uPA-silica-Particles (SP-uPA): a novel analytical system to investigate uPA-uPAR interaction and to test synthetic uPAR antagonists as potential cancer therapeutics.
The urokinase-type plasminogen activation system, including the serine protease uPA (urokinase-type plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPA-derived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SP-uPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPA-peptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 microm in diameter to which HMW-uPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SP-uPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)-labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMW-uPA, ATF) as well as highly effective, synthetic cyclic uPA-derived peptides (cyclo21,29[D-Cys21Cys29]-UPA21-30, cyclo21,29[D-Cys21Nle28Cys29]-uPA21-30, cyclo21,29[D-Cys(21)2-Nal24Cys29]-uPA21-30, and cyclo21,29[D-Cys21Orn23Thi24Thi25Cys29]-uPA21-30. The results obtained with the noncellular SP-uPA/uPAR system are highly comparable to those obtained with a cellular system involving FITC-uPA and the promyeloid cell line U937 as the source of uPAR.
Guthaus
Elke
E
Klinische Forschergruppe, Frauenklinik der TU München, Germany.
Bürgle
Markus
M
Schmiedeberg
Niko
N
Hocke
Stefan
S
Eickler
Alexandra
A
Kramer
Michael D
MD
Sweep
C G J Fred
CG
Magdolen
Viktor
V
Kessler
Horst
H
Schmitt
Manfred
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007118:Immunoassay; D007231:Infant, Newborn; D015394:Molecular Structure; D010316:Particle Size; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D012822:Silicon Dioxide; D012995:Solubility; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; C524898:PLAUR protein, human; D010456:Peptides, Cyclic; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D012822:Silicon Dioxide; D014568:Urokinase-Type Plasminogen Activator