RESUMEN
Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Neoplasias/metabolismo , Acetato CoA Ligasa/análisis , Acetato CoA Ligasa/genética , Acetilcoenzima A/metabolismo , Animales , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/metabolismo , Ratones , Neoplasias/química , Neoplasias/patología , Tomografía de Emisión de Positrones , Neoplasias de la Mama Triple Negativas/química , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, Kd = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and 3H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.
Asunto(s)
Receptores de GABA/análisis , Animales , Línea Celular Tumoral , Humanos , Ligandos , Microscopía Confocal , Modelos Moleculares , Imagen Molecular , Imagen Óptica , Unión Proteica , Ratas , Receptores de GABA/metabolismoRESUMEN
Improving response to epidermal growth factor receptor (EGFR)-targeted therapies in patients with advanced wild-type (WT) RAS colorectal cancer (CRC) remains an unmet need. In this preclinical work, we evaluated a new therapeutic combination aimed at enhancing efficacy by targeting cancer cell metabolism in concert with EGFR. We hypothesized that combined blockade of glutamine metabolism and EGFR represents a promising treatment approach by targeting both the "fuel" and "signaling" components that these tumors need to survive. To explore this hypothesis, we combined CB-839, an inhibitor of glutaminase 1 (GLS1), the mitochondrial enzyme responsible for catalyzing conversion of glutamine to glutamate, with cetuximab, an EGFR-targeted monoclonal antibody in preclinical models of CRC. 2D and 3D in vitro assays were executed following treatment with either single agent or combination therapy. The combination of cetuximab with CB-839 resulted in reduced cell viability and demonstrated synergism in several cell lines. In vivo efficacy experiments were performed in cell-line xenograft models propagated in athymic nude mice. Tumor volumes were measured followed by immunohistochemical (IHC) analysis of proliferation (Ki67), mechanistic target of rapamycin (mTOR) signaling (pS6), and multiple mechanisms of cell death to annotate molecular determinants of response. In vivo, a significant reduction in tumor growth and reduced Ki67 and pS6 IHC staining were observed with combination therapy, which was accompanied by increased apoptosis and/or necrosis. The combination showed efficacy in cetuximab-sensitive as well as resistant models. In conclusion, this therapeutic combination represents a promising new precision medicine approach for patients with refractory metastatic WT RAS CRC.
RESUMEN
PURPOSE: Pancreatic cancer is among the most aggressive malignancies and is rarely discovered early. However, pancreatic "incidentalomas," particularly cysts, are frequently identified in asymptomatic patients through anatomic imaging for unrelated causes. Accurate determination of the malignant potential of cystic lesions could lead to life-saving surgery or spare patients with indolent disease undue risk. Current risk assessment of pancreatic cysts requires invasive sampling, with attendant morbidity and sampling errors. Here, we sought to identify imaging biomarkers of high-risk pancreatic cancer precursor lesions. EXPERIMENTAL DESIGN: Translocator protein (TSPO) expression, which is associated with cholesterol metabolism, was evaluated in premalignant and pancreatic cancer lesions from human and genetically engineered mouse (GEM) tissues. In vivo imaging was performed with [18F]V-1008, a TSPO-targeted PET agent, in two GEM models. For image-guided surgery (IGS), V-1520, a TSPO ligand for near-IR optical imaging based upon the V-1008 pharmacophore, was developed and evaluated. RESULTS: TSPO was highly expressed in human and murine pancreatic cancer. Notably, TSPO expression was associated with high-grade, premalignant intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanIN) lesions. In GEM models, [18F]V-1008 exhibited robust uptake in early pancreatic cancer, detectable by PET. Furthermore, V-1520 localized to premalignant pancreatic lesions and advanced tumors enabling real-time IGS. CONCLUSIONS: We anticipate that combined TSPO PET/IGS represents a translational approach for precision pancreatic cancer care through discrimination of high-risk indeterminate lesions and actionable surgery.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Colesterol/genética , Neoplasias Pancreáticas/genética , Lesiones Precancerosas/genética , Receptores de GABA/genética , Animales , Animales Modificados Genéticamente/genética , Carcinoma in Situ/diagnóstico por imagen , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Páncreas/diagnóstico por imagen , Páncreas/patología , Quiste Pancreático/diagnóstico por imagen , Quiste Pancreático/patología , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Lesiones Precancerosas/diagnóstico por imagen , Lesiones Precancerosas/patologíaRESUMEN
Tumor vascular endothelium is rather resistant to the cytotoxic effects of radiation. The HIV protease inhibitors (HPI) amprenavir, nelfinavir, and saquinavir have previously been shown to sensitize tumor cells to the cytotoxic effects of radiation. Additionally, this class of drug has been shown to inhibit angiogenesis and tumor cell migration. Therefore, in the current study, we wanted to determine whether HPIs could enhance the effect of radiation on endothelial function. Our study shows that HPIs, particularly nelfinavir, significantly enhance radiations effect on human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelium. We show that pretreatment of HUVEC with nelfinavir results in enhanced cytotoxicity, including increased apoptosis, when combined with radiation. Moreover, using several functional assays, we show that combination treatment effectively blocks endothelial cell migration and organization. These findings were accompanied by attenuation of Akt phosphorylation, a known pathway for radioresistance. Last, in vivo analysis of tumor microvasculature destruction showed a more than additive effect for nelfinavir and radiation. This study shows that HPIs can enhance the effect of ionizing radiation on vascular endothelium. Therefore, the Food and Drug Administration-approved drug, nelfinavir, may be an effective radiosensitizer in the clinic.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Inhibidores de la Proteasa del VIH/farmacología , Nelfinavir/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de la radiación , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Células Cultivadas , Células Endoteliales/citología , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
PURPOSE: SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR isoforms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied. METHODS AND MATERIALS: For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 microM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation-induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ionizing radiation, a clonogenic survival assay was performed using 0-6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies. RESULTS: SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Furthermore, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control. CONCLUSIONS: SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer.
Asunto(s)
Adenocarcinoma/fisiopatología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Indoles/administración & dosificación , Neoplasias Pancreáticas/fisiopatología , Pirroles/administración & dosificación , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Adenocarcinoma/radioterapia , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Neoplasias Pancreáticas/radioterapia , SunitinibRESUMEN
Histone deacetylases (HDAC) have been identified as therapeutic targets due to their regulatory function in DNA structure and organization. LBH589 is a novel inhibitor of class I and II HDACs. We studied the effect of LBH589 and ionizing radiation (IR) on DNA repair in two human non-small cell lung cancer (NSCLC) cell lines (H23 and H460). gamma-H2AX foci present at DNA double-strand breaks (DSBs) were detected in the nuclei following 3 Gy irradiation for up to 6 hours. LBH589 administered before irradiation increased the duration of gamma-H2AX foci beyond 24 hours. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm, indicating that HDAC inhibition affects the nuclear localization of HDAC4. The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR. Annexin-propidium iodide flow cytometry assays, cell morphology studies, and cleaved caspase-3 Western blot analysis revealed a synergistic effect of LBH589 with IR in inducing apoptosis. Clonogenic survival showed a greater than additive effect when LBH589 was administered before irradiation compared with irradiation alone. In vivo tumor volume studies showed a growth delay of 20 days with combined treatment compared with 4 and 2 days for radiation or LBH589 alone. This study identifies HDAC4 as a biomarker of LBH589 activity and recognizes the ability of LBH589 to sensitize human NSCLC to radiation-induced DNA DSBs.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Neoplasias Pulmonares/patología , Proteínas Represoras/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de la radiación , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/efectos de la radiación , Histona Acetiltransferasas/metabolismo , Histonas/genética , Humanos , Ácidos Hidroxámicos/farmacología , Immunoblotting , Indoles , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Ratones , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Panobinostat , Factores de Tiempo , Trasplante HeterólogoRESUMEN
Curative cancer treatment regimens often require cranial irradiation, resulting in lifelong neurocognitive deficiency in cancer survivors. This deficiency is in part related to radiation-induced apoptosis and decreased neurogenesis in the subgranular zone of the hippocampus. We show that lithium treatment protects irradiated hippocampal neurons from apoptosis and improves cognitive performance of irradiated mice. The molecular mechanism of this effect is mediated through multiple pathways, including Akt/glycogen synthase kinase-3beta (GSK-3beta) and Bcl-2/Bax. Lithium treatment of the cultured mouse hippocampal neurons HT-22 induced activation of Akt (1.5-fold), inhibition of GSK-3beta (2.2-fold), and an increase in Bcl-2 protein expression (2-fold). These effects were sustained when cells were treated with lithium in combination with ionizing radiation. In addition, this combined treatment led to decreased expression (40%) of the apoptotic protein Bax. The additional genes regulated by lithium were identified by microarray, such as decorin and Birc1f. In summary, we propose lithium treatment as a novel therapy for prevention of deleterious neurocognitive consequences of cranial irradiation.
Asunto(s)
Trastornos del Conocimiento/prevención & control , Irradiación Craneana/efectos adversos , Cloruro de Litio/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/fisiopatología , Relación Dosis-Respuesta en la Radiación , Femenino , Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipocampo/efectos de la radiación , Cloruro de Litio/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de la radiación , Fármacos Neuroprotectores/administración & dosificación , Embarazo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Glutamina/metabolismo , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Sistema de Transporte de Aminoácidos ASC/química , Sistema de Transporte de Aminoácidos ASC/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Modelos Animales de Enfermedad , Glutamina/química , Glutamina/genética , Células HCT116 , Humanos , Ratones , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The tumor microenvironment, in particular, the tumor vasculature, as an important target for the cytotoxic effects of radiation therapy is an established paradigm for cancer therapy. We review the evidence that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated in endothelial cells exposed to ionizing radiation (IR) and is a molecular target for the development of novel radiation sensitizing agents. On the basis of this premise, several promising preclinical studies that targeted the inhibition of the PI3K/Akt activation as a potential method of sensitizing the tumor vasculature to the cytotoxic effects of IR have been conducted. An innovative strategy to guide cytotoxic therapy in tumors treated with radiation and PI3K/Akt inhibitors is presented. The evidence supports a need for further investigation of combined-modality therapy that involves radiation therapy and inhibitors of PI3K/Akt pathway as a promising strategy for improving the treatment of patients with cancer.
Asunto(s)
Endotelio Vascular/efectos de la radiación , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/radioterapia , Neovascularización Patológica/radioterapia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de la Angiogénesis/farmacología , Animales , Fosfatidilinositol 3-Quinasa Clase I , Terapia Combinada , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Indoles/uso terapéutico , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/etiología , Oxindoles , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de la radiación , Propionatos , Pirroles/uso terapéutico , Quinazolinas/uso terapéutico , Tolerancia a Radiación/fisiología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Sunitinib , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
PURPOSE: Src family kinases (SFK) have been identified as molecular targets. SU6656 is a small-molecle indolinone that specifically inhibits this family of kinases. METHODS AND MATERIALS: Human umbilical vein endothelial cells were used to study the effects of SFK inhibition. Western blot analysis was performed to determine the effect of SFK inhibition on the PI3K/Akt pathway and caspase cleavage. Apoptosis was studied by propidium iodide staining of nuclei. Angiogenesis was examined using capillary tubule formation in Matrigel. Tumor response was further studied in vivo using Lewis lung carcinoma cells implanted into the dorsal skin fold of mice in the window model and in the hind limb in the tumor volume model. RESULTS: Clonogenic survival of endothelial cells was decreased after the combined therapy of SU6656 and radiation compared with radiotherapy alone. Furthermore, SFK inhibition by SU6656 attenuated radiation-induced Akt phosphorylation and increased radiation-induced apoptosis and vascular endothelium destruction. In vivo, SU6656 administered before irradiation significantly enhanced radiation-induced destruction of blood vessels within the tumor windows and enhanced tumor growth delay when administered during fractionated irradiation. CONCLUSIONS: This study demonstrates the potential use of SFK inhibition to enhance the effects of ionizing radiation during radiotherapy.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/efectos de la radiación , Indoles/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Sulfonamidas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Apoptosis , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/radioterapia , Caspasas/metabolismo , Recuento de Células , Fosfatidilinositol 3-Quinasa Clase I , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Endotelio Vascular/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Venas UmbilicalesRESUMEN
The phosphatidylinositol 3'-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The delta isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110delta specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110delta is a therapeutic target to enhance radiation-induced tumor control.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/radioterapia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinazolinas/farmacología , Animales , Dominio Catalítico , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/efectos de la radiación , Humanos , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Neovascularización Patológica/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [18F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [18F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [18F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [18F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.
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Neoplasias Encefálicas/patología , Glioma/patología , Tomografía de Emisión de Positrones/métodos , Receptores de GABA/metabolismo , Acetanilidas/farmacología , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Radiofármacos/farmacología , Ratas , Sensibilidad y Especificidad , Análisis de Matrices Tisulares/métodosRESUMEN
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
UNLABELLED: Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume. METHODS: Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). RESULTS: Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure. CONCLUSION: We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.
Asunto(s)
Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/terapia , Didesoxinucleósidos , Terapia Molecular Dirigida , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Radioisótopos de Flúor , Humanos , Indoles/uso terapéutico , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones , Valor Predictivo de las Pruebas , Inhibidores de Proteínas Quinasas/uso terapéutico , Radiofármacos , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.
Asunto(s)
Pirazoles/síntesis química , Pirimidinas/síntesis química , Radiofármacos/síntesis química , Receptores de GABA/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Radioisótopos de Flúor , Humanos , Tomografía de Emisión de Positrones/métodos , Ratas , Relación Estructura-ActividadRESUMEN
UNLABELLED: There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-(18)F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ((18)F-DPA-714), as a translational probe for quantification of TSPO levels in glioma. METHODS: Glioma-bearing rats were imaged with (18)F-DPA-714 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of (18)F-DPA-714 (130-200 MBq/0.2 mL). Blood was collected to derive the arterial input function (AIF), with high-performance liquid chromatography radiometabolite analysis performed on selected samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of (3)H-PK 11195 with DPA-714 in vitro and displacement of (18)F-DPA-714 with an excess of DPA-714 in vivo. Immediately after imaging, tumor and healthy brain tissues were harvested for validation by Western blotting and immunohistochemistry. RESULTS: (18)F-DPA-714 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced (18)F-DPA-714 binding by greater than 60% on average. Tumor uptake of (18)F-DPA-714 was similar to another high-affinity TSPO imaging ligand, (18)F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline, and agreed with ex vivo assay of TSPO levels in tumor and healthy brain. CONCLUSION: These studies illustrate the feasibility of using (18)F-DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, (18)F-DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of (18)F-DPA-714 PET to serve as a novel predictive cancer imaging modality.
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Neoplasias Encefálicas/diagnóstico por imagen , Proteínas Portadoras/metabolismo , Radioisótopos de Flúor , Regulación Neoplásica de la Expresión Génica , Glioma/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Pirazoles , Pirimidinas , Receptores de GABA-A/metabolismo , Acetanilidas/metabolismo , Animales , Transporte Biológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Estudios de Factibilidad , Glioma/genética , Glioma/metabolismo , Masculino , Pirazoles/metabolismo , Pirimidinas/metabolismo , RatasRESUMEN
UNLABELLED: Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. METHODS: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70-100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. RESULTS: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. CONCLUSION: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.
Asunto(s)
Acetanilidas , Proteínas Portadoras/análisis , Radioisótopos de Flúor , Glioma/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/metabolismo , Receptores de GABA-A/análisis , Acetanilidas/metabolismo , Animales , Línea Celular Tumoral , Isoquinolinas/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas WistarRESUMEN
BACKGROUND: Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment. CONCLUSIONS/SIGNIFICANCE: This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/patología , Neoplasias/radioterapia , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Unión Competitiva , Biomarcadores/química , Biomarcadores/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/inmunología , Espacio Intracelular/metabolismo , Espacio Intracelular/efectos de la radiación , Metiltransferasas , Ratones , Neoplasias/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Dominios PDZ , Transporte de Proteínas/efectos de la radiaciónRESUMEN
PURPOSE: Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. MATERIALS AND METHODS: For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. RESULTS: Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). CONCLUSION: The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.