Domain 5 (D5) of kininogen inhibits endothelial cell adhesion, migration, proliferation and angiogenesis by inducing apoptosis and disrupting a signaling pathway initiated by binding to the urokinase receptor (uPAR).Objectives
Because tumor cells frequently overexpress uPAR, we hypothesized that D5 can directly inhibit proliferation of colon carcinoma cells.Methods and results
A recombinant fusion protein of D5 and glutathione S-transferase (GST-D5) but not GST at 280 nM inhibited proliferation of human colon carcinoma cells (HCT-116) in vitro by 75–86%. We found that treatment with GST-D5 did not affect the survival pathway, phosphatidylinositol 3-kinase or the apoptotic pathway. In contrast, the G1/S phase transition of the cell cycle was downregulated as evidenced by an increase of cells in G0/G1 and a decrease in cells in S by flow cytometry. We found a decrease in serine phosphorylation of the retinoblastoma protein Rb (p107) after incubation with GST-D5. Less E2F-1 transcription factor and p107 were released and fewer cells overcame the G1/S growth restriction point. Expression levels of cyclins D1, A and E were reduced as measured by densiometric analysis of western blots. Cyclin-dependent protein kinase activities were downregulated and p27, the cyclin-dependent kinase inhibitor, was activated by GST-D5.Conclusions
These findings indicate that D5 of high molecular weight kininogen interferes with the G1 to S phase transition, reducing the proliferation of human colon carcinoma cells.