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A., Gunsalus, I. C., Peisach, J., Blumberg, W., Orme-Johnson, W. H., & Beinart, H. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 1157-1 163. Van Gunsteren, W. F., & Karplus, M. (1981) Nature (Lon- don) 293, 677-678. Wagner, G. C., & Gunsalus, I. E. (1982) in The Biological Chemistry ofZron (Dunford, H. B., Dolphin, D., Raymond, K. N., & Sieker, L., Eds.) pp 405-412, Reidel, Boston. U.S.A. 73, 1078-1082. 2290-2295. U.S.A. 77, 6371-6375. Chemically Modified Heparins as Inhibitors of Heparan Sulfate Specific Endo-@-glucuronidase (Heparanase) of Metastatic Melanoma Cellst Tatsuro Irimura, Motowo Nakajima, and Garth L. Nicolson* Received February 11, 1986; Revised Manuscript Received April 14, 1986 Department of Tumor Biology, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 ABSTRACT: To determine the significance of the heparan sulfate (HS) degradative endo-&glucuronidase (heparanase) in tumor invasion and metastasis and to develop possible antimetastatic agents, we synthesized specific inhibitors of this enzyme. We previously found that heparanase activity correlates with the lung colonization abilities of murine B16 melanoma cells and is inhibited by heparin [Nakajima, M., Irimura, T., Di Ferrante, N., & Nicolson, G. L. (1984) J. Biol. Chem. 259,2283-22901. In this study, heparin was chemically modified in order to determine which portions of its structure are responsible for heparanase inhibitory activity and to obtain heparanase inhibitors that have minimal additional biological effects, such as anticoagulation. N-Sulfate groups and 0-sulfate in heparin were removed separately, and the resultant free amino groups were acetylated or resulfated. Heparin was also reduced at the carboxyl groups of uronic acid. The heparanase inhibitory activities of these heparin derivatives were examined by high-speed gel- permeation chromatography and by the use of radioactive HS immobilized on agarose beads. The results indicated that although N-sulfate and 0-sulfate groups on glucosamine residues, and carboxyl groups on uronic acid residues, are important for heparanase inhibition, they are not essential for full activity. When highly metastatic B 16-BL6 melanoma cells were incubated with N-acetylatyed N-desulfated heparin, N-resulfated N- and 0-desulfated heparin, or carboxyl-reduced heparin and injected intravenously to syngenic C57BL/6 mice, significant reductions in the numbers of experimental melanoma lung metastases occurred. %e malignancy of solid tumors can be explained, in part, by their abilities to invade and destroy normal tissues, including extracellular matrix and basement membranes (Nicolson, 1982, 1984; Liotta et al., 1983; Irimura et al., 1983b). Gly- cosaminoglycans, such as heparan sulfate (HS),’ are important constituents in these structures. Recently, we found that HS degradative activities of murine B16 melanoma sublines correlated with their metastatic lung colonization and invasive This study was supported by zyxwvutsrq U.S. Department of Health and Human Services Grants BRSG RR-5511-23 and R01-CA39319 to T.I. and Grant R01-CA42346 to G.L.N. and a grant from the National Foun- dation for Cancer Research to G.L.N. Abbreviations: DPBS, Dulbecco’s phosphate-buffered saline; HS, heparan sulfate; Mr, relative molecular weight; Tris-HCI, tris(hydroxy- methy1)aminomethane hydrochloride; EDTA, ethylenediaminetetraacetic acid. *Author to whom correspondence should be addressed. 0006-2960/86/0425-5322$01 .50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1986 American Chemical Society