Effect of His-Gly-Lys Motif Derived from Domain 5 of High Molecular Weight Kininogen on Suppression of Cancer Metastasis Both in Vitro and in Vivo

Masayasu Kawasaki,Toshinaga Maeda,Kazuyoshi Hanasawa,Iwao Ohkubo,Tohru Tani

doi:10.1074/jbc.m308790200

Masayasu Kawasaki, Toshinaga Maeda + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m308790200

Copy DOI

Abstract

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.

Highlights

High molecular weight kininogen (HK)1 is well known to be a cofactor in the intrinsic pathway of blood coagulation cascade
Inhibition of Cell Adhesion by Peptides Derived from D5H in Vitro—We have shown previously that peptide P-5 (His479– Lys493) derived from D5H inhibited VN-mediated haptotaxis and haptoinvasion of MG-63 cells [27]
D5H of HK inhibits the adhesion of many cell lines such as MG-63, Human umbilical vein endothelial cells (HUVEC), MC3T3 E-1, and platelets to VN [24, 26, 27, 45]

Summary

EXPERIMENTAL PROCEDURES

Materials—Growth factor-reduced matrix Matrigel and 24- and 96well tissue culture plates were obtained from BD Biosciences. Cells that had traversed into the Matrigel and attached to the lower surface of the membrane were fixed in 10% formaldehyde, stained with Giemsa staining solution, and counted in 5 randomly selected microscopic fields (ϫ100) per filter. These experiments were performed independently at least three times. Labeling of Cell Surface Membrane Proteins with Biotin—Four ϫ 107 MDA-MB-231 cells were detached in 0.05% trypsin, 1 mM EDTA, washed three times with ice-cold PBS (pH 8.0), and incubated in 2 ml of PBS containing 1 mg/ml sulfo-NHS-LC-biotin at 4 °C for 1 h [36]. The significance of differences in numbers of cells was determined using the Student’s two-tailed t test compared with the control

RESULTS

Determination of Active Amino Acid Residues in Peptides

Treatment with

DISCUSSION