RESUMEN
The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Glioma/metabolismo , Factor de Crecimiento de Hepatocito/inmunología , Proteínas Proto-Oncogénicas c-met/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Daño del ADN , Glioma/patología , Humanos , Ratones , Ratones Desnudos , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Transducción de Señal/efectos de la radiaciónRESUMEN
The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface alpha5beta1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1alpha) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1alpha mediated transcriptional activity as well as HIF-1alpha mediated angiogenic sprouting of ECs. HIF-1alpha plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1alpha activities.
Asunto(s)
Citocinas/metabolismo , Células Endoteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Hipoxia de la Célula , Línea Celular , Células Cultivadas , Citocinas/genética , Citoplasma/metabolismo , Células Endoteliales/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Integrina alfa5beta1/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/genética , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neovascularización Patológica , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Neoplasias Meníngeas/terapia , Meningioma/terapia , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quimioradioterapia/métodos , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Meningioma/metabolismo , Meningioma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Fosfatasa 2/metabolismoRESUMEN
In the event of a radiological or nuclear attack, advanced clinical countermeasures are needed for screening and medical management of the exposed population. In such a scenario, minimally invasive biomarkers that can accurately quantify radiation exposure would be useful for triage management by first responders. In this murine study, we evaluated the efficacy of a novel combination of radiation responsive proteins, Flt3 ligand (FL), serum amyloid A (SAA), matrix metalloproteinase 9 (MMP9), fibrinogen beta (FGB) and pentraxin 3 (PTX3) to predict the received dose after whole- or partial-body irradiation. Ten-week-old female C57BL6 mice received a single whole-body or partial-body dose of 18 Gy from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by cardiac puncture at 24, 48, 72 h and 1 week postirradiation. Plasma protein levels were determined via commercially available ELISA assay. A multivariate discriminant analysis was utilized to generate best-fit dose prediction models for whole-body exposures using the selected biomarker panel and its potential application to partial-body exposures was examined. The combination of values from FL, SAA, MMP9, FGB and PTX3 between 24 h and 1 week postirradiation yielded novel dose-response relationships. For day 1 postirradiation, the best-fit model yielded a predictive accuracy of 81% utilizing FL alone. The use of additional proteins did not enhance the model accuracy whereas, at day 2 postirradiation, the addition of PTX3 and FGB to FL increased the accuracy to 100%. At day 3 the use of FL and PTX3 yielded a predictive accuracy of 93% and at day 7 use of FL and SAA had an accuracy of 90%. Dose prediction of partial-body exposures based on the TBI model had a higher predictive accuracy when the percentage of the body exposed to radiation increased. Our findings indicate that this novel combination of radiation responsive biomarker proteins are an efficient method for predicting radiation exposure and are more accurate when used in concert compared to using any single biomarker protein alone.
Asunto(s)
Modelos Estadísticos , Exposición a la Radiación/análisis , Irradiación Corporal Total , Animales , Biomarcadores/metabolismo , Proteína C-Reactiva/metabolismo , Femenino , Fibrinógeno/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Componente Amiloide P Sérico/metabolismoRESUMEN
BACKGROUND: Epithelial to mesenchymal transition, and mimicking processes, contribute to cancer invasion and metastasis, and are known to be responsible for resistance to various therapeutic agents in many cancers. While a number of studies have proposed molecular signatures that characterize the spectrum of such transition, more work is needed to understand how the mesenchymal signature (MS) is regulated in non-epithelial cancers like gliomas, to identify markers with the most prognostic significance, and potential for therapeutic targeting. RESULTS: Computational analysis of 275 glioma samples from "The Cancer Genome Atlas" was used to identify the regulatory changes between low grade gliomas with little expression of MS, and high grade glioblastomas with high expression of MS. TF (transcription factor)-gene regulatory networks were constructed for each of the cohorts, and 5 major pathways and 118 transcription factors were identified as involved in the differential regulation of the networks. The most significant pathway - Extracellular matrix organization - was further analyzed for prognostic relevance. A 20-gene signature was identified as having prognostic significance (HR (hazard ratio) 3.2, 95% CI (confidence interval) = 1.53-8.33), after controlling for known prognostic factors (age, and glioma grade). The signature's significance was validated in an independent data set. The putative stem cell marker CD44 was biologically validated in glioma cell lines and brain tissue samples. CONCLUSIONS: Our results suggest that the differences between low grade gliomas and high grade glioblastoma are associated with differential expression of the signature genes, raising the possibility that targeting these genes might prolong survival in glioma patients.
Asunto(s)
Genómica , Glioma/genética , Glioma/patología , Mesodermo/patología , Adulto , Animales , Línea Celular Tumoral , Movimiento Celular , Estudios de Cohortes , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/diagnóstico , Glioma/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , PronósticoRESUMEN
Inhibition of angiogenesis has emerged as a key focus for the treatment of cancer, necessitating a better understanding of the downstream molecular targets of angiogenesis inhibitors. Endostatin, thrombospondin-1, fumagillin, and its synthetic derivative, TNP-470, are potent inhibitors of endothelial cell proliferation and migration in culture and of angiogenesis in vivo. To identify targets that mediate the effects of these inhibitors, we compared two-dimensional gel electrophoresis patterns from lysates of treated and untreated human endothelial cells. Among the proteins identified were cofilin and hsp27, two proteins involved in actin dynamics. Western blotting and immunofluorescence experiments confirmed that the phosphorylation states and subcellular localization of these two proteins were affected by all of the inhibitors tested and that treated cells had a more extensive network of actin stress fibers and more numerous focal adhesion plaques compared with untreated cells. Endothelial monocyte activating polypeptide II, another angiogenesis inhibitor, elicited the same response in the actin cytoskeleton and focal adhesions of endothelial cells. This more adherent phenotype may explain the shared ability of these inhibitors to block endothelial migratory signals. Starting with a proteomics approach, we have identified common effector molecules used by a panel of angiogenesis inhibitors that perturb the cytoskeleton to prevent endothelial migration.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Citoesqueleto/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Ciclohexanos , Citocinas/farmacología , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Endostatinas/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Ácidos Grasos Insaturados/farmacología , Humanos , Proteínas de Neoplasias/farmacología , O-(Cloroacetilcarbamoil) Fumagilol , Fosforilación/efectos de los fármacos , Proteínas de Unión al ARN/farmacología , Sesquiterpenos/farmacología , Fracciones Subcelulares/metabolismo , Trombospondina 1/farmacologíaRESUMEN
Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-ß/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.
Asunto(s)
Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/genética , Glioblastoma/genética , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/terapia , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína smad3/metabolismoRESUMEN
Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo. Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteína Forkhead Box M1/metabolismo , Glioblastoma/metabolismo , Tolerancia a Radiación , Factor de Transcripción STAT3/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/radioterapia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Forkhead Box M1/genética , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/radioterapia , Recombinación Homóloga , Humanos , Estimación de Kaplan-Meier , Mitosis/efectos de los fármacos , Péptidos/farmacología , Pronóstico , Unión Proteica , Transporte de Proteínas , Proteoma , Proteómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Aneuploidy has been recognized as a common characteristic of cancers. Aneuploidy frequently results from errors of the mitotic checkpoint, the major cell cycle control mechanism that acts to prevent chromosome missegregation. Mutation of the genes that control chromosome segregation during mitosis may explain the high rate of chromosomal instability and aneuploidy, a characteristic of most solid tumors, including glioblastomas (GBM)[1, 2]. Monopolar spindle 1 (MPS1), is an essential spindle assembly checkpoint kinase that is overexpressed in several human cancers [3-5]. In our previous publication, we have shown the role of MPS1 kinase in DNA repair and enhanced radiosensitivity in GBM[6]. Here, we provide methodological and analytical details of that study, to compare mRNA expression profile of siMPS1-silenced U251 cells with untransfected control, and siRNA control (siNeg) at 6, 24, and 48 hours after transfection. The raw data of this study is deposited in Gene Expression Omnibus under the accession number GSE57091.
RESUMEN
There is a need for minimally invasive biomarkers that can accurately and quickly quantify radiation exposure. Radiation-responsive proteins have applications in clinical medicine and for mass population screenings after a nuclear or radiological incident where the level of radiation exposure and exposure pattern complicate medical triage for first responders. In this study, we evaluated the efficacy of the acute phase protein serum amyloid A (SAA) as a biomarker for radiation exposure using plasma from irradiated mice. Ten-week-old female C57BL6 mice received a 1-8 Gy single whole-body or partial-body dose from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by mandibular or cardiac puncture at 6, 24, 48 and 72 h or 1-3 weeks postirradiation. SAA levels were determined using a commercially available ELISA assay. Data was pooled to generate SAA µg/ml threshold values correlating plasma SAA levels with radiation dose. SAA levels were statistically significant over control at all exposures between 2 and 8 Gy at 24 h postirradiation but not at 6, 48 and 72 h or 1-3 weeks postirradiation. SAA levels at 1 Gy were not significantly elevated over control at all time points. Total-body-irradiated (TBI) SAA levels at 24 h were used to generate a dose prediction model that successfully differentiated TBI mice into dose received cohorts of control/1 Gy and ≥ 2 Gy groups with a high degree of accuracy in a blind study. Dose prediction of partial-body exposures based on the TBI model correlated increasing predictive accuracy with percentage of body exposure to radiation. Our findings indicate that plasma SAA levels might be a useful biomarker for radiation exposure in a variety of total- and partial-body irradiation settings.
Asunto(s)
Monitoreo de Radiación/métodos , Proteína Amiloide A Sérica/análisis , Animales , Biomarcadores , Línea Celular , Femenino , Histonas/análisis , Humanos , Inflamación/sangre , Inflamación/etiología , Ratones , Ratones Endogámicos C57BL , Irradiación Corporal TotalRESUMEN
UNLABELLED: To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. IMPLICATIONS: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Reparación del ADN/efectos de la radiación , Glioblastoma/genética , Glioblastoma/radioterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/enzimología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Glioblastoma/enzimología , Humanos , Ratones Desnudos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Tolerancia a Radiación , Distribución Aleatoria , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Glioblastoma/tratamiento farmacológico , Mitosis/efectos de los fármacos , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Carga Tumoral/efectos de los fármacos , Animales , Western Blotting , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Línea Celular Tumoral , Terapia Combinada , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Inmunohistoquímica , Ratones Desnudos , Mitosis/efectos de la radiación , Proteína Fosfatasa 2/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Carga Tumoral/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
With the role of angiogenesis in tumor growth and progression firmly established, considerable effort has been directed to antiangiogenic therapy as a new modality to treat human cancers. Antiangiogenic agents have recently received much widespread attention but strategies for their optimal use are still being developed. Gene therapy represents an attractive alternative to recombinant protein administration for several reasons. This review evaluates the potential advantages of gene transfer for antiangiogenic cancer therapy and describes preclinical gene transfer work with endogenous angiogenesis inhibitors demonstrating the feasibility of effectively suppressing and even eradicating tumors in animal models. Additionally, we describe the advantages and disadvantages of currently available gene transfer vectors and update novel developments in this field. In conclusion, gene therapy holds great promise in advancing antiangiogenesis as an effective cancer therapy and will undoubtedly be evaluated in human clinical trials in the near future.
RESUMEN
Angiogenesis is an important natural process occurring in the body, both in health and in infirmity, that is controlled by angiogenesis-stimulating growth factors and angiogenesis inhibitors. Uncontrolled angiogenesis in a tumor can result in both tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are major players in many human malignancies and contribute directly to disease outcome. There is compelling evidence indicating that the beneficial effects of VEGF and VEGFR can be targeted as antiangiogenic therapy. Many of the agents have shown promising results in cell culture preclinical and animal models. Some of these agents have been tested in clinical trials as well. This review discusses the clinical significance of VEGF/VEGFR in human cancer, summarizes the more recent progress in the field, and further emphasizes the current development of agents that block VEGFR/VEGFR as angiogenesis inhibitors and the therapeutic significance of these agents in clinical trials.
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Inhibidores de la Angiogénesis/uso terapéutico , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention.
Asunto(s)
Astrocitos/efectos de la radiación , Transformación Celular Neoplásica/efectos de la radiación , Reparación del ADN , ADN/metabolismo , Transcripción Genética , Adulto , Animales , Astrocitos/citología , Astrocitos/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Corteza Cerebral/efectos de la radiación , Cromatina/química , Cromatina/metabolismo , Cromatina/efectos de la radiación , Evolución Clonal , ADN/química , Roturas del ADN de Doble Cadena , Feto , Rayos gamma , Expresión Génica , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Macaca fascicularis , Mutación , Cultivo Primario de CélulasRESUMEN
Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.
Asunto(s)
Proteínas de Ciclo Celular/genética , Glioblastoma/genética , Mitosis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Femenino , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Ratones , Ratones Desnudos , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Tiofenos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1RESUMEN
Exosomes are nanometer-sized lipid vesicles released ubiquitously by cells, which have been shown to have a normal physiological role, as well as influence the tumor microenvironment and aid metastasis. Recent studies highlight the ability of exosomes to convey tumor-suppressive and oncogenic mRNAs, microRNAs, and proteins to a receiving cell, subsequently activating downstream signaling pathways and influencing cellular phenotype. Here, we show that radiation increases the abundance of exosomes released by glioblastoma cells and normal astrocytes. Exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration. In particular, connective tissue growth factor (CTGF) mRNA and insulin-like growth factor binding protein 2 (IGFBP2) protein levels were elevated, and coculture of nonirradiated cells with exosomes isolated from irradiated cells increased CTGF protein expression in the recipient cells. Additionally, these exosomes enhanced the activation of neurotrophic tyrosine kinase receptor type 1 (TrkA), focal adhesion kinase, Paxillin, and proto-oncogene tyrosine-protein kinase Src (Src) in recipient cells, molecules involved in cell migration. Collectively, our data suggest that radiation influences exosome abundance, specifically alters their molecular composition, and on uptake, promotes a migratory phenotype.
RESUMEN
BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor-alpha (TNF-alpha) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF-alpha, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF-alpha targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno-associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF-alpha (AAVP-TNF-alpha). The activity of AAVP-TNF-alpha was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP-TNF-alpha infection of human melanoma cells resulted in high levels of TNF-alpha expression. Systemic administration of targeted AAVP-TNF-alpha to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF-alpha, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF-alpha specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF-alpha is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy.
Asunto(s)
Melanoma/irrigación sanguínea , Melanoma/terapia , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/terapia , Factor de Necrosis Tumoral alfa/genética , Animales , Línea Celular Tumoral , Dependovirus/genética , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos , Humanos , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/terapia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Under the direction and sponsorship of the National Cancer Institute, we report on the first pre-clinical trial of the Comparative Oncology Trials Consortium (COTC). The COTC is a novel infrastructure to integrate cancers that naturally develop in pet dogs into the development path of new human drugs. Trials are designed to address questions challenging in conventional preclinical models and early phase human trials. Large animal spontaneous cancer models can be a valuable addition to successful studies of cancer biology and novel therapeutic drug, imaging and device development. METHODOLOGY/PRINCIPAL FINDINGS: Through this established infrastructure, the first trial of the COTC (COTC001) evaluated a targeted AAV-phage vector delivering tumor necrosis factor (RGD-A-TNF) to alphaV integrins on tumor endothelium. Trial progress and data was reviewed contemporaneously using a web-enabled electronic reporting system developed for the consortium. Dose-escalation in cohorts of 3 dogs (n = 24) determined an optimal safe dose (5x10(12) transducing units intravenous) of RGD-A-TNF. This demonstrated selective targeting of tumor-associated vasculature and sparing of normal tissues assessed via serial biopsy of both tumor and normal tissue. Repetitive dosing in a cohort of 14 dogs, at the defined optimal dose, was well tolerated and led to objective tumor regression in two dogs (14%), stable disease in six (43%), and disease progression in six (43%) via Response Evaluation Criteria in Solid Tumors (RECIST). CONCLUSIONS/SIGNIFICANCE: The first study of the COTC has demonstrated the utility and efficiency of the established infrastructure to inform the development of new cancer drugs within large animal naturally occurring cancer models. The preclinical evaluation of RGD-A-TNF within this network provided valuable and necessary data to complete the design of first-in-man studies.