RESUMEN
Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.
Asunto(s)
Antígenos B7/genética , Antígenos B7/metabolismo , Inmunoconjugados/farmacología , Neoplasias/irrigación sanguínea , Animales , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Antígenos B7/inmunología , Benzodiazepinas/farmacología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Línea Celular Tumoral , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Inmunoconjugados/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Neoplasias/patología , Neoplasias/terapia , Oligopéptidos/farmacología , Pirroles/farmacología , ConejosRESUMEN
1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of (3) H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the ß-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the ß-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - ß-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound.
Asunto(s)
Antineoplásicos/farmacología , ARN Helicasas DEAD-box/metabolismo , Neoplasias/tratamiento farmacológico , Piperazinas/farmacología , Quinoxalinas/farmacología , beta Catenina/metabolismo , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , ARN Helicasas DEAD-box/química , Humanos , Neoplasias/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Citidina/análogos & derivados , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Administración Oral , Animales , Línea Celular Tumoral , Citidina/administración & dosificación , Citidina/uso terapéutico , Desoxicitidina/administración & dosificación , Desoxicitidina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.
Asunto(s)
Pulmón/efectos de los fármacos , Surfactantes Pulmonares/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Every organ in the body requires blood vessels for efficient delivery of oxygen and nutrients, but independent vascular beds are highly specialized to meet the individual needs of specific organs. The vasculature of the brain is tightly sealed, with blood-brain barrier (BBB) properties developing coincident with neural vascularization. G protein-coupled receptor 124 (GPR124) (tumor endothelial marker 5, TEM5), an orphan member of the adhesion family of G protein-coupled receptors, was previously identified on the basis of its overexpression in tumor vasculature. Here, we show that global deletion or endothelial-specific deletion of GPR124 in mice results in embryonic lethality associated with abnormal angiogenesis of the forebrain and spinal cord. Expression of GPR124 was found to be required for invasion and migration of blood vessels into neuroepithelium, establishment of BBB properties, and expansion of the cerebral cortex. Thus, GPR124 is an important regulator of neurovasculature development and a potential drug target for cerebrovascular diseases.
Asunto(s)
Barrera Hematoencefálica/embriología , Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/embriología , Embrión de Mamíferos/irrigación sanguínea , Receptores Acoplados a Proteínas G/fisiología , Animales , Barrera Hematoencefálica/metabolismo , Western Blotting , Cartilla de ADN/genética , Embrión de Mamíferos/metabolismo , Citometría de Flujo , Técnicas Histológicas , Hibridación in Situ , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5's ability to distinguish different forms of cell surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding protein, as intracellular factors able to alter TEM8 cell surface structure. Overexpression of either of these proteins in cells converted TEM8 from an SB5-exposed to an SB5-masked form and protected cells from SB5-saporin immunotoxins. Because the predominant form of TEM8 on the cell surface is not recognized by SB5, we also developed a new monoclonal antibody, called AF334, which is able to recognize both the SB5-exposed and the SB5-masked forms of TEM8. AF334-saporin selectively killed TEM8-positive cells independent of TEM8 cell surface structure. These studies reveal that TEM8 exists in different forms at the cell surface, a structure dependent on interactions with components of the actin cytoskeleton, and should aid in the rational design of the most effective diagnostic and therapeutic anti-TEM8 monoclonal antibodies.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Anticuerpos Monoclonales/inmunología , Western Blotting , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Separación Inmunomagnética , Inmunoprecipitación , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tumor endothelial marker 8 (TEM8) was initially identified as a gene overexpressed in the vasculature of human tumors and was subsequently identified as an anthrax toxin receptor. To assess the functional role of TEM8, we disrupted the TEM8 gene in mice by targeted homologous recombination. TEM8(-/-) mice were viable and reached adulthood without defects in physiologic angiogenesis. However, histopathologic analysis revealed an excess of extracellular matrix in several tissues, including the ovaries, uterus, skin, and periodontal ligament of the incisors, the latter resulting in dental dysplasia. When challenged with B16 melanoma, tumor growth was delayed in TEM8(-/-) mice, whereas the growth of other tumors, such as Lewis lung carcinoma, was unaltered. These studies show that host-derived TEM8 promotes the growth of certain tumors and suggest that TEM8 antagonists may have utility in the development of new anticancer therapies.
Asunto(s)
Melanoma Experimental/patología , Receptores de Péptidos/fisiología , Animales , Biomarcadores de Tumor , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Femenino , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Neovascularización Patológica/patología , Receptores de Superficie Celular , Receptores de Péptidos/biosíntesis , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genéticaRESUMEN
To unravel the normal vasculature transcriptome and determine how it is altered by neighboring malignant cells, we compared gene expression patterns of endothelial cells derived from the blood vessels of eight normal resting tissues, five tumors, and regenerating liver. Organ-specific endothelial genes were readily identified, including 27 from brain. We also identified 25 transcripts overexpressed in tumor versus normal endothelium, including 13 that were not found in the angiogenic endothelium of regenerating liver. Most of the shared angiogenesis genes have expected roles in cell-cycle control, but those specific for tumor endothelium were primarily cell surface molecules of uncertain function. These studies reveal striking differences between physiological and pathological angiogenesis potentially important for the development of tumor-specific, vascular-targeted therapies.
Asunto(s)
Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Animales , Antígenos CD/metabolismo , Antígenos B7 , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Endotelio Vascular/metabolismo , Perfilación de la Expresión Génica , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Regeneración Hepática , Ratones , Especificidad de Órganos , Receptores Inmunológicos/metabolismoRESUMEN
An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8-100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/diagnóstico , Proteínas no Estructurales Virales/inmunología , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Antivirales/sangre , Bovinos , Brotes de Enfermedades , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Fiebre Aftosa/epidemiología , Fiebre Aftosa/prevención & control , Fiebre Aftosa/virología , Cabras , Mongolia/epidemiología , Sensibilidad y Especificidad , Ovinos , Porcinos , Vacunación/veterinaria , Proteínas no Estructurales Virales/química , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
The infiltration of both monocyte and activated T cells in the skin is one of critical steps in the development of UVB-induced inflammation. Upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) on the surface of keratinocytes plays an important role in this process. In this study, we examined the molecular mechanism responsible for UVB-induced expression of ICAM-1 and subsequent monocyte adhesion by keratinocyte. We observed that (1) UVB induced protein and mRNA expression of ICAM-1 in a dose- and time-dependent manner in human keratinocyte cell HaCaT; (2) UVB induced the translocation of NF-kappaB and inhibition of NF-kappaB by NF-kappaB inhibitors suppressed UVB-induced mRNA and protein expression of ICAM-1; (3) UVB increased the intracellular level of reactive oxygen species (ROS) by HaCaT cells; (4) UVB-induced increase of intracellular ROS level was suppressed by pretreatment with diphenyl iodonium (DPI) and N-acetyl cysteine (NAC); and (5) inhibition of UVB-induced ROS production by DPI or NAC suppressed UVB-mediated translocation of NF-kappaB, expression of ICAM-1 and subsequent monocyte adhesion in HaCaT cells. These results suggest that UVB-induced ROS is involved in the translocation of NF-kappaB which is responsible for expression of ICAM-1 and subsequent increased monocyte adhesion in human keratinocyte.
Asunto(s)
Expresión Génica/efectos de la radiación , Queratinocitos/fisiología , Monocitos/fisiología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rayos Ultravioleta , Adhesión Celular/efectos de la radiación , Línea Celular Tumoral , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Monocitos/inmunología , Transducción de Señal , Piel/inmunología , Linfocitos T/fisiologíaRESUMEN
2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. However, the mechanisms by which TGHQ induces apoptosis are unclear, and they were the focus of the present investigation. TGHQ stimulated the rapid formation (30 min) of reactive oxygen species (ROS) in HL-60 cells, and co-treatment with catalase or the antioxidant N-acetylcysteine (NAC) completely blocked TGHQ-induced apoptosis, implicating a causative role for ROS in HL-60 cell death. Western blot analysis revealed the complete disappearance of pro-caspase 9 between 1 and 2 hours after exposure of HL-60 cells to TGHQ, concomitant with the appearance of cleaved caspase 9 and increases in caspase 9 activity. The appearance of two cleaved forms of caspase 3 occurred subsequent to increases in caspase 9 activity. Levels of the anti-apoptotic Bcl-2 protein remained constant during TGHQ-induced apoptosis of HL-60 cells, but Bcl-2 S70 phosphorylation decreased. In contrast, changes in the subcellular localization of the pro-apoptotic molecule Bax were observed, with a rapid (15-60 min) increase in the ratio of cytosolic to mitochondrial Bax. Cytochrome c release from mitochondria to the cytosol occurred after Bax translocation and the dephosphorylation of pS70 Bcl-2. However the mitochondrial inner transmembrane potential (deltapsi(m)) was maintained, even after cytochrome c was released from the mitochondria. Cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition pore (PTP), did not completely rescue HL-60 cells from apoptosis. Taken together, we conclude that TGHQ facilitates ROS production, alters the post-translational modification of Bcl-2 and subcellular localization of Bax, culminating in the release of cytochrome c and caspase activation.