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

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.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. Several lines of experiments have demonstrated that platelets can facilitate hematogenous dissemination of tumor cells (1). In this process, platelets act mainly through emboli complexes formed by the interaction of tumor cells and platelets. The formation of tumor cell-platelet emboli complexes in the blood stream not only provides a protective shield that masks them from the cytotoxic activity of natural killer cells, but also favors tumor cell adhesion to vascular endothelial cells (2-4).The interaction between tumor cells and platelets may involve several kinds of adhesion molecules. Selectins are a family of intercellular adhesion molecules that mediate the initial adhesion of leukocytes to the endothelia of blood vessels during inflammation (5-9). The family includes E-, P-, and L-selectin, and all three selectins can bind to sialylated, fucosylated, or, in some cases, sulfated glycans on glycoproteins, glycolipids, or proteoglycans (10). The tetrasaccharides [Neu5Ac␣2,3Gal␤1,4-(Fuc␣1,3)GlcNAc] (sLe x ) 1 and [Neu5Ac␣2,3Gal␤1,3(Fuc␣1,4)-GlcNAc] (sLe a ) have been identified as the minimal ligands for all three types of selectins. Recently, accumulating evidence indicates that P-selectin plays a crucial role during hematogenous metastasis, and P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, s...

Modified heparins inhibit integrin αIIbβ3 mediated adhesion of melanoma cells to platelets in vitro and in vivo

“…In the assay of enzyme treatments, A375 cells (10 7 cells/ml in IMDM) were simultaneously treated with 5 U/ml heparinases I, II and III in the presence of a mixture of protease inhibitors (10 mg/ ml leupeptin, 20 mg/ml aprotinin and 10 mM benzamidine) for 1 h at 37°C with end-to-end rotation. 17 …”

“…17 Briefly, the bottom of the chamber was a 35-mm tissue culture dish precoated with GP-CHO (or CHO) cells over night, or a 35-mm circular glass slide (treated with 4% APES) precoated with PRP (platelet was adjusted to 2 3 10 8 /ml) for 60 min at 37°C. To induce a IIb b 3 activation, GP-CHO cells were stimulated with 10 mM DTT for 20 min at 22°C, the glass slides coated with platelets were incubated with thrombin (1 U/ml, sigma) for 10 min at 37°C.…”

“…20 The prior work of our laboratory demonstrated that P-selectin could bind to human colon carcinoma cells. 17 To assess the role of platelet P-selectin in the firm adhesion of A375 cells to platelets, 9E1 was used to block P-selectin alone. We found that P-selectin blocking only slightly reduced the firm adhesion.…”

A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro

“…Heparin has weak inhibitory activity against platelet-collagen interactions which accounted for its non-specific interference with agonist-receptor interactions (Davies and Menys 1982). Some chemically modified heparins with low anticoagulant activities may also have potential value as therapeutic agents that block P-selectin-mediated cell adhesion (Wei et al 2004). In this study, we found that heparin directly inhibited platelet adhering to endothelial cells.…”

Old and new applications of non-anticoagulant heparin

“…[99] Astenose Adjuvant in cardiovascular intervention c [113] ROH Heparanase antiangiogenesis inhibitor b [114] ROH P selectin inhibitor [115] LAC-HP antimetastatic b [116] ROH Integrin-melanoma cell binding inhibitor a [117] ROH Antianemic as hepicdine inhibitor b [118] ROH Antinflammation-human elastase inhibitors a [118] gs-LMWH ORG 31733 HIV-1 and HIV-2 inhibitor a [120] SR 80258 Allergic airway response inhibitor b [121] Vasoflux Anticoagulant independent from AT activity, coadjuvant in myocardial infarction therapy d [108,109] NAC from Tinzaparin Antimetastatic b [122] gs-LMWH antimalarial, parasite adhesion, inhibitor b [44] DF01 -Tafoxiparin Labor pain attenuation d [111] M-402 Nocuparanib Metastasis and multiple pathway inhibitor d [110] DFX 232-Sevuparin Antimalarial, distrupting "Plasmodium falciparium" rosettes d [112]]-Preventive activity of vasoocclusion in sickle severe hereditary anemia d S-NACH Anticancer increasing tumor chemo-responsiveness c [35] gs-ULMWH RO-Fondaparinux Antinflammation b [123] Undersulfated RO ST1514" Antinflammatory -heparanase inhibitor b [124] Antiangiogenic, as FGF-2 and VEGF inhibitors b [125,126] N-acylated gs-heparins …”