Modified Heparin Inhibits P-selectin-mediated Cell Adhesion of Human Colon Carcinoma Cells to Immobilized Platelets under Dynamic Flow Conditions

Min Wei,Guihua Tai,Yanguang Gao,Na Li,Baiqu Huang,Yifa Zhou,Shui Hao,Xianlu Zeng

doi:10.1074/jbc.m312951200

Abstract

Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis.

Highlights

Hematogenous metastasis is a highly regulated and dynamic process in which the cancerous cells separate from the primary tumor, migrate across blood vessel walls into the bloodstream, and disperse throughout the body to generate new colonies
Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion
Our results showed that three human colon carcinoma cell lines strongly expressed heparan sulfate proteoglycans, which are involved in adhesion of these tumor cells as a ligand of P-selectin

Summary

EXPERIMENTAL PROCEDURES

Heparin and Its Derivatives—Porcine intestinal heparin (Mr ϭ 18,000 –20,000) was purchased from Sigma-Aldrich Inc. For the cell surface P-selectin binding assay, each aliquot (0.1 ml) of cells was incubated with 0.3 ␮g of human IgG or P-Fc at 22 °C for 20 min, followed by a FITC-conjugated Ab against human IgG at 22 °C for 1 h with end-to-end rotation. For cell surface P-selectin ligand experiments, each aliquot (0.1 ml) of cells was incubated with 1ϳ2 ␮g of mouse IgM, mouse IgG, CSLEX-1 mAb, 10E4 mAb, or KPL1 mAb at 22 °C for 30 min, followed by treatment with a FITC-conjugated Ab against mouse IgM or mouse IgG at 22 °C for 1 h with end-over-end rotation. After washing the platelet layer with Dulbecco’ s PBS, 0.1% BSA for ϳ2 min, tumor cells were perfused through the chamber for 3 min at the appropriate flow rates to obtain wall shear stresses of 0.3–1.2 dyn/cm at 22 °C using a syringe pump (Cole-Parmer Instrument Co.), thereby mimicking the fluid mechanical environment of the microcirculation and postcapillary venules. Probability values of p Ͻ .05 or p Ͻ .01 were selected to be statistically significant

RESULTS

Sulfate contenta

TABLE II Anticoagulant activities of modified heparins

DISCUSSION