Inhibition of urokinase receptor gene expression and cell invasion by anti-uPAR DNAzymes in osteosarcoma cells.
The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G)-pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by approximately 93% and approximately 84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis.
de Bock
Charles E
CE
Orthopaedic Research Institute, Department of Medicine, St George Hospital, University of New South Wales, Sydney, NSW, Australia.
Lin
Zhen
Z
Itoh
Takashi
T
Morris
David
D
Murrell
George
G
Wang
Yao
Y
D045744:Cell Line, Tumor; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009361:Neoplasm Invasiveness; D012516:Osteosarcoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator
D021881:DNA, Catalytic; D018076:DNA, Complementary; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator