 7th day (all P<0.05). The expression of NF-κB was elevated from the 3rd day and reached peak on the 7th day while decreased on the 14th day. There was no significant difference at every time point between the 2 groups (all P>0.05). The apoptosis index in the non-thermal group and the thermal group on the 3rd and 7th day were (28.60±1.14)% vs (21.80±1.92)% and (21.00±1.58)% vs (14.80±1.48)%, respectively. It was higher in the non-thermal group than that in the thermal group (both P<0.01).â© CONCLUSION: Both the thermal and the non-thermal effect of HIFU can induce apoptosis in transient zone, but the latter have a stronger effect.
Asunto(s)
Apoptosis , Ultrasonido Enfocado de Alta Intensidad de Ablación , Neoplasias Hepáticas/ultraestructura , Animales , Caspasa 3/metabolismo , Neoplasias Hepáticas/patología , FN-kappa B/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Conejos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.
Asunto(s)
Fraccionamiento Celular/métodos , Separación Celular/métodos , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestructura , Extracción en Fase Sólida/métodos , Agua/química , Animales , Línea Celular Tumoral , Dextranos/química , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/ultraestructura , Transición de Fase , Polietilenglicoles/químicaRESUMEN
Morphological detection of cancer cells in the rabbit VX2 allograft transplantation model is often difficult in a certain region such as serosal cavity where reactive mesothelial cells mimic cancer cells and both cells share common markers such as cytokeratins. Therefore, tagging VX2 cells with a specific and sensitive marker that easily distinguishes them from other cells would be advantageous. Thus, we tried to establish a successively transplantable, enhanced green fluorescent protein (EGFP)-expressing VX2 model. Cancer cells obtained from a conventional VX2-bearing rabbit were cultured in vitro and transfected with an EGFP-encoding vector, and then successively transplanted in Healthy Japanese White rabbits (HJWRs) (n = 8). Besides, conventional VX2 cells were transplanted in other HJWRs (n = 8). Clinicopathological comparison analyses were performed between the two groups. The success rate of transplantation was 100% for both groups. The sensitivity and specificity of EGFP for immunohistochemical detection of VX2 cells were 84.3 and 100%, respectively. No significant differences in cancer cell morphology, tumor size (P = 0.742), Ki-67 labeling index (P = 0.878), or survival rate (P = 0.592) were observed between the two. VX2 cells can be genetically altered, visualized by EGFP, and successively transplanted without significant alteration of morphological and biological properties compared to those of the conventional model.
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Proteínas Fluorescentes Verdes/metabolismo , Trasplante de Neoplasias/métodos , Neoplasias Experimentales/metabolismo , Células Tumorales Cultivadas/trasplante , Animales , Línea Celular Tumoral , Femenino , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microscopía de Contraste de Fase , Neoplasias Experimentales/genética , Neoplasias Experimentales/ultraestructura , Conejos , Análisis de Supervivencia , TransfecciónRESUMEN
Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.
Asunto(s)
Microscopía Electrónica/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neoplasias Experimentales/mortalidad , Neoplasias Experimentales/ultraestructura , Animales , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Ácidos Grasos Monoinsaturados/química , Ácidos Grasos Monoinsaturados/farmacología , Nanopartículas/química , Neoplasias Experimentales/patología , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacología , Animales , Fluorescencia , Células HeLa , Humanos , Liposomas , Masculino , Ratones , Ratones Desnudos , Microscopía Fluorescente/métodos , Neoplasias Experimentales/ultraestructura , Propiedades de SuperficieRESUMEN
A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1 ± 2.1 nm) generated high transversal relaxivity r2 (188 ± 3 m m(-1) s(-1) ) and saturation magnetization (94 emu g(-1) Fe). The USPIO nanoparticles were coated with PDA [2-(pyridyldithio)-ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF-7, A-549, HT-29 and HT-1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (-51.3 ± 6.7%) was observed for in vivo MR imaging of MCF-7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection.
Asunto(s)
Medios de Contraste/análisis , Dextranos/análisis , Integrinas/análisis , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/análisis , Nanocompuestos/análisis , Nanoconjugados/análisis , Oligopéptidos/análisis , Animales , Línea Celular/metabolismo , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/trasplante , Medios de Contraste/síntesis química , Disulfuros/análisis , Fluoresceína-5-Isotiocianato/análisis , Ratones , Ratones Desnudos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Neoplasias Experimentales/química , Neoplasias Experimentales/ultraestructura , Ácido Oléico/análisis , Oligopéptidos/síntesis química , Piridinas/análisisRESUMEN
Alterations in the biomechanical properties and cytoskeletal organization of cancer cells in addition to genetic changes have been correlated with their aggressive phenotype. In this study, we investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells, a mouse model for progressive ovarian cancer. We demonstrate that the elasticity of late-stage MOSE cells (0.549 ± 0.281 kPa) were significantly less than that of their early-stage counterparts (1.097 ± 0.632 kPa). Apparent cell viscosity also decreased significantly from early (144.7 ± 102.4 Pa-s) to late stage (50.74 ± 29.72 Pa-s). This indicates that ovarian cells are stiffer and more viscous when they are benign. The increase in cell deformability directly correlates with the progression of a transformed phenotype from a nontumorigenic, benign cell to a tumorigenic, malignant one. The decrease in the level of actin in the cytoskeleton and its organization is directly associated with the changes in cell biomechanical property. FROM THE CLINICAL EDITOR: The authors have investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells and demonstrated that ovarian cells are stiffer and more viscous when they are benign.
Asunto(s)
Transformación Celular Neoplásica/química , Células Epiteliales/química , Microtúbulos/química , Neoplasias Experimentales/química , Neoplasias Ováricas/química , Actinas/química , Actinas/ultraestructura , Animales , Elasticidad , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Femenino , Humanos , Ratones , Microscopía de Fuerza Atómica , Microtúbulos/ultraestructura , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Neoplasias Ováricas/patología , Neoplasias Ováricas/ultraestructura , ViscosidadRESUMEN
BACKGROUND AND OBJECTIVE: In vitro, arsenic trioxide (As(2)O(3)) can inhibit proliferation of many lymphoma cell lines. In clinic, it also can be used to treat many subtypes of lymphoma. But the dosage and administration ways are undetermined yet. In this research, we studied the antitumor effect of As(2)O(3) with different administration ways on T-cell lymphoma and observed the toxicity. METHODS: Murine T-cell lymphoma cell line EL4 was treated with As(2)O(3) of eight concentrations. Cell proliferation was detected by MTT assay. Cell apoptosis was evaluated by flow cytometry with Annexin-V-FITC/PI staining and observed under electroscope and fluorescent microscope. EL4 cells were inoculated into nude mice to establish lymphoma models. The effect of As(2)O(3) on lymphoma in nude mice was observed. RESULTS: As(2)O(3) inhibited the proliferation of EL4 cells with a 50% inhibition concentration (IC(50)) of 1.28 micromol/L at 72 h (p < 0.05). When treated with the same total dose of As(2)O(3) by 4 mg(kg d)(-1) for seven days or 2 mg(kg d)(-1) for 14 days, the inhibition rates of tumor growth in mice were equivalent (58.8% vs. 55.6%, p = 0.351). Apoptotic cells were increased and apoptotic bodies were observed in xenograft tumor tissues. Acute liver damage is the major toxicity. CONCLUSION: Shortening the administration course and increasing the daily dosage of As(2)O(3) can be considered as a reasonable model for treating advanced/refractory lymphomas.
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Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Proliferación Celular/efectos de los fármacos , Linfoma de Células T/tratamiento farmacológico , Óxidos/farmacología , Animales , Trióxido de Arsénico , Arsenicales/administración & dosificación , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Concentración 50 Inhibidora , Linfoma de Células T/patología , Linfoma de Células T/ultraestructura , Masculino , Ratones , Ratones Desnudos , Microscopía Electrónica , Microscopía Fluorescente , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Óxidos/administración & dosificación , Factores de Tiempo , Carga Tumoral/efectos de los fármacosRESUMEN
Hepatic oval cells are thought to represent facultative hepatic epithelial stem cells in liver in which damage inhibits hepatocyte proliferation and liver regeneration. The LE/6 hepatic stem cell line was derived from the liver of male Sprague-Dawley rats fed a choline-deficient diet containing 0.1% ethionine. They are histochemically characterized by their expression of hepatocytic (hepPar1), cholangiocytic cytokeratin (CK19), hepatic progenitor cell (OV-6), and hematopoietic stem cell (c-kit) markers. In this study, we transplanted LE/6 cells by subcutaneous injection into adult female nude mice, and examined their engraftment and differentiation potential in the subcutaneous microenvironment in vivo. Our results demonstrated that following subcutaneous transplantation, differentiation of LE/6 cells into mesenchymal tumor tissue (MTT) was associated with reduced E-cadherin expression, upregulation of E-cadherin repressor molecules (Snail proteins), and increased expression of vimentin and N-cadherin, all of these events are characteristic of the epithelial-mesenchymal transition (EMT).
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Transdiferenciación Celular , Trasplante de Células/métodos , Hígado/citología , Neoplasias Experimentales/patología , Animales , Western Blotting , Cadherinas/metabolismo , Células Cultivadas , Epitelio/patología , Femenino , Hibridación Fluorescente in Situ , Masculino , Mesenquimoma/metabolismo , Mesenquimoma/patología , Mesenquimoma/ultraestructura , Mesodermo/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Electrónica de Transmisión , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/ultraestructura , Ratas , Ratas Sprague-Dawley , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Trasplante Heterólogo , Vimentina/metabolismoRESUMEN
The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is administered systemically in drinking water, is the most comparable animal model to the development of human oral carcinoma. This is the first study to report the ultrastructural changes in this model. The most significant changes were observed in the carcinoma cells at the invasion front and included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasmalemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrangement. The microfilaments, in both appearance and organization, were consistent with contractile microfilaments. These observations may be the morphological reflection of the phenotypic modifications occurring within the carcinoma cells, approaching smooth muscle differentiation.
Asunto(s)
Neoplasias Experimentales/ultraestructura , Neoplasias de la Lengua/ultraestructura , 4-Nitroquinolina-1-Óxido , Citoesqueleto de Actina/ultraestructura , Animales , Membrana Basal/ultraestructura , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/ultraestructura , Caveolas/ultraestructura , Células Epiteliales/ultraestructura , Invasividad Neoplásica , Neoplasias Experimentales/inducido químicamente , Ratas , Neoplasias de la Lengua/inducido químicamenteRESUMEN
Tumour-associated lymphatics contribute to a key component of metastatic spread, however, the biological interaction of tumour cells with intratumoural and peritumoural lymphatics (ITLs and PTLs) has remained unclear. To address this important issue, we have focused on the morphological and molecular aspects of newly formed lymphatics (lymphangiogenesis) and pre-existing lymphatics in the intratumoural and peritumoural tissues by using a hybridoma-induced tumour model. In the present study, ITLs with very high vessel density within the tumour mass showed small and flattened contours that varied from non-solid-to-solid tumours, whereas PTLs were relatively disorganized and tortuous, and packed with a cluster of tumour cells at the tumour periphery. Lymphatic endothelial cells (LECs) both in ITLs and PTLs were expressed with LYVE-1 and podoplanin in various tumour tissues, in which initial lymphatics were extremely extended and dilated. The tumour cells were frequently detected adhering to or penetrating lymphatic walls, especially near the open junctions. In the metastatic tissues, lymphangiogenic vasculatures occurred within the tumour matrix, and collecting PTLs represented abnormal twisty valve leaflets. The Western blot and RT-PCR analysis showed local variations of LEC proliferating potentials and lymphatic involvement in metastasis by a distinct profile of the protein and mRNA expression by LYVE-1, podoplanin, Prox-1 and vascular endothelial growth factor-3 (VEGFR-3). These findings indicated that both ITLs and PTLs, including enlarged pre-existing and newly formed lymphatics, may play a crucial role in metastasis with an active tumour cell adhesion, invasion, migration and implantation.
Asunto(s)
Glicoproteínas/metabolismo , Vasos Linfáticos/patología , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/patología , Animales , Biomarcadores/metabolismo , Endotelio Linfático/metabolismo , Endotelio Linfático/patología , Proteínas de Homeodominio/metabolismo , Linfangiogénesis , Vasos Linfáticos/metabolismo , Vasos Linfáticos/ultraestructura , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/ultraestructura , Proteínas Supresoras de Tumor/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of malignant cells, but not in normal cells. This preferential toxicity to the abnormal cells renders TRAIL potentially a very powerful therapeutic weapon against cancer. However, a requirement for large quantities of TRAIL to suppress tumor growth in vivo is one of the major factors that has hindered it from being widely applied clinically. To overcome this, we constructed a replication-deficient adenovirus that carries a human full-length TRAIL gene (Ad-TRAIL) and tested its efficacy against a lung cancer model system in comparison to that of the recombinant soluble TRAIL protein. METHODS: To investigate the antitumor activity and therapeutic value of the Ad-TRAIL on the non-small cell lung cancer (NSCLC), four NSCLC cell lines, namely, YTMLC, GLC, A549, and H460 cells, were used. TRAIL protein expression was determined by Western blotting and flow cytometry. Cell viability was analyzed by proliferation assay, and DNA ladder and cell-cycle analysis were used to identify apoptosis. To further evaluate the effect of Ad-TRAIL in vivo, YTMLC cells were inoculated to the subcutis of nude mice. The Ad-TRAIL was subsequently administered into the established tumors. Tumor growth and the TRAIL toxicity were evaluated after treatment. RESULTS: YTMLC cells infected with Ad-TRAIL showed decreased cell viability and a higher percentage of apoptosis. Similar, Ad-TRAIL treatment also significantly suppressed tumor growth in vivo. CONCLUSIONS: TRAIL gene therapy provides a promising therapy for the treatment of NSCLC.
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Adenoviridae , Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/terapia , Terapia Genética , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/ultraestructura , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestructura , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/terapia , Neoplasias Experimentales/ultraestructura , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The oncolytic effects of encephalomyocarditis (EMC) virus were examined in human retinoblastoma cell (Y79) cultures which were infected with 10(4 )tissue culture infectious doses (TCIDs) of the E variant of EMC (EMC-E) virus. The TCIDs were used to titer the maximum effect of EMC virus on L-929 cells. In-vitro studies showed 90% cytopathic effect (CPE) at 24 h and 100% CPE at 52 h. The CPE was used to observe pathologic effects of the cells. In-vivo studies employing human retinoblastoma grown as a tumor in nude mice, revealed degeneration of 80% of the tumor cells at 3 days and total destruction at 4 days following inoculation with the EMC-E virus. The virus is highly neurotropic in mice, but is usually not pathogenic in man. These studies suggest a possible new direction in the treatment of retinoblastoma and other malignant tumors using the viral technology.
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Virus de la Encefalomiocarditis , Neoplasias Experimentales/terapia , Viroterapia Oncolítica/métodos , Retinoblastoma/terapia , Retinoblastoma/virología , Animales , Virus de la Encefalomiocarditis/clasificación , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Neoplasias Experimentales/virología , Virus Oncolíticos/clasificación , Retinoblastoma/patología , Retinoblastoma/ultraestructura , Células Tumorales CultivadasRESUMEN
Tumor blood vessels are biological targets for cancer therapy. In this study, a tumor vasculature targeting system that consisted of liposomes and lectin (WGA) was built. Liposomes were used to carry a number of liposome-friendly anti-tumoral agents along with WGA, a lectin which posseses a specific affinity for binding to inflamed endothelial cells. In order to target tumor vasculature, inflammation of endothelial cells was induced by radiation. Because ionizing radiation induces an inflammatory response in tumor vasculature, lectin-conjugates were utilized to determine whether radiation can be used to target drug delivery to tumor vessels. Wheat germ agglutinin (WGA) is one such lectin that binds to inflamed microvasculature. WGA was conjugated to liposomes containing cisplatin and administered to tumor bearing mice. Tumor growth delay was used to analyze the efficacy of cytotoxicity. FITC-conjugated WGA accumulated within irradiated tumor microvasculature. WGA was conjugated to liposomes and labeled with 111In. This demonstrated radiation-inducible tumor-selective binding. WGA-liposome-conjugates were loaded with Cisplatin and administered to mice bearing irradiated tumors. Tumors treated with a combination of liposome encapsulated cisplatin together with radiation showed a significant increase in tumor growth delay as compared to radiation alone. These findings demonstrate that ionizing radiation can be used to guide drug delivery to tumor microvasculature.
Asunto(s)
Terapia Combinada/métodos , Sistemas de Liberación de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Neoplasias Experimentales/radioterapia , Neoplasias/radioterapia , Terapia por Captura de Neutrón , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Cisplatino/farmacología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacología , Radioisótopos de Indio , Liposomas/química , Liposomas/metabolismo , Liposomas/farmacología , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/ultraestructura , Fármacos Sensibilizantes a Radiaciones/farmacología , Radiometría/efectos adversos , Radiometría/métodos , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Piel/ultraestructura , Células Tumorales Cultivadas , Aglutininas del Germen de Trigo/metabolismo , Aglutininas del Germen de Trigo/farmacologíaRESUMEN
Arsenic and cadmium (Cd(+2)) are human carcinogens, and epidemiological studies have implicated both pollutants in the development of urinary bladder cancer. Despite this epidemiological base, it is unknown if either Cd(+2) or arsenite (As(+3)) can directly cause the malignant transformation of human urothelial cells. The goal of this study was to determine if Cd(+2) and/or As(+3) are able to cause the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line UROtsa to long-term in vitro exposure to Cd(+2) and As(+3), with the endpoint being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. It was demonstrated that a long-term exposure to either 1 M Cd(+2) or 1 M As(+3) resulted in the selection of cells that were able to form colonies in soft agar and tumors when heterotransplanted into nude mice. The histology of the tumor heterotransplants produced by UROtsa cells malignantly transformed by Cd(+2) had epithelial features consistent with those of a classic transitional-cell carcinoma of the bladder. The histology of the tumor heterotransplants produced by cells malignantly transformed by As(+3) was unique in that the cells displayed a prominent squamoid differentiation.
Asunto(s)
Arsenicales/efectos adversos , Compuestos de Cadmio/efectos adversos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Experimentales/inducido químicamente , Vejiga Urinaria/patología , Urotelio/patología , Agar , Animales , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Línea Celular , Transformación Celular Neoplásica/patología , Transformación Celular Neoplásica/ultraestructura , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Desnudos/genética , Trasplante de Neoplasias/métodos , Trasplante de Neoplasias/patología , Neoplasias Experimentales/patología , Neoplasias Experimentales/ultraestructura , Factores de Tiempo , Urotelio/efectos de los fármacosRESUMEN
Spleen macrophages, as most active elements of the mononuclear phagocyte system, were studied using light and electron microscopy in experimental rats and mice with differenet types of malignant neoplasia, including chemically induced carcinogenesis, transplantable tumor growth and in leukosis. In chemically induced carcinogenesis macrophage phagocytic activity was reduced, morphologically, the cellular surface smoothing, cytoplasm organell reduction and nuclear pyknotic changes were found. In animals with transplanted tumors, high activity of spleen macrophages was detected. In animals with leukosis, macrophages are characterized by reduced phagocytic activity, smoothed cellular surface and a variable number of lysosomes. The results obtained support the concept of high reactivity of the cells of mononuclear phagocyte system in neoplasia.
Asunto(s)
Macrófagos/ultraestructura , Neoplasias Experimentales/ultraestructura , Bazo/citología , Animales , Macrófagos/fisiología , Ratones , Trasplante de Neoplasias , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Fagocitosis , RatasRESUMEN
Differences between soft X-ray imaging and microcomputed tomography (micro-CT) in the microangiographic depiction of small vessels in tumors were compared to evaluate the tumors' 3D microangioarchitecture and the progress of growth-related neovascularization. VX2 carcinomas transplanted to the auricles of 24 rabbits randomly assigned to three groups were examined after 1, 3, and 7 days. Eight rabbits without transplants were the controls. Barium sulfate was injected into the auricular artery, and conventional soft X-ray images and micro-CT microangiograms of auricle specimens were studied. Micro-CT detected vessels with diameters of less than 50 microm, and imaging from several angles clearly showed the network of tumor vessels. Moreover, micro-CT confirmed the 3D process of growth-related neovascularization. There were no significant differences between the present findings and those of our previously published microscopic study. Micro-CT should prove useful for evaluating the 3D microarchitecture of tumors and for clear imaging of tumor angiogenesis.