RESUMEN
The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:
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Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Carbohidratos de la Dieta/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Butiratos/metabolismo , Proliferación Celular , Transformación Celular Neoplásica , Pólipos del Colon/metabolismo , Pólipos del Colon/microbiología , Pólipos del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/microbiología , Reparación de la Incompatibilidad de ADN , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Inflamación/genética , Inflamación/metabolismo , Inflamación/microbiología , Ratones , Ratones Endogámicos C57BL , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/metabolismo , Organismos Libres de Patógenos Específicos , beta Catenina/metabolismoRESUMEN
BACKGROUND: A high rate of glycolysis leading to elevated lactate content has been linked to poor clinical outcomes in patients with head and neck and cervical cancer treated with radiotherapy. Although the biological explanation for this relationship between lactate and treatment response remains unclear, there is a continued interest in evaluating strategies of targeting metabolism to enhance the effectiveness of radiotherapy. The goal of this study was to investigate the effect of metabolic-targeting through HIF-1α inhibition and the associated changes in glycolysis, oxygen consumption and response on the efficacy of high-dose single-fraction radiotherapy (HD-SFRT). METHODS: HIF-1α wild-type and HIF-1α knockdown FaDu and ME180 xenograft tumors were grown in the hind leg of mice that were placed in an environmental chamber and exposed to different oxygen conditions (air-breathing and hypoxia). Ex vivo bioluminescence microscopy was used to measure lactate and ATP levels and the hypoxic fraction was measured using EF5 immunohistochemical staining. The oxygen consumption rate (OCR) in each cell line in response to in vitro hypoxia was measured using an extracellular flux analyzer. Tumor growth delay in vivo was measured following HD-SFRT irradiation of 20 Gy. RESULTS: Targeting HIF-1α reduced lactate content, and increased both oxygen consumption and hypoxic fraction in these tumors after exposure to short-term continuous hypoxia. Tumors with intact HIF-1α subjected to HD-SFRT immediately following hypoxia exposure were less responsive to treatment than tumors without functional HIF-1α, and tumors irradiated under air breathing conditions regardless of HIF-1α status. CONCLUSIONS: Blocking the HIF1 response during transient hypoxic stress increased hypoxia, reduced lactate levels and enhanced response to HD-SFRT. This strategy of combining hypofractionated radiotherapy with metabolic reprogramming to inhibit anaerobic metabolism may increase the efficacy of HD-SFRT through increased oxygen consumption and complementary killing of radiosensitive and hypoxic, radioresistant cells.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Ácido Láctico/metabolismo , Neoplasias/metabolismo , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Animales , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de la radiación , Femenino , Técnicas de Silenciamiento del Gen , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Neoplasias/patología , Neoplasias/radioterapia , Neovascularización Patológica , Dosis de Radiación , Carga Tumoral/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor-dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.
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Caveolina 1/metabolismo , Receptores ErbB/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Transducción de Señal , Regulación hacia Arriba , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caveolas/metabolismo , Caveolas/ultraestructura , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Secuencia Conservada/genética , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , ARN Polimerasa II/metabolismo , Elementos de Respuesta/genética , Transcripción Genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.
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Algoritmos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía de Polarización/métodos , Refractometría/métodos , Fenómenos Fisiológicos de la Piel , Piel/citología , Animales , Estudios de Factibilidad , Luz , Ratones , Dispersión de RadiaciónRESUMEN
BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) has been demonstrated to modulate inflammatory processes with evidence suggesting that treatment protocol, such as wavelength, total energy, and number of treatments determine the clinical efficacy. In this study, the effects of LLLT mediated by different wavelengths and continuous versus pulsed delivery mode were quantified in a transgenic murine model with the luciferase gene under control of the inducible nitric oxide synthase (iNOS) expression. STUDY DESIGN/MATERIALS AND METHODS: LLLT modulated iNOS gene expressed in the acute Zymosan-induced inflammation model is quantified using transgenic mice (FVB/N-Tg(iNOS-luc)). Here an energy density of 5 J cm(-2) at either 635, 660, 690, and 905 nm in continuous wave mode and at 905 nm for short pulse delivery were evaluated. Age of the animals was determined as additional modulating the inflammatory response and the LLLT efficacy for some treatment protocols. RESULTS: Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression for some LLLT protocols. Intensity and time course of inducible nitric oxide expression was found to not only depend on wavelength, but also on the mode of delivery, continuous, or pulsed irradiation. CONCLUSION: LLLT exhibit different effects in induced inflammatory process according to different wavelengths and wave mode. Upregulation of iNOS gene following 905 nm pulsed wave suggests a different mechanism in activating the inflammatory pathway response when compared to the continuous wave.
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Inflamación/enzimología , Inflamación/radioterapia , Terapia por Luz de Baja Intensidad , Óxido Nítrico Sintasa de Tipo II/efectos de la radiación , Envejecimiento/patología , Animales , Inflamación/inducido químicamente , Inflamación/diagnóstico , Articulación de la Rodilla/enzimología , Articulación de la Rodilla/patología , Articulación de la Rodilla/efectos de la radiación , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Distribución Aleatoria , Regulación hacia Arriba , ZimosanRESUMEN
Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.
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Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Aumento de la Imagen/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Fotoquimioterapia/métodos , Porfirinas/administración & dosificación , Tomografía de Coherencia Óptica/métodos , Animales , Ratones , Ratones Desnudos , VerteporfinaRESUMEN
See page 841 To explore systemic utilization of Epstein-Barr virus (EBV)-specific transcriptionally targeted adenoviruses, three vectors were constructed to examine kinetics, specificity, and biodistribution: adv.oriP.luc, expressing luciferase under EBV-specific control; adv.SV40luc, expressing luciferase constitutively; and adv.oriP.E1A.oriP.luc, a conditionally replicating adenovirus, expressing both luciferase and E1A. Bioluminescence imaging (BLI) was conducted on tumor-bearing severe combined immunodeficient (SCID) mice (C666-1, EBV-positive human nasopharyngeal cancer) treated intravenously (i.v.) with 3 × 108 infectious units (ifu) of the adenoviral vectors. At 72 hours, adv.oriPluc demonstrated an 8.4-fold higher tumor signal than adv.SV40luc; adv.oriP.E1A.oriP.luc was 26.7-fold higher; however, a significant liver signal was also observed, necessitating further action to improve biodistribution. Several compounds were examined to this end, including norepinephrine, serotonin, clodronate liposomes, and STI571, to determine whether any of these measures could improve adenoviral biodistribution. Each of these interventions was assessed using BLI in mice i.v. injected with adv.oriP.luc. STI571 achieved the highest increase in tumor-to-liver ratio (TLR; 6.6-fold), which was associated with a 59% reduction in tumor interstitial fluid pressure (IFP) along with a decrease in platelet-derived growth factor-ß receptor (PDGFßR) activation. This study reports the favorable modulation by STI571 of the biodistribution of adenoviral vectors, providing a potential approach to improving therapeutic outcome.
RESUMEN
Polymeric micelles are emerging as an effective drug delivery system for hydrophobic photosensitizers in photodynamic therapy (PDT). The objective of this study was to investigate the formulation of hydrophobic protoporphyrin IX (PpIX) with MePEG(5000)-b-PCL(4100) [methoxy poly (ethylene glycol)-b-poly (caprolactone)] diblock copolymers and to compare their PDT response to that of free PpIX. The photophysical and photochemical properties of the polymeric PpIX micelles were studied by measuring absorbance and fluorescence spectra, PpIX-loading efficiency and stability, the micelle particle size and morphology, as well as singlet oxygen luminescence and lifetime. The spherical micelles have a high PpIX-loading efficiency of 82.4% and a narrow size distribution with a mean diameter of 52.2 +/- 6.4 nm. The cellular uptake of PpIX in RIF-1 cells using PpIX micelles was approximately two-fold higher than that for free PpIX. Free PpIX and PpIX formulated in micelles exhibited similar subcellular localization in or around the cellular plasma membrane, as demonstrated using fluorescence microscopy. In vitro PDT results showed that the PpIX micelles have markedly increased photocytotoxicity over that with free PpIX, by nearly an order of magnitude at the highest light dose used. The micelles alone had no evident phototoxicity or dark toxicity. These findings suggest that MePEG(5000)-b-PCL(4100) diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.
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Interacciones Hidrofóbicas e Hidrofílicas , Poliésteres/química , Polietilenglicoles/química , Protoporfirinas/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica de Transmisión , Fotoquimioterapia , Protoporfirinas/farmacología , EspectrofotometríaRESUMEN
This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (lambda = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm(-2), 200 s, 5 J cm(-2)) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.
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Artritis/radioterapia , Terapia por Luz de Baja Intensidad , Mediciones Luminiscentes/métodos , Óxido Nítrico Sintasa/metabolismo , Factores de Edad , Animales , Artritis/inducido químicamente , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Miembro Posterior , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Transgénicos , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/genética , ZimosanRESUMEN
Laser technology has been studied as a potential replacement to the conventional dental drill. However, to prevent pulpal cell damage, information related to the safety parameters using high-power lasers in oral mineralized tissues is needed. In this study, the heat distribution profiles at the surface and subsurface regions of human dentine samples irradiated with a Nd:YAG laser were simulated using Crank-Nicolson's finite difference method for different laser energies and pulse durations. Heat distribution throughout the dentin layer, from the external dentin surface to the pulp chamber wall, were calculated in each case, to investigate the details of pulsed laser-hard dental tissue interactions. The results showed that the final temperature at the pulp chamber wall and at the dentin surface are strongly dependent on the pulse duration, exposure time, and the energy contained in each pulse.
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Temperatura Corporal/fisiología , Temperatura Corporal/efectos de la radiación , Dentina/fisiología , Dentina/efectos de la radiación , Calor , Rayos Láser , Modelos Biológicos , Anisotropía , Simulación por Computador , Relación Dosis-Respuesta en la Radiación , Humanos , Temperatura , Conductividad TérmicaRESUMEN
Photodynamic therapy (PDT) is a promising modality for the treatment of solid tumors that combines a photosensitizing agent and light to produce cytotoxic reactive oxygen species that lead to tumor cell death. The recent introduction of bioluminescence imaging (BLI), involving the use of the luciferase gene (luc) transferred into target tumor cells, followed by systemic administration of luciferin and detection of the emitted visible chemiluminescence photons, offers the potential for longitudinal imaging of tumor growth and therapeutic response in single animals. We demonstrate in this study the first results of the use of BLI to assess the response of an intracranial brain tumor model (9L rat gliosarcoma) to aminolevulinic acid (ALA)-mediated PDT. Complementary in vitro experiments with the luciferase-transfected 9L cells show that the decrease in the luminescent signal after PDT correlates with cell kill. In vivo imaging shows a decrease in the BLI signal from the tumor after ALA-PDT treatment, followed by tumor regrowth. Furthermore, preliminary studies using cells transfected with a hypoxia-responsive vector show an increase in bioluminescence within 4 h after Photofrin-mediated PDT, demonstrating the ability to observe stress-gene responses. These results suggest that BLI can be used to provide spatiotemporal information of intracranial brain tumor responses after PDT and may serve as a valuable response-endpoint measure.
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Ácido Aminolevulínico/uso terapéutico , Gliosarcoma/tratamiento farmacológico , Gliosarcoma/patología , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/metabolismo , Ácido Aminolevulínico/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Femenino , Mediciones Luminiscentes , Fármacos Fotosensibilizantes/farmacología , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We have developed and tested a novel conjugation of the clinically used prodrug aminolevulinic acid with 2-deoxyglucosamine as a novel probe (ALA-2DG) for fluorescence imaging and photodynamic therapy. PROCEDURES: ALA-2DG was successfully synthesized, and the mechanisms of probe uptake, PpIX synthesis, and photodynamic therapy efficacy were evaluated in vitro and in vivo. RESULTS: ALA-2DG led to PpIX synthesis in tumor cells in vitro and in tumor in vivo. Competitive and inhibitory assays in vitro showed a reduction of this PpIX synthesis that was not observed when cells were incubated with ALA itself, indicating that intracellular uptake of ALA-2DG occurs by GLUT-mediated active transport. Initial photodynamic therapy studies confirmed the efficacy of ALA-2DG as a photodynamic sensitizer. CONCLUSIONS: The in vitro assays suggest that ALA-2DG is taken up by cells via glucose transporters. Initial studies in oral cancer demonstrated the applicability of ALA-2DG for in vivo imaging and its potential as an alternative to ALA-PpIX-based fluorescence diagnostics and photodynamic therapy, providing higher tumor specificity.
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Ácido Aminolevulínico/química , Glucosa/análogos & derivados , Fotoquimioterapia , Cirugía Asistida por Computador , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Ácido Aminolevulínico/síntesis química , Animales , Línea Celular Tumoral , Fluorescencia , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa/química , Humanos , Masculino , Mesocricetus , Ratones SCID , Floretina/farmacología , Protoporfirinas/metabolismo , RatasRESUMEN
Head and neck cancer (HNC) is the eighth most common malignancy worldwide, comprising a diverse group of cancers affecting the head and neck region. Despite advances in therapeutic options over the last few decades, treatment toxicities and overall clinical outcomes have remained disappointing, thereby underscoring a need to develop novel therapeutic approaches in HNC treatment. Uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified from an RNA interference-based high-throughput screen as a tumor-selective radiosensitizing target for HNC. UROD knockdown plus radiation induced caspase-mediated apoptosis and cell cycle arrest in HNC cells in vitro and suppressed the in vivo tumor-forming capacity of HNC cells, as well as delayed the growth of established tumor xenografts in mice. This radiosensitization appeared to be mediated by alterations in iron homeostasis and increased production of reactive oxygen species, resulting in enhanced tumor oxidative stress. Moreover, UROD was significantly overexpressed in HNC patient biopsies. Lower preradiation UROD mRNA expression correlated with improved disease-free survival, suggesting that UROD could potentially be used to predict radiation response. UROD down-regulation also radiosensitized several different models of human cancer, as well as sensitized tumors to chemotherapeutic agents, including 5-fluorouracil, cisplatin, and paclitaxel. Thus, our study has revealed UROD as a potent tumor-selective sensitizer for both radiation and chemotherapy, with potential relevance to many human malignancies.
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Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/enzimología , Neoplasias de Cabeza y Cuello/radioterapia , Fármacos Sensibilizantes a Radiaciones/metabolismo , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Uroporfirinógeno Descarboxilasa/genética , Uroporfirinógeno Descarboxilasa/metabolismo , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Homeostasis , Humanos , Hierro/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Estrés Oxidativo , Interferencia de ARNRESUMEN
Ovarian cancer is known as the silent killer for being asymptomatic until late stages. Current first-line treatment consists of debulking surgery followed by i.v. chemotherapeutics administered intermittently, which leads to insufficient drug concentrations at tumor sites, accelerated tumor proliferation rates, and drug resistance, resulting in an overall median survival of only 2 to 4 years. For these reasons, more effective treatment strategies must be developed. We have investigated a localized, continuous chemotherapy approach in tumor models of human and murine ovarian cancers using the antineoplastic agent docetaxel. We show here that continuous docetaxel therapy is considerably more efficacious than intermittent therapy, resulting in a greater decrease in tumor burden and ascites fluid accumulation. Immunohistochemical analyses show that continuous chemotherapy abrogates tumor cell proliferation and angiogenesis to the tumor microenvironment, leading to greater tumor cell death than intermittent docetaxel therapy. Overall, our results show greater therapeutic advantages of continuous over intermittent chemotherapy in the treatment of ovarian cancer.
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Antineoplásicos/uso terapéutico , Taxoides/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ascitis/complicaciones , Ascitis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Docetaxel , Femenino , Humanos , Inmunohistoquímica , Inyecciones Intraperitoneales , Ratones , Neovascularización Patológica/complicaciones , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Taxoides/administración & dosificación , Taxoides/farmacología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-kappaB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-kappaB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel-Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-kappaB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-kappaB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-kappaB-survival signature.
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Carcinoma de Células Renales/terapia , Virus de la Encefalomiocarditis/fisiología , Neoplasias Renales/terapia , Virus Oncolíticos/fisiología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Virus de la Encefalomiocarditis/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones SCID , FN-kappa B/metabolismo , Virus Oncolíticos/genética , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Trasplante Heterólogo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.
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Vasos Sanguíneos/efectos de los fármacos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Animales , Vasos Sanguíneos/fisiología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Histocitoquímica , Degeneración Macular , Ratones , Ratones Desnudos , Microscopía Confocal , Espectrometría de Fluorescencia , VerteporfinaRESUMEN
Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-alpha subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-alpha is carried out by the von Hippel-Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-alpha for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL-HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-alpha or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-alpha while preserving its E3 enzymatic activity. Using speckle variance-optical coherence tomography (sv-OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of 'bio-tailored' enzymes in the treatment of HIF-overexpressing tumours.
Asunto(s)
Bioingeniería , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Adenoviridae/genética , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/terapia , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Neoplasias Renales/genética , Neoplasias Renales/patología , Neoplasias Renales/terapia , Ratones , Necrosis , Neovascularización Patológica/terapia , Plásmidos/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Transcripción Genética/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Er:YAG laser has been studied as a potential tool for restorative dentistry due to its ability to selectively remove oral hard tissue with minimal or no thermal damage to the surrounding tissues. The purpose of this study was to evaluate in vitro the tensile bond strength (TBS) of an adhesive/composite resin system to human enamel surfaces treated with 37% phosphoric acid, Er:YAG laser (lambda=2.94 microm) with a total energy of 16 J (80 mJ/pulse, 2Hz, 200 pulses, 250 ms pulse width), and Er:YAG laser followed by phosphoric acid etching. Analysis of the treated surfaces was performed by scanning electron microscopy (SEM) to assess morphological differences among the groups. TBS means (in MPa) were as follows: Er:YAG laser + acid (11.7 MPa) > acid (8.2 MPa) > Er:YAG laser (6.1 MPa), with the group treated with laser+acid being significantly from the other groups (p=0.0006 and p= 0.00019, respectively). The groups treated with acid alone and laser alone were significantly different from each other (p=0.0003). The SEM analysis revealed morphological changes that corroborate the TBS results, suggesting that the differences in TBS means among the groups are related to the different etching patterns produced by each type of surface treatment. The findings of this study indicate that the association between Er:YAG laser and phosphoric acid can be used as a valuable resource to increase bond strength to laser-prepared enamel.
Asunto(s)
Grabado Ácido Dental/métodos , Recubrimiento Dental Adhesivo , Esmalte Dental/ultraestructura , Grabado Dental/métodos , Materiales Dentales/química , Láseres de Estado Sólido/uso terapéutico , Ácidos Fosfóricos/química , Bisfenol A Glicidil Metacrilato/química , Resinas Compuestas/química , Esmalte Dental/efectos de la radiación , Recubrimientos Dentinarios/química , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Estrés Mecánico , Temperatura , Resistencia a la Tracción , Factores de Tiempo , Agua/químicaRESUMEN
Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) Asunto(s)
Gliosarcoma/metabolismo
, Luciferasas/metabolismo
, Transgenes/genética
, Adenosina Trifosfato/metabolismo
, Animales
, Hipoxia de la Célula
, Línea Celular Tumoral
, Gliosarcoma/genética
, Luciferasas/genética
, Mediciones Luminiscentes
, Oxígeno
, Ratas
, Transfección
RESUMEN
We report on imaging of microcirculation by calculating the speckle variance of optical coherence tomography (OCT) structural images acquired using a Fourier domain mode-locked swept-wavelength laser. The algorithm calculates interframe speckle variance in two-dimensional and three-dimensional OCT data sets and shows little dependence to the Doppler angle ranging from 75 degrees to 90 degrees . We demonstrate in vivo detection of blood flow in vessels as small as 25 microm in diameter in a dorsal skinfold window chamber model with direct comparison with intravital fluorescence confocal microscopy. This technique can visualize vessel-size-dependent vascular shutdown and transient vascular occlusion during Visudyne photodynamic therapy and may provide opportunities for studying therapeutic effects of antivascular treatments without on exogenous contrast agent.