RESUMEN
At diagnosis, 10 % of breast cancer patients already have locally advanced or metastatic disease; moreover, metastasis eventually develops in at least 40 % of early breast cancer patients. Osteolytic bone colonization occurs in 80-85 % of metastatic breast cancer patients and is thought to be an early step in metastatic progression. Thus, breast cancer displays a strong preference for metastasis to bone, and most metastatic breast cancer patients will experience its complications. Our prior research has shown that the α5ß1 integrin fibronectin receptor mediates both metastatic and angiogenic invasion. We invented a targeted peptide inhibitor of activated α5ß1, Ac-PHSCN-NH2 (PHSCN), as a validated lead compound to impede both metastatic invasion and neovascularization. Systemic PHSCN monotherapy prevented disease progression for up to 14 months in Phase I clinical trial. Here, we report that the next-generation construct, Ac-PhScN-NH2 (PhScN), which contains D-isomers of histidine (h) and cysteine (c), is greater than 100,000-fold more potent than PHSCN at blocking basement membrane invasion. Moreover, PhScN is also up to 10,000-fold more potent than PHSCN at inhibiting lung extravasation and colonization in athymic mice for both MDA-MB-231 metastatic and SUM149PT inflammatory breast cancer cells. Furthermore, we show that systemic treatment with 50 mg/kg PhScN monotherapy reduces established intratibial MDA-MB-231 bone colony progression by 80 %. Thus, PhScN is a highly potent, well-tolerated inhibitor of both lung colonization and bone colony progression.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Integrina alfa5beta1/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Animales , Antineoplásicos/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Oligopéptidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Primary tumors often give rise to disseminated tumor cells (DTC's), which acquire full malignancy after invading distant site(s). Thus, DTC's may be a productive target for preventing prostate cancer metastasis progression. Our prior research showed that PHSCN peptide (Ac-PHSCN-NH2) targets activated α5ß1 integrin to prevent invasion and metastasis in preclinical adenocarcinoma models, and disease progression in Phase I clinical trial. Here, we report that D-stereoisomer replacement of histidine and cysteine in PHSCN produces a highly potent derivative, Ac-PhScN-NH2 (PhScN). PhScN was 27,000- to 150,000-fold more potent as an inhibitor of basement membrane invasion by DU 145 and PC-3 prostate cancer cells. A large increase in invasion-inhibitory potency occurred after covalent modification of the sulfhydryl group in PHSCN to prevent disulfide bond formation; while the potency of covalently modified PhScN was not significantly increased. Thus PhScN and PHSCN invasion inhibition occurs by a noncovalent mechanism. These peptides also displayed similar cell surface binding dissociation constants (Kd), and competed for the same site. Consistent with its increased invasion-inhibitory potency, PhScN was also a highly potent inhibitor of lung extravasation and colonization in athymic nude mice: it was several hundred- or several thousand-fold more potent than PHSCN at blocking extravasation by PC-3 or DU 145 cells, and 111,000- or 379,000-fold more potent at inhibiting lung colonization, respectively. Furthermore, systemic 5 mg/kg PhScN monotherapy was sufficient to cause complete regression of established, intramuscular DU 145 tumors. PhScN thus represents a potent new family of therapeutic agents targeting metastasis by DTC's to prevent parallel progression in prostate cancer.
Asunto(s)
Adenocarcinoma/prevención & control , Aminoácidos/farmacología , Integrina alfa5beta1/antagonistas & inhibidores , Neoplasias Pulmonares/prevención & control , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/prevención & control , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RADIOTHERAPY IS USED IN THE MANAGEMENT OF PANCREATIC CANCER BECAUSE OF ITS HIGH PROPENSITY FOR LOCOREGIONAL RELAPSE: one third of patients succumb to localized disease. Thus, strategies to improve the efficacy of radiotherapy in pancreatic cancer are important to pursue. We used naturally serum-free, selectively permeable basement membranes and confocal microscopy of fluorescent antibody-stained human Panc-1, MiaPaCa-2, and BxPC-3 pancreatic cancer cell lines to investigate the effects of ionizing radiation on α(5)ß(1) integrin fibronectin receptor expression and on α(5)ß(1)-mediated invasion. We report that radiation rapidly induces pancreatic cancer cell invasion, and that radiation-induced invasion is caused by up-regulation of α(5)ß(1) integrin fibronectin receptors by transcriptional and/or postendocytic recycling mechanisms. We also report that radiation causes α(5)ß(1) up-regulation in Panc-1, MiaPaCa-2, and BxPC-3 tumor xenografts and that upregulated α(5)ß(1) colocalizes with upregulated early or late endosomes in Panc-1 or BxPC-3 tumors, respectively, although it may colocalize significantly with both endosome types in MiaPaCa-2 tumors. Our results suggest that systemic inhibition of α(5)ß(1)-mediated invasion might be an effective way to reduce radiation-induced pancreatic cancer cell invasion, thereby improving the efficacy of radiotherapy.
RESUMEN
The α5ß1 integrin fibronectin receptor is an attractive therapeutic target in breast cancer because it plays key roles in invasion and metastasis. While its inactive form is widely expressed, activated α5ß1 occurs only on tumor cells and their associated vasculature. The PHSCN peptide has been shown to bind activated α5ß1 preferentially, thereby blocking invasion in vitro, and inhibiting growth, metastasis and tumor recurrence in preclinical models. Moreover in a recent Phase I clinical trial, systemic PHSCN monotherapy was well tolerated, and metastatic disease failed to progress for 4-14 months in 38% of patients receiving it. A significantly more potent PHSCN derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP) has recently been developed. We report that it is 1280- to 6700-fold more potent than the PHSCN peptide at blocking α5ß1 mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion of naturally occurring basement membranes in vitro. Chou-Talalay analysis of these data suggested that invasion inhibition by the PHSCN dendrimer was highly synergistic. We also report that, consistent with its enhanced invasion-inhibitory potency, the PHSCN dendrimer is 700- to 1100-fold more effective than the PHSCN peptide at preventing SUM-149 PT and MDA-MB-231 extravasation in the lungs of athymic, nude mice. Our results also show that many extravasated SUM-149 PT and MDA-MB-231 cells go on to develop into metastatic colonies, and that pretreatment with the PHSCN dendrimer is more than 100-fold more effective at reducing lung colony formation. Since many patients newly diagnosed with breast cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy that can prevent metastatic progression by blocking invasion could be very beneficial.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Dendrímeros/farmacología , Integrina alfa5beta1/antagonistas & inhibidores , Neoplasias Pulmonares/secundario , Oligopéptidos/farmacología , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular , Dendrímeros/metabolismo , Dendrímeros/uso terapéutico , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Concentración 50 Inhibidora , Integrina alfa5beta1/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica/prevención & control , Oligopéptidos/uso terapéuticoRESUMEN
Activated alpha5beta1 integrin occurs specifically on tumor cells and on endothelial cells of tumor-associated vasculature, and plays a key role in invasion and metastasis. The PHSCN peptide (Ac-PHSCN-NH(2)) preferentially binds activated alpha5beta1, to block invasion in vitro, and inhibit growth, metastasis and tumor recurrence in preclinical models of prostate cancer. In Phase I clinical trial, systemic Ac-PHSCN-NH(2) monotherapy was well tolerated, and metastatic disease progression was prevented for 4-14 months in one-third of treated patients. We have developed a significantly more potent derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP). Using in vitro invasion assays with naturally serum-free basement membranes, we observed that the PHSCN dendrimer was 130- to 1900-fold more potent than the PHSCN peptide at blocking alpha5beta1-mediated invasion by DU 145 and PC-3 human prostate cancer cells, whether invasion was induced by serum, or by the Ac-PHSRN-NH(2) peptide, under serum-free conditions. The PHSCN dendrimer was also approximately 800 times more effective than PHSCN peptide at preventing DU 145 and PC-3 extravasation in the lungs of athymic mice. Chou-Talalay analysis suggested that inhibition of both invasion in vitro and extravasation in vivo by the PHSCN dendrimer are highly synergistic. We found that many extravasated DU 145 and PC-3 cells go onto develop into metastatic colonies, and that a single pretreatment with the PHSCN dendrimer was 100-fold more affective than the PHSCN peptide at reducing lung colony formation. Since many patients newly diagnosed with prostate cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy, like the PHSCN dendrimer, which prevents metastatic progression by inhibiting invasion, could be very beneficial.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Dendrímeros/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Oligopéptidos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Dendrímeros/metabolismo , Dendrímeros/farmacología , Progresión de la Enfermedad , Humanos , Concentración 50 Inhibidora , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
A current focus of proteomics research is the establishment of acceptable confidence measures in the assignment of protein identifications in an unknown sample. Development of new algorithmic approaches would greatly benefit from a standard reference set of spectra for known proteins for the purpose of testing and training. Here we describe an openly available library of mass spectra generated on an ABI 4700 MALDI TOF/TOF from 246 known, individually purified and trypsin-digested protein samples. The initial full release of the Aurum Dataset includes gel images, peak lists, spectra, search result files, decoy database analysis files, FASTA file of protein sequences, manual curation, and summary pages describing protein coverage and peptides matched by MS/MS followed by decoy database analysis using Mascot, Sequest, and X!Tandem. The data are publicly available for use at ProteomeCommons.org.
Asunto(s)
Mapeo Peptídico/métodos , Proteómica , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Bases de Datos Factuales , Humanos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Estándares de ReferenciaRESUMEN
Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Miopatías Estructurales Congénitas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Secuencia de Aminoácidos , Sitios de Unión , Enfermedad de Charcot-Marie-Tooth/genética , Cristalografía por Rayos X , Humanos , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación Missense , Miopatías Estructurales Congénitas/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras , Alineación de SecuenciaRESUMEN
Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M(r) electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.