Many advanced tumors overexpress and secrete the S100A4 protein that is known to promote angiogenesis and metastasis development. The mechanisms of this effect and the endothelial receptor for S100A4 are both still unknown. Here we report that extracellular S100A4 interacts with annexin II, an endothelial plasminogen co-receptor. Co-localization and direct binding of S100A4 and annexin II were demonstrated, and the binding site was identified in the N-terminal region of annexin II. S100A4 alone or in a complex with annexin II accelerated tissue plasminogen activator-mediated plasminogen activation in solution and on the endothelial cell surface through interaction of the S100A4 Cterminal lysines with the lysine-binding domains of plasminogen. A synthetic peptide corresponding to the N terminus of annexin II prevented S100A4-induced plasmin formation in the endothelial cell culture. Local plasmin formation induced by circulating S100A4 could contribute to tumor-induced angiogenesis and metastasis formation that makes this protein an attractive target for new anti-cancer and anti-angiogenic therapies.

Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptormediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K + depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-bcyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, a(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes a(2,3)-, but not with Sambucus nigra agglutinin that recognizes a(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal a(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.The plasma membrane of the cell is a complex, carefully regulated, dynamic structure that is compartmentalized into cell surface domains such as lipid rafts, caveolae, and clathrincoated pits (1). Lipid rafts are postulated to be dynamic nanodomains ranging in size from 10 to 200 nm that are enriched in cholesterol, sphingolipids, and certain proteins and that play an important role in assembling signaling complexes (2-4). Caveolae are invaginated lipid raft domains (50 -150 nm) whose stability at the plasma membrane is attributable to the formation of stable oligomers of their coat protein, caveolin-1 (5). Caveolae are thought to serve as concentrators of various signal transduction machineries. Clathrin-coated pits (100 -150 nm) internalize rapidly upon formation at the plasma membrane, and their lateral cell surface mobility is enhanced by actin cytoskeleton depolymerization (6).The epidermal growth factor receptor (EGFR), 3 a 170-kDa transmembrane glycoprotein, is one of four members of the ErbB family of receptor tyrosine kinases involved in oncogene signaling. The initial step in receptor activation involves binding of the EGF peptide to the extracellular domain leading to dimerization or activation of pre-existing dimers (7,8). Following ligand binding, the EGFR is auto-phosphorylated in several tyrosine residues of the intracellular domain, creating high affinity sites for various adaptor molecules that transmit the mitogenic signal to the Ras/MAPK signal transduction pathway (9). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2, which regulate transcription of molecules that are linked to cell proliferation, survival, a...

/npsi/ctrl?action=rtdoc&an=21270647&lang=en http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?action=rtdoc&an=21270647&lang=fr READ THESE TERMS AND CONDITIONS CAREFULLY BEFORE USING THIS WEBSITE.http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/jsp/nparc_cp.jsp?lang=en Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions?Contact the NRC Publications Archive team at PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. For the publisher's version, please access the DOI link below./ Pour consulter la version de l'éditeur, utilisez le lien DOI ci-dessous.http://doi.org/10.1021/am302951k ACS Applied Materials and Interfaces, 5, 8, pp. 2870-2880, 2013 ABSTRACT: We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS 2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc) 3 ) as an In source, the growth temperature was as low as 160°C and the feed molar ratios were 1Cu-to-1In-to-4S. Amazingly, the resulting CuInS 2 NCs in toluene exhibit quantum yield (QY) of ∼23% with photoemission peaking at ∼760 nm and full width at half maximum (FWHM) of ∼140 nm. With a mean size of ∼3.4 nm (measured from the vertices to the bases of the pyramids), they are pyramidal in shape with a crystal structure of tetragonal chalcopyrite. In situ 31 P NMR (monitored from 30°C to 100°C) and in situ absorption at 80°C suggested that the Cu precursor should be less reactive toward SDPP than the In precursor. For our in vitro and in vivo imaging applications, CuInS 2 /ZnS core−shell QDs were synthesized; afterwards, dihydrolipoic acid (DHLA) or 11-mercaptoundecanoic acid (MUA) were used for ligand exchange and then bio-conjugation was performed. Two single-domain antibodies (sdAbs) were used. One was 2A3 for in vitro imaging of BxPC3 pancreatic cancer cells. The other was EG2 for in vivo imaging of a Glioblastoma U87MG brain tumour model. The bioimaging data illustrate that the CuInS 2 NCs from our SDPP-based low-temperature noninjection appr...

The multidrug resistant (MDR) transporter P-glycoprotein (Pgp) is constitutively expressed in normal tissues, where its spatial distribution defines it as an important element reducing the systemic exposure and tissue access of potentially harmful xenobiotics. We sought to determine whether P-gp is functionally expressed within alveolar epithelium of lung, in particular within the predominant cell type of this barrier, the alveolar epithelial (AE) type I cell. By immunohistochemistry, MDR-1/ mdr-1 P-gp was localized to luminal membranes of AE type I epithelium within normal human and rat lung tissue. Using a primary rat cell culture model affording study of AE type II to AE type I differentiation, we observed increased expression (reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunoflow cytometry techniques) of mdr-1a and mdr-1b P-gp in the cultures as they adopted an AE type I phenotype; freshly isolated AE type II cells were negative for mdr-1/P-gp. The functionality of P-gp within the AE cultures was demonstrated by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE type I monolayers (basal-to-apical permeability was 3-fold that of apical-to-basal permeability), which was found to be comparable with the P-gp transport barrier presented by Caco-2 cell monolayers. The implications of localizing P-gp within alveolar epithelium is of significance to studies of fundamental respiratory cell biology as well as to further clarifying the nature of the barrier to xenobiotic transfer from alveolar airspace to pulmonary interstitium and capillary blood.P-Glycoprotein (P-gp) is a member of the ATP-binding cassette superfamily of membrane transport proteins that mediates the vectorial movement across cell membranes of a wide range of physicochemically diverse solutes (Stouch and Gudmundsson, 2002). In humans, two P-gp-related genes have been cloned and subsequently termed MDR1 and MDR3 (for review, see Ambudkar et al., 1999). The MDR1/P-gp gene product is recognized in particular to actively efflux from a cell a diverse range of cytotoxic drugs, a characteristic that is an important facet in the multidrug resistant (MDR) cell phenotype. Current evidence suggests that the MDR3/P-gp gene product does not contribute to an MDR phenotype.In rodents, three P-gp-related genes have been identified and designated mdr-1a, mdr-1b, and mdr-

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New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [125I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.