Domain 5 of kininogen inhibits proliferation of human colon cancer cell line (HCT-116) by interfering with G1/S in the cell cycle

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Abstract

Background

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.

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