Modulation of the Heparanase-inhibiting Activity of Heparin through Selective Desulfation, Graded N-Acetylation, and Glycol Splitting

Giangiacomo Torri,Benito Casu,Marta Perez,Claudio Pisano,Rivka Ishai-Michaeli,Israel Vlodavsky,Annamaria Naggi,Sergio Penco,Giuseppe Giannini,Giuseppe Cassinelli

doi:10.1074/jbc.m414217200

Abstract

Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified D-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones.

Highlights

Heparanase is an endo-␤-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM)
Heparanase enzymatic activity participates in degradation and remodeling of the extracellular matrix (ECM), facilitating, among other activities, cell invasion associated with cancer metastasis, angiogenesis, and inflammation [1,2,3, 9]
The two glycosaminoglycans have different locations in tissues: whereas HS is a component of the ECM and of the surface of most cells, heparin is stored in granules of mast cells and co-released with histamine into the circulation upon cellular degranulation mainly in cases of allergic and inflammatory reactions and anaphylactic stress

Summary

Introduction

Heparanase is an endo-␤-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). In addition to its intimate involvement in the egress of cells from the blood stream, heparanase activity releases from the ECM and tumor microenvironment a multitude of HS-bound growth factors, cytokines, chemokines, and enzymes that affect cell and tissue function, most notably angiogenesis [17, 18] These observations, the anticancerous effect of heparanase gene silencing (ribozyme and small interfering RNA) [19] and of heparanaseinhibiting molecules (non-anticoagulant species of heparin and other sulfated polysaccharides) [20, 21], and the unexpected identification of a predominant functional heparanase [1,2,3] suggest that the enzyme is a promising target for development of new anticancer drugs. Exogenous heparin is widely used as an anticoagulant and antithrombotic drug and is of increasing interest for novel therapeutical applications (24 –27)

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Conclusion