@article {2396, title = {Binding and Uptake of RGD-Containing Ligands to Cellular alpha(v)beta(3) Integrins}, journal = {International Journal of Peptide Research and Therapeutics}, volume = {15}, number = {1}, year = {2009}, note = {ISI Document Delivery No.: 430WTTimes Cited: 2Cited Reference Count: 34Cressman, Sonya Sun, Ying Maxwell, E. Jane Fang, Ning Chen, David D. Y. Cullis, Pieter R.}, month = {Mar}, pages = {49-59}, type = {Article}, abstract = {The cyclic peptide, cRGDf[N(me)]V, binds to the alpha(v)beta(3) integrin and can disrupt binding of the integrin to its natural ligands in the extracellular matrix. In this work, the ability of a water-soluble, fluorescently labeled variant of the RGD-containing peptide (cRGDfK-488) to bind to integrins on human umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis was characterized. This information was compared to the binding and uptake properties of an alpha(v)beta(3) integrin-specific monoclonal antibody, LM609X. The specificity of the RGD-containing peptide is assessed by comparison with control peptide that does not bind to the alpha(v)beta(3) integrin, cRADfK-488. Using a high purity construct, it is shown that the RGD ligand exhibits dissociation constants in the micromolar range whereas LM609X exhibits dissociation constants in the nanomolar range. However, the RGD ligand showed greater uptake following incubation at temperatures which permit endocytosis. A 7.4-fold increase in uptake of the RGD peptide was observed following a 1 h incubation with HUVEC at 37 degrees C (an endocytosis permissive temperature), as compared to that at 4 degrees C (an endocytosis prohibitive temperature). In contrast, only a 1.9-fold increase in cell-associated fluorescence was observed for similar incubations with LM609X. Results from fluorescence microscopy supports the notion that the RGD peptide is rapidly endocytosed at 37 degrees C as compared to LM609X. These results are discussed with regard to previous work indicating that RGD ligands enter cells by integrin-independent pathways. These studies provide well-controlled measures of how RGD ligands stimulate endocytosis. This may be of considerable interest for intracellular delivery of ligand-associated drugs in anti-angiogenic applications.}, keywords = {alpha(v)beta(3) integrins, ALPHA-V-BETA-3, ANGIOGENESIS, ANTAGONISTS, ANTITUMOR EFFICACY, CELLS, Endocytosis, EXPRESSION, IN-VIVO, PEPTIDES, RGD, TARGETED DRUG-DELIVERY, TUMOR VASCULATURE}, isbn = {1573-3149}, url = {://000265022900007}, author = {Cressman, S. and Sun, Y. and Maxwell, E. J. and Fang, N. and Chen, D. D. Y. and Cullis, P. R.} } @article {2270, title = {Capillary electrophoresis frontal analysis for characterization of alpha(v)beta(3) integrin binding interactions}, journal = {Analytical Chemistry}, volume = {80}, number = {9}, year = {2008}, note = {ISI Document Delivery No.: 295JXTimes Cited: 8Cited Reference Count: 33Sun, Ying Cressman, Sonya Fang, Ning Cullis, Pieter R. Chen, David D. Y.}, month = {May}, pages = {3105-3111}, type = {Article}, abstract = {The specific binding characteristics of alpha(v)beta(3) integrins with an arginine-glycine-aspartic-acid (RGD) containing fluorescently labeled cyclic peptide is investigated with capillary electrophoresis-frontal analysis method. The new algorithm used to calculate the binding constants and binding stoichiometry was derived without the assumptions made in the commonly used Scatchard Plot method, thus enabling the determination of specific binding parameters in the presence of nonspecific binding. The a,6,3 integrin, a membrane protein, was studied in solution, without the need of immobilization or any other kind of modification. An RGD containing fluorescently labeled cyclic pentapeptide is used as the ligand with both specific and nonspecific binding characteristics, and an arginine-alanine-aspartic-acid (RAD) containing peptide is used as the control for nonspecific binding. While a typical specific binding isotherm has a shape of a rectangular hyperbola, a nonspecific binding isotherm is linear in the same ligand concentration region. A 1:2 specific binding stoichiometry was revealed with the second binding having a similar affinity compared to the first binding event.}, keywords = {ANGIOGENESIS, BEHAVIOR, CONSTANTS, CYCLIC RGD PEPTIDES, DRUG-PROTEIN-BINDING, INTEGRIN ALPHA(V)BETA(3), PRINCIPLES, RECOGNITION, TRANSDUCTION}, isbn = {0003-2700}, url = {://000255471900011}, author = {Sun, Y. and Cressman, S. and Fang, N. and Cullis, P. R. and Chen, D. D. Y.} } @article {1505, title = {Identification of sokotrasterol sulfate as a novel proangiogenic steroid}, journal = {Circulation Research}, volume = {99}, number = {3}, year = {2006}, note = {ISI Document Delivery No.: 070DTTimes Cited: 3Cited Reference Count: 39Murphy, Siun Larrivee, Bruno Pollet, Ingrid Craig, Kyle S. Williams, David E. Huang, Xin-Hui Abbott, Megan Wong, Fred Curtis, Cameron Conrads, Thomas P. Veenstra, Timothy Puri, Mira Hsiang, York Roberge, Michel Andersen, Raymond J. Karsan, Aly}, month = {Aug}, pages = {257-265}, type = {Article}, abstract = {The potential to promote neovascularization in ischemic tissues using exogenous agents has become an exciting area of therapeutics. In an attempt to identify novel small molecules with angiogenesis promoting activity, we screened a library of natural products and identified a sulfated steroid, sokotrasterol sulfate, that induces angiogenesis in vitro and in vivo. We show that sokotrasterol sulfate promotes endothelial sprouting in vitro, new blood vessel formation on the chick chorioallantoic membrane, and accelerates angiogenesis and reperfusion in a mouse hindlimb ischemia model. We demonstrate that sulfation of the steroid is critical for promoting angiogenesis, as the desulfated steroid exhibited no endothelial sprouting activity. We thus developed a chemically synthesized sokotrasterol sulfate analog, 2 beta,3 alpha,6 alpha-cholestanetrisulfate, that demonstrated equivalent activity in the hindlimb ischemia model and resulted in the generation of stable vessels that persisted following cessation of therapy. The function of sokotrasterol sulfate was dependent on cyclooxygenase-2 activity and vascular endothelial growth factor induction, as inhibition of either cyclooxygenase-2 or vascular endothelial growth factor blocked angiogenesis. Surface expression of alpha(v)beta(3) integrin was also necessary for function, as neutralization of alpha(v)beta(3) integrin, but not beta(1) integrin, binding abrogated endothelial sprouting and antiapoptotic activity in response to sokotrasterol sulfate. Our findings indicate that sokotrasterol sulfate and its analogs can promote angiogenesis in vitro and in vivo and could potentially be used for promoting neovascularization to relieve the sequelae of vasoocclusive diseases.}, keywords = {ACTIVATION, ANGIOGENESIS, apoptosis, ARTERIOGENESIS, ENDOTHELIAL-CELLS, endothelium, IN-VITRO, INHIBITION, INTEGRIN ALPHA(V)BETA(3), ischemia, MARINE NATURAL-PRODUCTS, NF-KAPPA-B}, isbn = {0009-7330}, url = {://000239501200009}, author = {Murphy, S. and Larrivee, B. and Pollet, I. and Craig, K. S. and Williams, D. E. and Huang, X. H. and Abbott, M. and Wong, F. and Curtis, C. and Conrads, T. P. and Veenstra, T. and Puri, M. and Hsiang, Y. and Roberge, M. and Andersen, R. J. and Karsan, A.} } @article {1198, title = {Strongylophorine-26, a Rho-dependent inhibitor of tumor cell invasion that reduces actin stress fibers and induces nonpolarized lamellipodial extensions}, journal = {Molecular Cancer Therapeutics}, volume = {4}, number = {5}, year = {2005}, note = {ISI Document Delivery No.: 926DETimes Cited: 6Cited Reference Count: 37}, month = {May}, pages = {772-778}, type = {Article}, abstract = {Strongylophorine-26, a new meroditerpenoid, was recently identified as an inhibitor of cancer cell invasion. This study was undertaken to characterize its mechanism of action. We find that strongylophorine-26 inhibits the motility of MDA-MB-231 breast carcinoma cells on a plastic surface. Upon addition of strongylophorine-26, rapid cell contraction and depolarization occurred, followed by spreading and flattening of the entire cell. Treated cells exhibited increased membrane ruffling throughout and extended lamellipodia in all directions. Strongylophorine-26 induced a decrease in actin stress fibers, a dramatic increase in the size and number of focal adhesions, and the appearance of a dense meshwork of actin filaments around the cell periphery. Strongylophorine-26 caused a transient activation of the small GTPase Rho and treatment with the Rho inhibitor C3 exoenzyme abrogated the anti-invasive activity of strongylophorine-26. These effects are distinct from those of many motility and angiogenesis inhibitors that seem to act by a common mechanism involving the induction of actin stress fibers. This difference in mechanism of action sets strongylophorine-26 apart as an experimental anticancer agent and indicates that pharmacologic inhibition of cell migration may be achieved by mechanisms not involving the stabilization of actin stress fibers.}, keywords = {ACTIVATION, ANGIOGENESIS, CYTOSKELETON, FAMILY, FOCAL ADHESIONS, KINASE, MIGRATION, MOTILITY, PROTEIN, SMALL GTPASE}, isbn = {1535-7163}, url = {://000229102300009}, author = {McHardy, L. M. and Warabi, K. and Andersen, R. J. and Roskelley, C. D. and Roberge, M.} } @article {941, title = {The tumor invasion inhibitor dihydromotuporamine C activates RHO, remodels stress fibers and focal adhesions, and stimulates sodium-proton exchange}, journal = {Cancer Research}, volume = {64}, number = {4}, year = {2004}, note = {ISI Document Delivery No.: 778MKTimes Cited: 23Cited Reference Count: 45}, month = {Feb}, pages = {1468-1474}, type = {Article}, abstract = {The motuporamines are macrocyclic alkaloids that inhibit tumor cell invasion by an, as yet, unknown mechanism. A structure-activity study recently identified dihydromotuporamine C (dhMotC) as a highly active and readily synthesized analogue. Here, we show that dhMotC causes subtle cytoskeletal alterations in highly invasive MDA231 breast tumor cells that include an increase in the thickness and number of cytoplasmic actin stress fibers. Experiments with serum-starved Swiss 3T3 fibroblasts showed that micromolar concentrations of dhMotC that inhibit tumor cell invasion induce the formation of new stress fibers and large focal adhesion complexes that are dispersed around the entire cell periphery. dhMotC treatment of Swiss 3T3 cells also initiates a strong, long-lived activation of the small GTP-binding protein Rho, and it stimulates Rho kinase-dependent sodium-proton exchanger activity. Liposome-mediated cell loading of C3 exoenzyme prevents dhMotC-mediated Rho activation and stress fiber formation in 3T3 cells. C3 exoenzyme loading also reestablishes elongated MDA231 breast tumor cell invasion in the presence of dhMotC. Taken together, these results indicate that the ability to activate Rho is one important determinant of the anti-invasive activity of dhMotC.}, keywords = {ANGIOGENESIS, CANCER, CELL-MIGRATION, FARNESYLTRANSFERASE INHIBITORS, GROWTH, GTP-BINDING PROTEIN, GTPASES, KINASE, MEMBRANE PROTRUSION, PROGRESSION}, isbn = {0008-5472}, url = {://000189245200037}, author = {McHardy, L. M. and Sinotte, R. and Troussard, A. and Sheldon, C. and Church, J. and Williams, D. E. and Andersen, R. J. and Dedhar, S. and Roberge, M. and Roskelley, C. D.} }