RESUMEN
BACKGROUND: Flow diverters carry the risk of thromboembolic complications (TEC). We tested a coating with covalently bound heparin that activates antithrombin to address TEC by locally downregulating the coagulation cascade. We hypothesized that the neuroimaging evidence of TEC would be reduced by the coating. METHODS: 16 dogs were implanted with overlapping flow diverters in the basilar artery, separated into two groups: heparin-coated (n=9) and uncoated (n=7). Following implantation, high-frequency optical coherence tomography (HF-OCT) was acquired to quantify acute thrombus (AT) formation on the flow diverters. MRI was performed postoperatively and repeated at 1, 2, 3, 4, and 8 weeks, consisting of T1-weighted imaging, time-0f-flight (ToF), diffusion weighted imaging (DWI), susceptibility weighted imaging (SWI), and fluid attenuated inversion recovery (FLAIR) sequences. Neurological examinations were performed throughout the 8-week duration of the study. RESULTS: The mean AT volume on coated devices was lower than uncoated (0.014 vs 0.018 mm3); however, this was not significant (P=0.3). The mean number of foci of magnetic susceptibility artifacts (MSAs) on SWI was significantly different between the uncoated and coated groups at the 1-week follow-up (P<0.02), and remained statistically different throughout the duration of the study. The AT volume showed a direct linear correlation with the MSA count and 80% of the variance in the MSA could be explained by the AT volume (P<0.001). Pathological analysis showed evidence of ischemic injury at locations of MSA. CONCLUSIONS: Heparin-coated flow diverters significantly reduced the number of new MSAs after 1 week follow-up, showing the potential to reduce TEC.
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BACKGROUND: Neurointerventionalists use in-vitro vascular models to train for worst-case scenarios and test new devices in a simulated use environment to predict clinical performance. According to the Food and Drug Administration (FDA), any neurovascular navigation device should be able to successfully navigate two 360-degree turns and two 180-degree turns at the distal portion of the anatomical model. Here, we present a device benchmarking vascular model that complies with FDA recommendations. METHODS: Our vascular model was assembled from quantitative characterization of 49 patients who underwent CT angiography either for acute ischemic stroke caused by large vessel occlusion or for aneurysm treatment. Following complete characterization of these data, the vascular segments were 3D reconstructed from CT angiograms of 6 selected patients that presented with challenging anatomy. The curvature and total rotational angle were calculated for each segment and the anatomical parts that complied with FDA recommendations were fused together into a single in-vitro model. RESULTS: The model was constructed containing two common carotid branches arising from a type two aortic arch and the dimensions of the overall model exceeded the recommendations of the FDA. Two experienced neurointerventionalists tested the model for navigation difficulty using several devices on an in-vitro perfusion system and concluded that the model provided a realistic, challenging scenario. CONCLUSIONS: This model provides a first prototype designed according to FDA recommendations of cumulative angle while also integrating an aggregation of actual patient-specific anatomy. The availability of this clinically relevant benchmark model presents a potential standardized approach for neurovascular device testing.
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BACKGROUND: The first-pass complete recanalization by mechanical thrombectomy (MT) for the treatment of stroke remains limited due to the poor integration of the clot within current devices. Aspiration can help retrieval of the main clot but fails to prevent secondary embolism in the distal arterial territory. The dense meshes of extracellular DNA, recently described in stroke-related clots, might serve as an anchoring platform for MT devices. We aimed to evaluate the potential of a DNA-reacting surface to aid the retention of both the main clot and small fragments within the thrombectomy device to improve the potential of MT procedures. METHODS: Device-suitable alloy samples were coated with 15 different compounds and put in contact with extracellular DNA or with human peripheral whole blood, to compare their binding to DNA versus blood elements in vitro. Clinical-grade MT devices were coated with two selected compounds and evaluated in functional bench tests to study clot retrieval efficacy and quantify distal emboli using an M1 occlusion model. RESULTS: Binding properties of samples coated with all compounds were increased for DNA (≈3-fold) and decreased (≈5-fold) for blood elements, as compared with the bare alloy samples in vitro. Functional testing showed that surface modification with DNA-binding compounds improved clot retrieval and significantly reduced distal emboli during experimental MT of large vessel occlusion in a three-dimensional model. CONCLUSION: Our results suggest that clot retrieval devices coated with DNA-binding compounds can considerably improve the outcome of the MT procedures in stroke patients.
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Accidente Cerebrovascular , Trombosis , Humanos , Resultado del Tratamiento , Trombectomía/métodos , Trombosis/terapia , Accidente Cerebrovascular/cirugía , Aleaciones , ADNRESUMEN
BACKGROUND: High-frequency optical coherence tomography (HF-OCT) is an intravascular imaging method that allows for volumetric imaging of flow diverters in vivo. OBJECTIVE: To examine the hypothesis that a threshold for both volume and area of communicating malapposition can be predictive of early aneurysm occlusion. METHODS: Fifty-two rabbits underwent elastase aneurysm formation, followed by treatment with a flow diverter. At the time of implant, HF-OCT was acquired to study the rate and degree of communicating malapposition. Treated aneurysms were allowed to heal for either 90 or 180 days and euthanized following catheter angiography. Healing was dichotomized into aneurysm remnant or neck remnant/complete occlusion. Communicating malapposition was measured by HF-OCT using a semi-automatic algorithm able to detect any points where the flow diverter was more than 50 µm from the vessel wall. This was then summed across image slices to either a volume or area. Finally, a subsampled population was used to train a statistical classifier for the larger dataset. RESULTS: No difference in occlusion rate was found between device type or follow-up time (p=0.28 and p=0.67, respectively). Both volume and area of malapposition were significantly lower in aneurysms with a good outcome (p<0.001, both). From the statistical model, a volume of less than 0.56 mm3 or a normalized area less than 0.69 as quantified by HF-OCT was predictive of occlusion (p<0.001, each). CONCLUSIONS: HF-OCT allows for measurements of both volume and area of malapposition and, from these measurements, an accurate prediction for early aneurysm occlusion can be made.
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Procedimientos Endovasculares , Aneurisma Intracraneal , Animales , Conejos , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/terapia , Tomografía de Coherencia Óptica/métodos , Stents , Procedimientos Endovasculares/métodos , Angiografía Cerebral/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: High-frequency optical coherence tomography (HF-OCT) is an intra-vascular imaging technique capable of assessing device-vessel interactions at spatial resolution approaching 10 µm. We tested the hypothesis that adequately deployed Woven EndoBridge (WEB) devices as visualized by HF-OCT lead to higher aneurysm occlusion rates. METHODS: In a leporine model, elastase-induced aneurysms (n=24) were treated with the WEB device. HF-OCT and digital subtraction angiography (DSA) were performed following WEB deployment and repeated at 4, 8, and 12 weeks. Protrusion (0-present, 1-absent) and malapposition (0-malapposed, 1-neck apposition >50%) were binary coded. A device was considered 'adequately deployed' by HF-OCT and DSA if apposed and non-protruding. Aneurysm healing on DSA was reported using the 4-point WEB occlusion score: A or B grades were considered positive outcome. Neointimal coverage was quantified on HF-OCT images at 12 weeks and compared with scanning electron microscopy (SEM). RESULTS: Adequate deployment on HF-OCT correlated with positive outcome (P=0.007), but no statistically significant relationship was found between good outcome and adequate deployment on DSA (P=0.289). Absence of protrusion on HF-OCT correlated with a positive outcome (P=0.006); however, malapposition alone had no significant relationship (P=0.19). HF-OCT showed a strong correlation with SEM for the assessment of areas of neointimal tissue (R²=0.96; P<0.001). More neointimal coverage of 78%±32% was found on 'adequate deployment' cases versus 31%±24% for the 'inadequate deployment' cases (P=0.001). CONCLUSION: HF-OCT visualizes features that can determine adequate device deployment to prognosticate early aneurysm occlusion following WEB implantation and can be used to longitudinally monitor aneurysm healing progression.
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Angiografía de Substracción Digital/métodos , Embolización Terapéutica/métodos , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/terapia , Stents Metálicos Autoexpandibles , Tomografía de Coherencia Óptica/métodos , Animales , Embolización Terapéutica/instrumentación , Aneurisma Intracraneal/inducido químicamente , Masculino , Elastasa Pancreática/toxicidad , Conejos , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate in situ decellularization of a large animal model of saccular aneurysm as a strategy for achieving aneurysmal growth and lasting inflammation. METHODS: 18 New Zealand White rabbits were randomized 2:1 to receive endoluminal sodium dodecyl sulfate infusion (SDS, 1% solution, 45 min) following elastase or elastase-only treatment (control). All aneurysms were measured by digital subtraction angiography every 2 weeks. Every 2 weeks, three of the rabbits (two elastase + SDS, one control) underwent MRI, followed by contrast injection with myeloperoxidase (MPO)-sensing contrast agent. MRI was repeated 3 hours after contrast injection and the enhancement ratio (ER) was calculated. Following MRI, aneurysms were explanted and subjected to immunohistopathology. RESULTS: During follow-up MRI, the average ER for SDS-treated animals was 1.63±0.20, compared with 1.01±0.06 for controls (p<0.001). The width of SDS-treated aneurysms increased significantly in comparison with the elastase aneurysms (47% vs 20%, p<0.001). Image analysis of thin sections showed infiltration of MPO-positive cells in decellularized aneurysms and surroundings through the 12-week observation period while control tissue had 5-6 times fewer cells present 2 weeks after aneurysm creation. Immunohistochemistry demonstrated the presence of MPO-positive cells surrounding decellularized lesions at early time points. MPO-positive cells were found in the adventitia and in the thrombi adherent to the aneurysm wall at later time points. CONCLUSIONS: In situ decellularization of a large animal model of saccular aneurysms reproduces features of unstable aneurysms, such as chronic inflammation (up to 12 weeks) and active aneurysm wall remodeling, leading to continued growth over 8 weeks.
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Aneurisma/diagnóstico por imagen , Modelos Animales de Enfermedad , Endotelio Vascular/diagnóstico por imagen , Remodelación Vascular/fisiología , Aneurisma/patología , Angiografía de Substracción Digital/métodos , Animales , Endotelio Vascular/patología , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Inflamación/diagnóstico por imagen , Inflamación/patología , Imagen por Resonancia Magnética/métodos , Masculino , Conejos , Distribución AleatoriaRESUMEN
PURPOSE: The implantation of flow diverters requires administration of dual anti-platelet therapy, posing the potential for complications. The p48MW HPC (phenox, Bochüm, Germany) hydrophilic-coated flow diverting stent is designed to be anti-thrombotic, thus opening the potential for single anti-platelet therapy. We deploy a novel intravascular high-resolution imaging technique, high-frequency optical coherence tomography (HF-OCT), to study in an animal model the acute thrombus formation on coated p48MW devices versus uncoated control devices. METHODS: Three pigs were implanted with 4 flow diverters each, two test hydrophilic-coated devices, and two control uncoated devices (p48MW). Each pig was treated with a different anti-platelet regime: no anti-platelet therapy, aspirin only, aspirin and clopidogrel. Twenty minutes after the flow diverter was implanted, an HF-OCT data set was acquired. Acute clot formed on the flow diverter at each covered side branch was measured from the HF-OCT slices. Factors considered to be important were the device type (pHPC versus bare metal), aspirin, clopidogrel, and vessel location. A linear model was constructed from the significant factors. RESULTS: Both coating (p < 0.001) and aspirin (p = 0.003) were significantly related to reduction in clot burden, leading to an approximate 100-fold and 50-fold reduction in clot, respectively. CONCLUSIONS: This study shows the power of HF-OCT not only in the detection of clot but also the quantification of clot burden. In an animal model, the pHPC-coated p48MW significantly reduced acute thrombus formation over jailed side branches as compared to the bare metal p48MW that was nearly eliminated when combined with aspirin administration.
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Aspirina/uso terapéutico , Clopidogrel/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Stents , Trombosis/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Enfermedad Aguda , Animales , Materiales Biocompatibles Revestidos , Modelos Animales de Enfermedad , Porcinos , Trombosis/prevención & controlRESUMEN
The small GTPase Ran orchestrates pleiotropic cellular responses of nucleo-cytoplasmic shuttling, mitosis and subcellular trafficking, but whether deregulation of these pathways contributes to disease pathogenesis has remained elusive. Here, we generated transgenic mice expressing wild type (WT) Ran, loss-of-function Ran T24N mutant or constitutively active Ran G19V mutant in pancreatic islet ß cells under the control of the rat insulin promoter. Embryonic pancreas and islet development, including emergence of insulin(+) ß cells, was indistinguishable in control or transgenic mice. However, by one month after birth, transgenic mice expressing any of the three Ran variants exhibited overt diabetes, with hyperglycemia, reduced insulin production, and nearly complete loss of islet number and islet mass, in vivo. Deregulated Ran signaling in transgenic mice, adenoviral over-expression of WT or mutant Ran in isolated islets, or short hairpin RNA (shRNA) silencing of endogenous Ran in model insulinoma INS-1 cells, all resulted in decreased expression of the pancreatic and duodenal homeobox transcription factor, PDX-1, and reduced ß cell proliferation, in vivo. These data demonstrate that a finely-tuned balance of Ran GTPase signaling is essential for postnatal pancreatic islet development and glucose homeostasis, in vivo.
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Diabetes Mellitus/etiología , Embrión de Mamíferos/citología , Hiperglucemia/etiología , Hiperinsulinismo/etiología , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Proteína de Unión al GTP ran/fisiología , Animales , Células Cultivadas , ADN/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/metabolismo , Hiperglucemia/patología , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Técnicas para Inmunoenzimas , Insulina/sangre , Insulina/genética , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Ratas , Proteína de Unión al GTP ran/antagonistas & inhibidoresRESUMEN
Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.
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Mitocondrias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Respuesta de Proteína Desplegada , Animales , Apoptosis , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Guanidinas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Estrés Fisiológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
PURPOSE: This study aimed to characterize the preclinical activity of the first class of combinatorial, mitochondria-targeted, small molecule heat shock protein-90 (Hsp90) inhibitors, gamitrinibs, in models of hormone-refractory, drug-resistant, localized, and bone metastatic prostate cancer in vivo. EXPERIMENTAL DESIGN: Mitochondrial permeability transition, apoptosis, and changes in metabolic activity were examined by time-lapse videomicroscopy, multiparametric flow cytometry, MTT, and analysis of isolated mitochondria. Drug-resistant prostate cancer cells were generated by chronic exposure of hormone-refractory PC3 cells to the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG). The effect of gamitrinibs on s.c. or intratibial prostate cancer growth was studied in xenograft models. Bone metastatic tumor growth and bone parameters were quantified by micro-computed tomography imaging. RESULTS: In the NCI 60-cell line screening, gamitrinibs were active against all tumor cell types tested, and efficiently killed metastatic, hormone-refractory, and multidrug-resistant prostate cancer cells characterized by overexpression of the ATP binding cassette transporter P-glycoprotein. Mechanistically, gamitrinibs, but not 17-AAG, induced acute mitochondrial dysfunction in prostate cancer cells with loss of organelle membrane potential, release of cytochrome c, and caspase activity, independently of proapoptotic Bcl-2 proteins Bax and Bak. Systemic administration of gamitrinibs to mice was well tolerated, and inhibited s.c. or bone metastatic prostate cancer growth in vivo. CONCLUSIONS: Gamitrinibs have preclinical activity and favorable safety in models of drug-resistant and bone metastatic prostate cancer in vivo.
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Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas/farmacología , Mitocondrias/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Pirimidinas/farmacología , Quinonas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas/síntesis química , Lactamas/química , Lactamas Macrocíclicas/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones SCID , Peso Molecular , Neoplasias de la Próstata/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Quinonas/síntesis química , Quinonas/química , Células Tumorales Cultivadas , Microtomografía por Rayos XRESUMEN
Despite conventional treatment strategies glioblastoma, the most common malignant primary brain tumor, has a bad prognosis with median survival times of 12-15 months. In this study, the efficacy of sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, on glioblastoma cells was evaluated both in vitro and in vivo. Treatment of established or patient-derived glioblastoma cells with low concentrations of sorafenib caused a dramatic dose dependent inhibition of proliferation (IC(50), 1.5 microM) and induction of apoptosis and autophagy. Sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (Stat3) and expression of cyclins, D and E. In contrast, AKT was not modulated by sorafenib. Most important, systemic delivery of sorafenib was well tolerated, and significantly suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis and autophagy, and reduction of angiogenesis. Furthermore, intracranial growth inhibition by sorafenib was accompanied by a significant reduction in ph-Stat3 (Tyr 705) levels. In summary, sorafenib has potent anti-glioma activity in vitro and in vivo.
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Antineoplásicos/farmacología , Bencenosulfonatos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Piridinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Bencenosulfonatos/administración & dosificación , Bencenosulfonatos/efectos adversos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/administración & dosificación , Piridinas/efectos adversos , Distribución Aleatoria , Sorafenib , Resultado del TratamientoRESUMEN
Drug discovery for complex and heterogeneous tumors now aims at dismantling global networks of disease maintenance, but the subcellular requirements of this approach are not understood. Here, we simultaneously targeted the multiple subcellular compartments of the molecular chaperone heat shock protein-90 (Hsp90) in a model of glioblastoma, a highly lethal human malignancy in urgent need of fresh therapeutic strategies. Treatment of cultured or patient-derived glioblastoma cells with Shepherdin, a dual peptidomimetic inhibitor of mitochondrial and cytosolic Hsp90, caused irreversible collapse of mitochondria, degradation of Hsp90 client proteins in the cytosol, and tumor cell killing by apoptosis and autophagy. Stereotactic or systemic delivery of Shepherdin was well tolerated and suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis, and reduction of angiogenesis in vivo. These data show that disabling Hsp90 cancer networks in their multiple subcellular compartments improves strategies for drug discovery and may provide novel molecular therapy for highly recalcitrant human tumors.
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Glioblastoma/metabolismo , Glioblastoma/terapia , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Adulto , Anciano , Animales , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Peptidil-Prolil Isomerasa F , Ciclofilinas/metabolismo , Femenino , Glioblastoma/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Fragmentos de Péptidos/farmacología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.
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Proteínas Inhibidoras de la Apoptosis/metabolismo , Invasividad Neoplásica , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Interferente Pequeño , SurvivinRESUMEN
Despite progress in the management of breast cancer, the molecular underpinnings of clinically aggressive subtypes of the disease are not well-understood. Here, we show that activation of Notch developmental signaling in estrogen receptor (ER)-negative breast cancer cells results in direct transcriptional up-regulation of the apoptosis inhibitor and cell cycle regulator survivin. This response is associated with increased expression of survivin at mitosis, enhanced cell proliferation, and heightened viability at cell division. Conversely, targeting Notch signaling with a peptidyl gamma-secretase inhibitor suppressed survivin levels, induced apoptosis, abolished colony formation in soft agar, and inhibited localized and metastatic tumor growth in mice, without organ or systemic toxicity. In contrast, ER+ breast cancer cells, or various normal cell types, were insensitive to Notch stimulation. Therefore, ER- breast cancer cells become dependent on Notch-survivin signaling for their maintenance, in vivo. Therapeutic targeting of this pathway may be explored for individualized treatment of patients with clinically aggressive, ER- breast cancer.
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Adenocarcinoma/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Neoplasias/fisiología , Receptores Notch/fisiología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis , Ratones , Ratones SCID , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/fisiología , Receptores Notch/genética , Transducción de Señal/genética , Survivin , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor cells often become resistant to DNA damage-based therapy; however, the underlying mechanisms are not yet understood. Here, we show that tumor cells exposed to DNA damage counteract cell death by releasing the antiapoptotic protein, survivin, from mitochondria. This is independent of p53, and requires activated checkpoint kinase 2 (Chk2), a putative tumor suppressor. Molecular or genetic targeting of Chk2 prevents the release of survivin from mitochondria, enhances DNA damage-induced tumor cell apoptosis, and inhibits the growth of resistant in vivo tumors. Therefore, activated Chk2 circumvents its own tumor-suppressive functions by promoting tumor cell survival. Inhibiting Chk2 in combination with DNA-damaging agents may provide a rational approach for treating resistant tumors.
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Supervivencia Celular/fisiología , Daño del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Adenocarcinoma , Animales , Apoptosis , Neoplasias de la Mama , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Neoplasias del Colon , Activación Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Transfección , Trasplante HeterólogoRESUMEN
BACKGROUND: Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in signaling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of Hsp90 are being examined as cancer therapeutic agents, but the molecular mechanism of their anticancer activity is still unclear. We investigated Hsp90 as a therapeutic target for acute myeloid leukemia (AML) by use of the Hsp90 inhibitor shepherdin (a novel peptidyl antagonist of the interaction between Hsp90 and survivin, which is a regulator of cell proliferation and cell viability in cancer). METHODS: We studied protein interactions by molecular dynamics simulations and conducted competition experiments by use of enzyme-linked immunosorbent assay (ELISA). Shepherdin[79-83], a novel variant carrying the survivin sequence from Lys-79 through Gly-83, or its scrambled peptide was made permeable to cells by adding the antennapedia helix III carrier sequence. Apoptosis, Hsp90 client protein expression, and mitochondrial dysfunction were evaluated in AML types (myeloblastic, monocytic, and chronic myelogenous leukemia in blast crisis), patient-derived blasts, and normal mononuclear cells. Effects of shepherdin on tumor growth were evaluated in AML xenograft tumors in mice (n = 6). Organ tissues were examined histologically. RESULTS: Shepherdin[79-83] bound to Hsp90, inhibited formation of the survivin-Hsp90 complex, and competed with ATP binding to Hsp90. Cell-permeable shepherdin[79-83] induced rapid (within 30 minutes) and complete (with concentrations inducing 50% cell death of 24-35 microM) killing of AML types and blasts, but it did not affect normal mononuclear cells. Shepherdin[79-83] made contact with unique residues in the ATP pocket of Hsp90 (Ile-96, Asp-102, and Phe-138), did not increase Hsp70 levels in AML cells, disrupted mitochondrial function within 2 minutes of treatment, and eliminated the expression of Hsp90 client proteins. Shepherdin[79-83] abolished growth of AML xenograft tumors (mean of control group = 1698 mm3 and mean of treated group = 232 mm3; difference = 1466 mm3, 95% confidence interval = 505.8 to 2426; P = .008) without systemic or organ toxicity and inhibited Hsp90 function in vivo. CONCLUSIONS: Shepherdin is a novel Hsp90 inhibitor with a unique mechanism of anticancer activity.