Decoy Molecules Based on PNA-DNA Chimeras and Targeting Sp1 Transcription Factors Inhibit the Activity of Urokinase-type Plasminogen Activator Receptor (uPAR) Promoter.

The expression levels of urokinase-type plasminogen activator receptor (uPAR) are correlated with metastatic potential in human cancer cell lines of melanoma, breast, lung and colon. Therefore, targeting of uPAR could have practical implications in the treatment of neoplastic diseases. Since the expression of uPAR is regulated at the level of transcription in part by Sp1, we designed and tested transcription factors decoy molecules targeting Sp1 with the aim of inhibiting uPAR gene expression. In this study we determined the activity of decoy molecules based on peptide nucleic acids (PNA)-DNA chimeras mimicking Sp1 binding sites. The results obtained firmly indicate that Sp1-binding molecules based on PNA-DNA-PNA (PDP) chimeras are powerful decoys, as they efficiently inhibit the interactions between Sp1 and the uPAR promoter elements. These data were obtained by determining the effects of DNA/DNA, DNA/PDP, PDP/DNA and PDP/PDP molecules in EMSA experiments. The effects of PNA-DNA-PNA based Sp1 decoys were also analysed in a luciferase assay using a firefly luciferase gene reporter under the control of the human uPAR promoter. In these experiments Hep G2 cells, plated at 80% confluency, were transfected either with DNA/DNA or with DNA/PDP, PDP/DNA and PDP/PDP chimeras complexed with Lipofectamine. After 8 hours, Hep G2 cells were transfected with uPAR-firefly luciferase reporter vector. After 16 hours cell lysates were isolated for determining luciferase activity. The results obtained demonstrate that PNA-DNA-PNA based decoy molecules are potent inhibitors of the transcriptional activity of the uPAR promoter. Advantages of these molecules include stability, possible delivery and efficient uptake by target cells. Our results suggest that these molecules warrant attention for the design of novel anti-metastatic drugs.