Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
J Biomol Screen ; 18(10): 1246-59, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24108119

ABSTRACT

Phenotypic screening seeks to identify substances that modulate phenotypes in a desired manner with the aim of progressing first-in-class agents. Successful campaigns require physiological relevance, robust screening, and an ability to deconvolute perturbed pathways. High-content analysis (HCA) is increasingly used in cell biology and offers one approach to prosecution of phenotypic screens, but challenges exist in exploitation where data generated are high volume and complex. We combine development of an organotypic model with novel HCA tools to map phenotypic responses to pharmacological perturbations. We describe implementation for angiogenesis, a process that has long been a focus for therapeutic intervention but has lacked robust models that recapitulate more completely mechanisms involved. The study used human primary endothelial cells in co-culture with stromal fibroblasts to model multiple aspects of angiogenic signaling: cell interactions, proliferation, migration, and differentiation. Multiple quantitative descriptors were derived from automated microscopy using custom-designed algorithms. Data were extracted using a bespoke informatics platform that integrates processing, statistics, and feature display into a streamlined workflow for building and interrogating fingerprints. Ninety compounds were characterized, defining mode of action by phenotype. Our approach for assessing phenotypic outcomes in complex assay models is robust and capable of supporting a range of phenotypic screens at scale.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Drug Evaluation, Preclinical/methods , Cells, Cultured , Cluster Analysis , Coculture Techniques , High-Throughput Screening Assays , Human Umbilical Vein Endothelial Cells , Humans , Multivariate Analysis , Neovascularization, Pathologic/drug therapy , Phenotype
2.
Mol Cancer Ther ; 12(9): 1715-27, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23861347

ABSTRACT

Continued androgen receptor (AR) expression and signaling is a key driver in castration-resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed, and therefore remains a target for the treatment of progressive disease. Here, we describe the biological characterization of AZD3514, an orally bioavailable drug that inhibits androgen-dependent and -independent AR signaling. AZD3514 modulates AR signaling through two distinct mechanisms, an inhibition of ligand-driven nuclear translocation of AR and a downregulation of receptor levels, both of which were observed in vitro and in vivo. AZD3514 inhibited testosterone-driven seminal vesicle development in juvenile male rats and the growth of androgen-dependent Dunning R3327H prostate tumors in adult rats. Furthermore, this class of compound showed antitumor activity in the HID28 mouse model of CRPC in vivo. AZD3514 is currently in phase I clinical evaluation.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Antineoplastic Agents/pharmacology , Prostatic Neoplasms, Castration-Resistant/pathology , Pyridazines/pharmacology , Receptors, Androgen/metabolism , Seminal Vesicles/drug effects , Abiraterone Acetate , Androgen Receptor Antagonists/metabolism , Androstadienes/pharmacology , Animals , Antineoplastic Agents/metabolism , Benzamides , Cell Line, Tumor , Disease Models, Animal , Down-Regulation , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Male , Mice , Mice, Nude , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Pyridazines/chemical synthesis , Pyridazines/metabolism , Rats , Rats, Wistar , Receptors, Androgen/genetics , Seminal Vesicles/growth & development , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
3.
Int J Oncol ; 39(1): 271-8, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21537841

ABSTRACT

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Piperidines/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Endothelial Cells/drug effects , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, SCID , Neoplasms/physiopathology , Phenotype , Vascular Endothelial Growth Factor Receptor-2/metabolism , Xenograft Model Antitumor Assays
4.
Mol Cancer Ther ; 10(5): 861-73, 2011 May.
Article in English | MEDLINE | ID: mdl-21441409

ABSTRACT

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-ß. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-ß and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-ß phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-ß was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-ß.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Animals , COS Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Chlorocebus aethiops , HEK293 Cells , Humans , Ligands , Lung/drug effects , Mice , Mice, Nude , NIH 3T3 Cells , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Quinazolines/chemistry , Rats , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Stem Cell Factor/metabolism , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/antagonists & inhibitors
5.
Angiogenesis ; 13(4): 337-47, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20953695

ABSTRACT

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.


Subject(s)
Antibodies, Neutralizing/pharmacology , Neovascularization, Pathologic/prevention & control , Pregnancy Proteins/immunology , Angiogenesis Inhibitors/pharmacology , Capillaries/drug effects , Capillaries/growth & development , Capillaries/metabolism , Cells, Cultured , Coculture Techniques , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Endothelial Cells/drug effects , Endothelial Cells/physiology , Humans , Models, Theoretical , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Organ Culture Techniques , Placenta Growth Factor , Pregnancy Proteins/antagonists & inhibitors , Pregnancy Proteins/pharmacology , Protein Multimerization/drug effects , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factors/metabolism
6.
Cancer Res ; 65(10): 4389-400, 2005 May 15.
Article in English | MEDLINE | ID: mdl-15899831

ABSTRACT

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a promising therapeutic approach that aims to stabilize the progression of solid malignancies by abrogating tumor-induced angiogenesis. This may be accomplished by inhibiting the kinase activity of VEGF receptor-2 (KDR), which has a key role in mediating VEGF-induced responses. The novel indole-ether quinazoline AZD2171 is a highly potent (IC50 < 1 nmol/L) ATP-competitive inhibitor of recombinant KDR tyrosine kinase in vitro. Concordant with this activity, in human umbilical vein endothelial cells, AZD2171 inhibited VEGF-stimulated proliferation and KDR phosphorylation with IC50 values of 0.4 and 0.5 nmol/L, respectively. In a fibroblast/endothelial cell coculture model of vessel sprouting, AZD2171 also reduced vessel area, length, and branching at subnanomolar concentrations. Once-daily oral administration of AZD2171 ablated experimental (VEGF-induced) angiogenesis in vivo and inhibited endochondral ossification in bone or corpora luteal development in ovary; physiologic processes that are highly dependent upon neovascularization. The growth of established human tumor xenografts (colon, lung, prostate, breast, and ovary) in athymic mice was inhibited dose-dependently by AZD2171, with chronic administration of 1.5 mg per kg per day producing statistically significant inhibition in all models. A histologic analysis of Calu-6 lung tumors treated with AZD2171 revealed a reduction in microvessel density within 52 hours that became progressively greater with the duration of treatment. These changes are indicative of vascular regression within tumors. Collectively, the data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. AZD2171 is being developed clinically as a once-daily oral therapy for the treatment of cancer.


Subject(s)
Neoplasms/drug therapy , Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Bone Development/drug effects , Cell Proliferation/drug effects , Corpus Luteum/drug effects , Corpus Luteum/growth & development , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Extracellular Matrix Proteins , Female , Humans , Mice , Myosin Heavy Chains , Neoplasms/blood supply , Neoplasms/pathology , Nonmuscle Myosin Type IIB , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacokinetics , Proteins/antagonists & inhibitors , Quinazolines/pharmacokinetics , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Xenograft Model Antitumor Assays
SELECTION OF CITATIONS
SEARCH DETAIL
...