Identification of Pigment Epithelium-Derived Factor Protein Forms with Distinct Activities on Tumor Cell Lines

Purpose. Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines. Methods. Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells. Results. Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. Conclusion. Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF

Dexamethasone Increases Pigment Epithelium-Derived Factor in Perfused Human Eyes

To investigate the effects of dexamethasone (DEX) on pigment epithelium-derived factor (PEDF) cDNA and secreted protein in human trabecular meshwork (TM)

Equilibrium-Fluctuation Analysis for Interaction Studies between Natural Ligands and Single G Protein-Coupled Receptors in Native Lipid Vesicles

G protein-coupled receptors (GPCRs) constitute the most versatile family of cell-membrane receptors and have been increasingly identified as important mediators of many physiological functions. They also belong to one of the most central drug target classes, but current screening technologies are limited by the requirements of overexpression and stabilization of GPCRs. This calls for sensitivity-increased detection strategies preferably meeting single-molecule detection limits. This challenge is here addressed by employing total internal reflection fluorescence microscopy to characterize the interaction kinetics between CXCR3, a GPCR involved in inflammatory responses, and two of its chemokine ligands, CXCL10 and CXCL11. Fluorescence labeling of the lipid membrane, rather than the membrane protein itself, of GPCR-containing native vesicles, and immobilization of the corresponding ligand on the surface, enabled determination of the interaction kinetics using single-molecule equilibrium-fluctuation analysis. With a limit of detection of GPCR-containing vesicles in the low picomolar concentration regime, the results demonstrate the possibility to use inhibition in solution screening of high affinity ligands/drug candidates, which due to target-binding depletion of the inhibiting compounds is demanding using assays with more moderate detection limits

E-selectin ligands recognised by heca452 induce drug resistance in myeloma, which is overcome by the e-selectin antagonist, gmi-1271

Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance