STRUCTURAL REQUIREMENTS FOR INHIBITION OF MELANOMA LUNG COLONIZATION BY HEPARANASE INHIBITING SPECIES OF HEPARIN

Heparanase activity correlates with metastatic potentials of lymphoma, melanoma and mammary adenocarcinoma cell lines. We investigated the a bility of various modified species of heparin and size homogeneous oli gosaccharides derived from depolymerized heparin to inhibit: a) hepara nase-mediated degradation of heparan sulfate (HS) in the extracellular matrix (ECM) deposited by cultured endothelial cells, and b) lung col onization of B16-BL6 melanoma cells in C(57)BL mice. For this purpose, melanoma cells or conditioned medium were incubated with metabolicall y sulfate-labeled subendothelial ECM in the absence and presence of he parin, heparin fragment or nonanticoagulant species of heparin. Labele d HS degradation fragments released into the incubation medium were an alyzed by gel filtration over Sepharose 6B. The B16-BL6 melanoma cells were also tested for lung colonization following their intravenous ad ministration to C(57)BL mice, in the absence and presence of the vario us species of heparin. Inhibition of both heparanase and melanoma lung colonization depended on the size and degree of sulfation of the hepa rin molecule, the position of sulfate groups, and the occupancy of the N position of the hexosamines. Inhibition of heparanase was best achi eved by heparin species containing 16 sugar units or more and having s ulfate groups at both the N and O positions. Low sulfate oligosacchari des were less effective heparanase inhibitors than medium and high sul fate fractions of the same size saccharide. While O-desulfation abolis hed the heparanase inhibiting effect of heparin, O-sulfated, N-substit uted (e.g., N-acetyl or N-hexanoyl) species of heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about greater than or equal to 4,000 daltons. Potent inhibitors of heparanase activity were also efficient inhibitors of t umor invasion and lung colonization The antimetastatic and anticoagula nt activities of heparin were unrelated, as indicated by using heparin fractions with high and low affinity for antithrombin III. These hepa rins differ about 200-ford in their anticoagulant activity, but expres sed similar high antiheparanase and antimetastatic activities. It appe ars that heparanase-inhibiting species of heparin interfere with the p assing of tumor cells across the capillary wall, as they significantly inhibited metastasis even when injected up to 3 h after lodgment. Str uctural requirements for inhibition of heparanase activity and lung co lonization of melanoma cells by species of heparin were different from those identified for a) release of ECM-bound basic fibroblast growth factor (bFGF), and b) stimulation of bFGF receptor binding and mitogen ic activity. These results indicate that various nonanticoagulant spec ies of heparin and other polyanionic molecules differing in size, sulf ation, and substituted groups can be designed to elicit specific effec ts resulting in inhibition of cell invasion in tumor metastasis and au toimmunity, or stimulation of neovascularization and wound healing.