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J. Biol. Chem., Vol. 280, Issue 13, 12103-12113, April 1, 2005
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From the
G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo, 81, 20133 Milan, Italy, ¶Sigma-Tau Research Department, 00040 Pomezia, Rome, Italy, ||Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel, and **Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel
Received for publication, December 17, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-glucuronidase that cleaves heparan sulfate (HS)1 chains at a limited number of sites (1–3). Cloning of the heparanase cDNA by several groups (1–6) suggests that a single functional HS-degrading endoglycosidase is expressed in mammalian cells. The enzyme is synthesized as a latent 65-kDa precursor that undergoes proteolytic cleavage, yielding 8- and 50-kDa subunits that heterodimerize to form a highly active enzyme (7, 8). 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 (1–3, 9). Heparanase upregulation has been documented in a variety of human tumors correlating, in some cases, with increased vascular density and poor postoperative survival (10–13). Heparanase overexpression has also been noted in several other pathologies such as cirrhosis (14), nephrosis (15), and diabetes (16). 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 heparanase-inhibiting molecules (non-anticoagulant species of heparin and other sulfated polysaccharides) (20, 21), and the unexpected identification of a predominant functional heparanase (1–3) suggest that the enzyme is a promising target for development of new anticancer drugs.
HS and the structurally related heparin are present in most animal species. They are glycosaminoglycans constituted by repeating disaccharide units of a uronic acid (either D-glucuronic acid (GlcA) or L-iduronic acid (IdoA)) and D-glucosamine (either GlcNAc or D-glucosamine N-sulfate (GlcNSO3)) and bear sulfate substituents in various positions (22–25). Although derived from the common biosynthetic precursor N-acetylheparosan (-GlcA-GlcNAc)n, HS and heparin have different structures: HS is less sulfated and more heterogeneous than heparin. The two glycosaminoglycans also 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. On the other hand, exogenous heparin is widely used as an anticoagulant and antithrombotic drug and is of increasing interest for novel therapeutical applications (24–27).
As an analog of the natural substrate of heparanase, heparin is commonly considered to be a potent inhibitor of heparanase (20, 21, 28–31). This activity is attributed, in part, to its high affinity interaction with the enzyme and limited degradation, serving as an alternative substrate. Early reports (20, 21, 30, 31) showed that heparin and some chemically modified species of heparin as well as other sulfated polysaccharides (22, 32) that inhibit tumor cell heparanase also inhibit experimental metastasis in animal models, while other related compounds that lack heparanase-inhibiting activity fail to exert an antimetastatic effect (20–22, 30–32). Regardless of the mode of action, heparin and low molecular weight heparin (LMWH) were reported to exert a beneficial effect in cancer patients (33), stimulating research on the potential use of modified, non-anticoagulant species of heparin and HS in cancer therapy.
Screening of heparin derivatives has permitted the identification of some of the structural features of heparin associated with inhibition of the enzyme. As a general trend, the heparanase-inhibiting activity increases with increasing degrees of O-sulfation. However, N-sulfates seems to exert little effect since they can be replaced by N-acyl (N-acetyl, N-succinyl, or N-hexanoyl) groups without substantial loss of inhibitory activity (20, 34). No significant differences were found between the currently used unfractionated heparins and low molecular weight heparins and a tetradecasaccharidic fragment (34). 2-O-Desulfated derivatives were shown to retain the inhibitory activity, whereas N-desulfated, N-acetylated derivatives displayed a reduced activity (35). In the present study, relationships between structure and heparanase-inhibiting activity of heparin were studied using a larger number of heparins and heparin derivatives, including some with various degrees of 6-O-sulfation of GlcN and 2-O-sulfation of IdoA residues as well as "glycol-split" derivatives obtained by controlled periodate oxidation/borohydride reduction of natural (36) or partially 2-O-desulfated heparins (37, 38). Glycol splitting of C-2–C-3 bonds of nonsulfated uronic acid residues was suggested to interfere with the biological interactions of heparin by providing flexible joints between protein binding sequences (37–39). When framing heparin sequences that bind FGF-2, glycol-split residues were shown not to impair the binding to FGF-2. However, they prevented activation of FGF-2 and FGF-2-induced angiogenic activity (37, 38). The present study shows that glycol splitting enhances the heparanase-inhibiting activity of heparin. Based on the observation that N-acetyl groups do not prevent and may even assist recognition by heparanase (40, 41) and taking into account that N-acetylheparin, as opposed to heparin, does not release angiogenic factors from ECM (34), we prepared and tested heparins with various degrees of N-acetylation/N-sulfation together with some of their glycol-split derivatives. N-Acetylated, glycol-split heparins were shown to inhibit heparanase more efficiently than the corresponding non-glycol-split N-acetylated heparins.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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w, by GPC-HPLC (43)) were: H-1, 14,200; H-2, 18,100; H-3, 19,600; H-4, 18,800; H-5, 18,200; H-6, 23,200; and H-7, 21,600. Sample desalting was carried out by dialysis against water with 1000-Da cut-off tubes or by fractionation on a 2.5 x 100-cm Sephadex G-25 column (Amersham Biosciences) using 10% ethanol in water as eluent and UV detection at 210 nm. Molecular weight determinations were performed by GPC-HPLC on a Viscotex instrument equipped with a VE1121 pump, Rheodyne valve (100 µl), and triple detector array 302 equipped with IR, viscosimeter, and 90° light-scattering systems. Two 300 x 7.8-mm TSK GMPWXL Viscotek columns were used with 0.1 M NaNO3 as eluent (flow, 0.6 ml/min). Samples were dissolved in the eluent solution at the concentration of 15 mg/ml (43).
NMR spectra were recorded at 500 MHz for 1H and 125 MHz for 13C with a Bruker AMX spectrometer equipped with a 5-mm 1H/X inverse probe. The spectra were obtained at 45 °C from D2O solutions (15 mg/0.5 ml D2O, 99.99% D). Chemical shifts, given in parts per million down field from sodium-3-(trimethylsilyl)propionate, were measured indirectly with reference to acetone in D2O (
2.235 for 1H and 30.20 for 13C). The 13C NMR spectra were recorded at 300 or 400 MHz with a Bruker AC-300 or AMX-400 spectrometer.
Recombinant Human Heparanase
Recombinant enzymatically active heparanase was purified from heparanase-transfected Chinese hamster ovary cells (4). Briefly Chinese hamster ovary cells were harvested with trypsin and centrifuged, and the cell pellet was suspended in 20 mM citrate-phosphate buffer pH 5.4. The suspension was subjected to four cycles of freeze/thaw (–70/37 °C, 5 min each), the cell extract was centrifuged (18,000 rpm, 15 min, 2–8 °C), and the supernatant was collected and filtered through a 0.45-µm filter. The filtrate was applied onto a Source 15 S column (Amersham Biosciences) equilibrated with 20 mM phosphate buffer, pH 6.8. The column was washed (20 mM phosphate buffer, pH 6.8, followed by 20 mM phosphate buffer, pH 8.0), and heparanase was eluted with a linear gradient (0–35%) of 8 column volumes of 1.5 M NaCl in 20 mM phosphate buffer, pH 8.0. Active fractions were pooled and applied onto a Fractogel EMD SO–3 (Merck) column equilibrated with 20 mM citratephosphate buffer, pH 5.4. Heparanase was eluted with a linear gradient (0–22%) of 1 column volume followed by 10 column volumes (22–25%) of 1.5 M NaCl in 20 mM phosphate buffer, pH 8.0. Finally heparanase eluted from the Fractogel column was applied onto a HiTrap heparin column (Amersham Biosciences) equilibrated with 20 mM phosphate buffer, pH 8.0, and eluted with a linear gradient of 1 column volume (0–20%) and 15 column volumes (20–28%) of 1.5 M NaCl in 20 mM phosphate buffer, pH 8.0. Eluted fractions were analyzed by gradient SDS-PAGE, stained with Gelcode® (Pierce), and pooled according to their purity. An at least 90% pure, highly active heparanase preparation was obtained, containing the active 50- and 8-kDa heparanase subunits and, to a lower extent, the 65-kDa proheparanase (8). Active recombinant human heparanase was also produced in insect cells as described previously (7). The construct encoding the 8- and 50-kDa heparanase subunits was kindly provided by Dr. E. McKenzie (Oxford GlycoSciences Ltd., Abingdon, Oxon, UK) (7). Similar results were obtained with both preparations.
Preparation of Heparin Derivatives
6-O-Desulfated Heparins
Procedure A—An extensively 6-O-desulfated heparin also partially (
15%) 2-O-desulfated (716OdeS-H(A) where the superscript denotes the degree of 6-O-desulfation), w 16,000, was prepared according to Nagasawa et al. (44), starting from the pyridinium salt of heparin H-1, under solvolytic conditions (10 mg/ml in Me2SO:water 9:1) at 100 °C for 2.5 h followed by resulfation of free amino groups with sulfur trioxidetrimethylamine complex in alkaline aqueous medium (45).
Procedure B—6-O-Desulfated-heparins (776OdeS-H(B), w 19,000; 736OdeS-H(B), w 17,700; and 466OdeS-H(B), w 20,400) were prepared according to Matsuo et al. (46) by O-desulfation through activation with N-methyl-N-(trimethylsilyl)trifluoroacetamide or N,O-bis (trimethylsilyl)acetamide without N-desulfation. Heparin H-1 (200 mg) was converted into its pyridinium salt and soaked in pyridine (20 ml). After addition of 4 ml of N-methyl-N-(trimethylsilyl)trifluoroacetamide, the solution was heated for 4 h at 80 °Cto yield 736OdeS-H or for 8 h at 60 °C to yield 776OdeS-H. Heparin (H-1) was converted into its pyridinium salt and soaked in pyridine (30 ml). After addition of 6 ml of N,O-bis(trimethylsilyl)acetamide, the solution was heated for 2 h at 60 °C to yield 466OdeS-H.
2-O-Desulfated Heparins
Procedure A—2-O-Desulfated heparin in the IdoA form (H, IdoA(A), w 17,700) was prepared according to Jaseja et al. (47). Heparin (500 mg) was simply dissolved in 500 ml of 0.1 M NaOH, and the solution was frozen and lyophilized. The residue dissolved in 500 ml of distilled water was dialyzed, and the product was isolated by evaporation under reduced pressure. Its 13C NMR spectrum closely corresponded to the one reported in the literature (48), indicating an essentially complete conversion of the original IdoA2SO3 residues into IdoA residues.
Procedure B—2-O-Desulfated heparin in the GalA form (H, GalA(B), w 12,600) was prepared by a modification of methods used by Jaseja et al. (47) and Rej and Perlin (49) essentially as described previously (48). Heparin (500 mg) was dissolved in 10 ml of 1 M NaOH and then heated at 85 °C for 1 h. After cooling below 30 °C, the solution was brought to pH 7 with 0.1 M HCl and heated at 70 °C for 48 h to give (after cooling, dialysis, and freeze-drying) the GalA derivative with a typical 13C NMR spectrum (48).
N-Acetylated Heparins
N-Acetylated heparins (xNAH, where the superscript x denotes the degree of N-acetylation as referred to total GlcN) were prepared by time-controlled N-desulfation under solvolytic conditions (44). Briefly the pyridinium salt of heparin was stirred at 20–25 °C in Me2SO:water (9:1) for different times (30, 60, 90, 100, and 120 min and 8 h) to obtain intermediates with different degrees of N-desulfation, which upon N-acetylation with acetic anhydride in alkaline aqueous medium (NaHCO3, 4 °C, 2 h) (50) gave 29NAH (w 22,000), 39NAH (w 21,000), 50NAH (w 21,000), 58NAH (w 21,000), and 70NAH (w 22,000), 92NAH (w 13,700), and 100NAH (w 15,700).
Glycol-split Heparins and Glycol-split N-Acetylated Heparins
Glycol-split heparins and glycol-split N-acetylated heparins were prepared by exhaustive periodate oxidation and borohydride reduction of heparin and N-acetylheparins, respectively, without (36) or with (37, 38) prior partial 2-O-desulfation. For the first series of glycol-split N-acetylheparins, 250-mg samples of H-1, 29NAH, 39NAH, 50NAH, 70NAH, and 100NAH were dissolved in 6 ml of H2O, and 6 ml of 0.1 M NaIO4 were added to the solutions. The solutions were stirred at 4 °C for 16 h in the dark. The reactions were stopped by adding 1 ml of ethylene glycol, and the solutions were dialyzed through 1000-Da cut-off tubes for 16 h. Solid sodium borohydride (60 mg) was added to the retentate solutions in several portions under stirring. After 2–3 h the pH was adjusted to 4 with 0.1 M HCl, and the solutions were neutralized with 0.1 M NaOH. After desalting and dialysis, the final products were recovered by freeze-drying to yield RO-H (w 15,700), 26NA,RO-H (w 17,000), 40NA,RO-H (w 16,000), 53NA,RO-H (w 11,250), 67NA,RO-H (w 15,000), and 100NA,RO-H (w 20,200). For the second series of N-acetylated, glycol-split heparins (NAH,gs), 250-mg samples of H-1, 29NAH, 39NAH, 58NAH, and 70NAH were dissolved in 5 ml of 1 M NaOH and then heated at 60 °C for 30 min. After cooling below 30 °C, the solutions were brought to pH 7 with 0.1 M HCl and heated at 70 °C for 48 h to give (after cooling, dialysis, and freeze-drying) partial conversion of IdoA2SO3 to GalA. Products were treated as described above to yield the corresponding glycol-split derivatives H,52gs (w 11,000), 29NAH,60gs (w 6,000), 43NAH,60gs (w 8,500), 57NAH,64gs (w 9,500), and 70NAH,59gs (w 9,300). The glycol-splitting (gs) percentages were evaluated by integration of the anomeric 13C NMR signals at 106.5 ppm (A) and at 102 ppm (B), corresponding to the split uronic acid residues and 2-O-sulfated iduronic acid residues, respectively; gs = (A/(A + B)) x 100. Products obtained without generation of additional nonsulfated uronic acid residues had a content of glycol-split residues (mainly arising from GlcA) of 24 ± 1% and are designated as "reduced oxyheparins" (RO-H) (36). Products obtained by glycol splitting of both the preexisting and the newly generated nonsulfated uronic acids (IdoA or GalA) were designated as H,xgs (or NAH,xgs if derived from N-acetylheparins) where the superscript x indicates the percentage of glycol-split uronic acid.
Low Molecular Weight Derivatives
Low molecular weight derivatives of H-1, H,44gs, and 50NAH,25gs (50NA,RO-H) were prepared by nitrous acid depolymerization of the corresponding polysaccharides (51). A solution of polysaccharide (4 g) was dissolved in 65 ml of H2O and cooled at 4 °C. After the addition of 75 mg of NaNO2 the pH was adjusted to 2 with 0.1 M HCl. The solution was stirred at 4 °C for 20 min, and then the pH was brought to 7. Solid NaBH4 (1 g) was added in several portions under stirring. After 2–3 h, the pH was adjusted to 4 with 0.1 M HCl, and the solution was neutralized with 0.1 M NaOH. The products (low molecular weight H-1, 6,500; LMWH,49gs, 6,300; LMWH,49gs, 3,000; and 50NA,RO-H, 5,400) obtained by precipitation with 3 volumes of ethanol were dissolved in water and recovered by freeze-drying. The depolymerization degrees and the corresponding molecular weight values were determined by integration of the 13C NMR signals at 98–107 and 82, 85, and 87 ppm, corresponding to total C-1 and C-2, C-3, and C-5 of the anhydromannitol unit, respectively. The percentage of glycol splitting, expressed as glycol-split residues referred to total uronic acids, was evaluated by integration of the 13C NMR signals at 106.5 and 102 ppm, corresponding to C-1 of the split uronic residues and 2-O-sulfated iduronic residues, respectively.
Cells
Cultures of bovine corneal endothelial cells were established from steer eyes and maintained in Dulbecco's modified Eagle's medium (1 g of glucose/liter) supplemented with 5% newborn calf serum, 10% fetal calf serum, and 1 ng/ml FGF-2 as described previously (4, 52). Confluent cell cultures were dissociated with 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS) and subcultured at a split ratio of 1:8 (52).
Preparation of Dishes Coated with ECM
Bovine corneal endothelial cells were plated into 35-mm tissue culture dishes at an initial density of 2 x 105 cells/ml and cultured as described above except that 4% dextran T-40 was included in the growth medium (4, 52). On day 12, the subendothelial ECM was exposed by dissolving the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH4OH followed by four washes with PBS (52). The ECM remained intact, free of cellular debris, and firmly attached to the entire area of the tissue culture dish. To produce sulfate-labeled ECM, Na 352SO4 (Amersham Biosciences) was added (25 µCi/ml) on days 2 and 5 after seeding, and the cultures were incubated with the label without a medium change and processed as described previously (4, 52). Nearly 80% of the ECM radioactivity was incorporated into HSPG.
Heparanase Inhibition Activity
Heparin species were tested for their ability to inhibit heparanase using metabolically sulfate-labeled ECM as a substrate (28, 29). Briefly sulfate-labeled ECM coating the surface of 35-mm culture dishes was incubated (4 h, 37 °C, pH 6.0) with recombinant human heparanase (40 ng/ml) in the absence and presence of three concentrations (0.2, 1.0, 5.0 µg/ml) of each heparin species. The reaction mixture contained 50 mM NaCl, 1 mM dithiothreitol, 1 mM CaCl2, and 10 mM phosphate-citrate buffer, pH 6.0. To evaluate the occurrence of proteoglycan degradation, the incubation medium was collected and applied for gel filtration on Sepharose 6B columns (0.9 x 30 cm). Fractions (0.2 ml) were eluted with PBS at a flow rate of 5 ml/h and counted for radioactivity. The excluded volume (Vo) was marked by blue dextran, and the total included volume (Vt) was marked by phenol red. Nearly intact HSPGs are eluted from Sepharose 6B just after the void volume (Kav < 0.2, fractions 1–10), while HS degradation fragments are eluted toward the Vt of the column (peak II, 0.5 < Kav < 0.8, fractions 15–35) (4, 28, 29). Labeled fragments eluted in peak II were shown to be degradation products of HS as they were 5–6-fold smaller than intact HS chains of HSPGs, resistant to further digestion with papain and chondroitinase ABC, and susceptible to deamination by nitrous acid (29). Heparanase activity = Kav x total cpm in peak II. Recovery of labeled material applied on the column ranged from 85 to 95% in different experiments. Each experiment was performed at least three times, and the variation in elution positions (Kav values) did not exceed ±15%.
Release of ECM-bound FGF-2
ECM-coated wells (4-well plates) were incubated with iodinated FGF-2 (1–2 x 105 cpm/ng, 1.5–2.5 x 104 cpm/0.25 ml/well, 3 h, 24 °C), and the unbound FGF-2 was removed by four washes with PBS containing 0.02% gelatin (34). The ECM was then incubated (3 h, 24 °C) with the various heparins and modified heparins, and aliquots (0.25 ml) of the incubation medium were counted in a 
-counter to determine the amount of released material. The remaining ECM was washed twice with PBS and solubilized with 1 N NaOH, and the radioactivity was counted in a -counter (34). The percentage of released 125I-FGF-2 was calculated from the total ECM-associated radioactivity. "Spontaneous" release of 125I-FGF-2 in the presence of incubation medium alone was 7–12% of the total ECM-bound FGF-2 (34). Each experiment was performed three to five times, yielding similar results.
Stimulation of FGF-2 Mitogenic Activity
A cytokine-dependent, heparan sulfate-deficient lymphoid cell line (BaF3) engineered to express fibroblast growth factor receptor-1 (53, 54) was applied to investigate the effect of heparin derivatives on FGF-2-mediated cell proliferation. These cells (clone F32) respond to FGF-2 only in the presence of exogenously added heparin, HS, or some modified species of heparin. Briefly F32 cells (2 x 104/well) were plated into 96-well microtiter plates in the presence of 2.5 or 5.0 ng/ml FGF-2 and increasing concentrations of the test compound in a total volume of 250 µl. Forty-eight hours later, 1 µCi of [3H]thymidine was added per well, and the cells were incubated for an additional 6 h and collected with a cell harvester. Incorporated thymidine was determined by liquid scintillation counting using a TopCount microplate counter (53, 54).
Gel Permeation Analysis of NAH and 50% Glycol-split Heparin (H,52gs) before and after Digestion with Heparanase
2 mg of each compound were incubated for 48 h at 37 °C in 40 mM phosphate-citrate buffer, pH 5.8, with or without 4 µg of recombinant heparanase in a total volume of 50 µl. The samples were lyophilized, then redissolved in 0.5 ml of water, and analyzed by GPC-HPLC using 300 x 7.8-mm TSK PW 2000 and PW 3000 columns.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Fig. 1 shows the general scheme for the preparation of derivatives of heparin, N-acetylheparins, and the corresponding glycol-split derivatives. Scheme 1 shows the formulas of heparin and heparin derivatives. The structure of a heparin chain is schematized as composed of N-acetylated (NA), N-sulfated (NS), and mixed NA/NS domains; the prevalent sequences are those of the trisulfated disaccharide (formula 1) (24). For simplicity, only chains containing the antithrombin binding region are presented, although this region is contained in only about one-third of the chains of heparin. The prevalent structure of 6-O-desulfated heparins is shown in formula 2, and that of 2-O-desulfated heparins retaining the IdoA configuration is shown in formula 3. Formula 4 shows the prevalent structure of heparins that underwent inversion of configuration to GalA during 2-O-desulfation of IdoA. Fully N-acetylated heparins are represented by the general formula 5. Partially N-acetylated heparins have different percentages of N-acetyl groups; the NSO3 groups are the complement to 100% N-substitution. Glycol splitting is depicted in Fig. 1 in two different ways. Fig. 1A refers to heparin and fully N-acetylated heparin (NAH) glycol-split without any previous modification of their structures. The corresponding glycol-split products are referred to as RO-H (36) and "N-acetyl-reduced oxyheparins" (NA,RO-H), respectively. Fig. 1B refers to heparin and NAH (in the example, 50% N-acetylated heparin 50NAH) that were glycol-split after removal of 2-OSO3 groups to reach a total content of 50% nonsulfated uronic acid residues; the nonmodified residues were IdoA2SO3 (37). De-O-sulfation of one of two IdoA residues followed by glycol splitting gave heparins with prevalent structure 6, corresponding to sequences of polypentasulfated trisaccharides separated by glycol-split uronic acid residues (37). Low molecular weight species of heparin and representative heparin derivatives were obtained by controlled nitrous acid depolymerization (51) of heparin, 50% glycol-split heparin, and the RO derivative of 50% N-acetylated heparin.
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70% inhibition at 1 µg/ml). No significant differences in inhibitory activity were found among H-1 and other heparin preparations from pig mucosa, beef mucosa, and beef lung (data not shown) despite significant differences in their sulfation patterns (detailed under "Experimental Procedures"). Also activity differences found between the parent heparin and its low molecular weight species as well as between glycol-split 50NAH and its low molecular weight species were not significant. On the other hand, well defined significant differences in heparanase-inhibiting activity were associated with specific chemical modifications of heparin. As illustrated in Fig. 3, whereas either 6-O-desulfation or 2-O-desulfation with retention of IdoA configuration had little or no effect on the heparanase-inhibitory activity of heparin, 2-O-desulfation with change of configuration of the IdoA residues to GalA markedly decreased the inhibitory activity of heparin.
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50%. On the other hand, glycol splitting markedly increased the heparanase-inhibiting activity of both heparins and N-acetylated heparins and restored the inhibitory effect lost upon N-acetylation of heparin (Fig. 4 and Table I). This effect is illustrated in Fig. 4 and Table I for N-acetylated heparins of the RO type (i.e. 25% glycol-split), which almost completely inhibited the heparanase activity (to less than 10% of the control at 1 µg/ml and to 20–30% at 0.2 µg/ml) irrespective of their degree of N-acetylation. Glycol splitting extended to newly generated nonsulfated IdoA/GalA residues in heparin and N-acetylated heparins gave products showing high heparanase-inhibitory activity. The dose dependence of the heparanase-inhibitory activity is illustrated in Fig. 5 for heparin, fully N-acetylated heparin, and its RO derivative. IC50 values calculated from the corresponding curves are >5 µg/ml for NAH, 0.4 µg/ml for H-1, and 0.2 µg/ml for 100NA,RO-H.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-glycosidic linkage of a GlcA residue, which must be flanked by N-sulfated or N-acetylated 
-linked GlcN residues. At least one OSO3 group is essential for efficient recognition by the enzyme (40, 41). The three-dimensional structure of heparanase is not yet known in detail. Translation of the primary structure of heparanase over another endo--glycosidase (-xylanase) shows clusters of basic amino acid residues at least one of which is conceivably implicated in binding to sulfate groups of the substrate (55). Our preliminary studies, applying point mutations and deletions as well as synthetic peptides, identified amino acid residues 158–171 as the predominant HS binding domain of the heparanase molecule.2 HS/heparin and derived oligosaccharides must have a minimal octasaccharidic size to be good substrates for heparanase (40, 41). However, the enzyme also can be efficiently inhibited by shorter but more extensively sulfated oligosaccharides such as maltohexaose polysulfate and phosphomannopentaose polysulfate (PI-88) (22). Heparin, although less sulfated than these persulfated oligosaccharides, is a good inhibitor of heparanase activity (28) and is active in experimental metastasis models as well (20–22). The inhibitory activity of heparin is lost upon extensive O-desulfation and/or a decrease in chain length below a tetradecasaccharidic size (20, 34).
Previous reports on the effects of selective O-desulfation on the heparanase-inhibiting activity of heparin (35) are essentially confirmed by the present study. In fact, removal of either the 6-O-sulfate group on the glucosamine residue or the 2-O-sulfate group on the iduronic acid residue only slightly reduced the inhibitory activity of heparin (Fig. 3).
The decrease in inhibitory activity observed in the present study for extensively 6-O-desulfated heparin prepared by the solvolytic method appears to be associated with a concomitant partial 2-O-desulfation. On the other hand, the consistently lower heparanase-inhibiting activity of partially 2-O-desulfated derivative obtained with procedure B (involving a change of configuration from IdoA to GalA) as compared with that of the 2-O-desulfated derivative obtained by procedure A (with retention of the IdoA configuration) is likely associated with different conformational properties of IdoA and GalA. IdoA residues have been demonstrated to be endowed with a unique conformational flexibility ("plasticity"). Such a plasticity, associated with different equienergetic conformations of IdoA residues, all coexisting in a rapid dynamic equilibrium, can currently explain the better protein binding capacity and associated biological properties of IdoA-containing sequences as compared with the more rigid GlcA-containing ones (56). Based on simple conformational criteria, GalA is expected to have very much the same conformational rigidity as GlcA.
Replacement of N-sulfate groups with N-acetyl groups does not completely suppress the heparanase-inhibitory activity of heparin (34); its activity is reduced to about one-third (35) as confirmed by the present study. However, it is noteworthy that a substantial decrease in heparanase-inhibiting activity was only observed for degrees of N-acetylation higher than about 50%. As shown in Table I and illustrated in Fig. 4, for degrees of N-acetylation lower than 40%, the inhibitory activity remained essentially the same as that of heparin. This is taken as an indication that only one-half of the NSO3 groups of heparin are essential for complete inhibition of heparanase. The accepted conformation of the NS domains of heparin is represented by helices where sets of three sulfate groups (NSO3, 2SO3, and 6SO3) alternate on each side of the chain (57). The observation that only one of two N-sulfate groups is required to inhibit heparanase and the assumption that the N-desulfation/N-acetylation reaction occurs randomly along the NS domains would accordingly suggest that heparin and its derivatives with unmodified backbones bind the enzyme only from one side of the chain.
The effect of glycol-splitting of heparin on heparanase inhibition is largely new. Lapierre et al. (35) reported that periodate oxidation/borohydride reduction of nonsulfated uronic acids of heparin, leading to a product corresponding to H,25gs of the present study, did not impair the inhibitory activity of heparin, a finding taken by these authors as an indication that nonsulfated IdoA is not essential for the activity. The present results on heparins subjected to glycol splitting only at the level of preexisting nonsulfated uronic acids (RO-H) (36) and of both the preexisting and the newly generated ones (such as H,52gs) (37, 38) indicate that in fact glycol splitting resulted in a marked general increase in the inhibition of the heparanase activity by heparin species. A reasonable explanation of such an effect is that formation of glycol-split residues, involving elimination of conformational constraints with formation of three additional degrees of rotational freedom per each split residue, generates flexible joints that separate from each other heparin sequences containing IdoA2SO3 residues, thus facilitating the docking of these sequences to sites essential for heparanase activity. The proposed "extra flexibility" (39) induced by these joints reinforces the binding driving influence of the already existing intrinsic conformational plasticity of iduronate residues. More notably, glycol splitting also increased the heparanase-inhibiting activity of N-acetylated heparins, even that of fully N-acetylated heparin, whose heparanase binding capacity is very weak when their backbone is unmodified.
Glycol splitting involves substantial loss of the anticoagulant activity of heparin (36). It is now clear that the main reason for such an effect is the cleavage of C-2–C-3 bonds of the GlcA residue of the pentasaccharidic sequence (Fig. 1, formula 7). This residue in its unmodified form is essential for binding to antithrombin, and whenever it is glycol-split (as in sequence 8), the heparin affinity for antithrombin is completely lost (Ref. 24 and references therein).
The heparanase inhibiting properties of glycol-split, extensively N-acetylated heparins cannot be explained by the model discussed previously where only sulfate groups (perhaps in addition to the uronate carboxyl groups) on the same side of the heparin helix are involved in binding to the enzyme. Such a model is compatible with heparanase binding of heparins that contain no more than 50% N-acetyl groups. Retention of strong inhibitory activity upon removal all NSO3 groups and their substitution with nonpolar N-acetyl groups followed by glycol splitting of nonsulfated uronic acids implies that efficient docking to heparanase, favored by the flexible joints generated by glycol splitting, occurs with a conformation different from that adopted by heparin with a degree of N-acetylation lower than 50%.
Conformational polymorphism is not uncommon in heparin sequences bound to proteins. Thus, x-ray studies showed that heparin sequences may bind FGF-1 in more than one, equally favored conformation (58). Increasing evidence is being accumulated that the plasticity of iduronate residues combined with some rotational freedom of both uronic acid and amino sugar residues around the glycosidic linkages favors several possibilities of binding to basic domains of proteins (59). As expected (37, 39), glycol splitting appears to further increase the molecular flexibility of heparin chains as determined by small angle x-ray scattering (60) and NMR spectroscopy supported by molecular modeling studies (61).
It appears that heparin contains both recognition/cleavage and inhibition sites for heparanase and that its inhibition of the enzyme also involves competition (40). Size profiling of HS degradation products by heparanase in the presence of N-acetylheparin (Fig. 6A) showed that the fully N-acetylated derivative NAH is also cleaved by the enzyme. On the contrary, the gel filtration profile obtained in the presence of glycol-split heparin is practically superimposable to that obtained in the absence of the enzyme (Fig. 6B), indicating that modification of GlcA residues totally abolishes cleavage by the enzyme.
HS assembles ligands and receptors into ternary signaling complexes, best exemplified by the FGF-fibroblast growth factor receptor-heparin complex (62, 63). Following cleavage by heparanase, the multitude of polypeptides sequestered and regulated by HS (17) become bioavailable (1, 18), and this requires a tight regulation of their activity, applying, among other approaches, modified species of heparin and HS. The present results on the effect of glycol splitting on the ability of heparins to release FGF-2 from ECM extend previous observations on the effect of O-sulfation and N-acetylation on this property (34). As illustrated in Fig. 7, glycol-split derivatives of both heparin (including LMWH) and NAH did not substantially modify the ability to displace FGF-2 from ECM. In other words, while both native and glycol-split heparins efficiently released FGF-2 from ECM, their N-acetylated counterparts exhibited a markedly reduced ability to displace FGF-2, reflecting the essential involvement of N-sulfate groups in this interaction. This observation supports the finding that glycol-split heparins bind FGF-2 (37) and vascular endothelial growth factor (64) with very much the same affinity as unmodified heparins. However, since H,50gs inhibits dimerization of FGF-2 (37), it is conceivable that the ECM-bound growth factor is released by glycol-split heparins in an inactive form. Moreover we demonstrated that unlike native heparin and LMWH, NAH and even more so glycol-split NAH (100NAH,25gs, i.e. NA,RO-H) failed to stimulate FGF-2-mediated proliferation of HS-deficient lymphoid cells (Fig. 8). The remarkable heparanase-inhibitory activity of N-acetylated, glycol-split heparins together with the low levels of FGF-2 that they release from ECM and their inability to stimulate the mitogenic activity of FGF-2 indicates this class of chemically modified heparins as potential antiangiogenic and antimetastatic agents. These compounds also markedly inhibit wound angiogenesis in transgenic mice overexpressing the heparanase gene (65). Furthermore our preliminary experiments show that some of these heparin derivatives effectively abolish experimental lung colonization of intravenously administered B16-BL6 mouse melanoma cells (Ref. 66).
Retrospective analyses suggest that treatment of venous thromboembolism in cancer patients with LMWHs is associated with additional benefits in terms of their survival (33). The experiments presented in this study were undertaken to develop heparin-based molecules for efficient inhibition of heparanase activity. Polysulfated chains such as those of heparin are expected to envelop the basic clusters of heparanase and compete with its binding to HS. Confirming previous findings (34), we showed that such an activity is retained by a low molecular weight heparin. Moreover we also demonstrated such a retention for three representative low molecular weight glycol-split derivatives. Further studies are planned to determine, for each type of derivative, the shortest chains as well as the shortest IdoA2SO3-containing sequences still showing significant inhibition of the enzyme and to elucidate whether the carboxylate groups of glycol-split residues participate in binding and inactivation of heparanase.
In conclusion, we applied desulfation strategies and controlled glycol splitting to remove sulfate groups not necessarily involved in heparanase recognition and inhibition and to improve the molecular flexibility and biological interactions of heparin. Generation of specific heparanase-inhibiting compounds such as those described in this study is important not only as a proof of concept but also as a promising approach to develop heparin-based anticancer lead compounds devoid of side effects.
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To whom correspondence should be addressed. E-mail: casu{at}ronzoni.it.
1 The abbreviations used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; ECM, extracellular matrix; FGF-2, basic fibroblast growth factor; FGF-1, acidic fibroblast growth factor; IdoA, L-iduronic acid; GlcA, D-glucuronic acid; GlcN, D-glucosamine; GlcNAc, N-acetyl-D-glucosamine; GlcNSO3, D-glucosamine N-sulfate; LMWH, low molecular weight heparin; GPC-HPLC, gel permeation chromatography-high performance liquid chromatography; NA, N-acetylated; NAH, N-acetylheparin; NS, N-sulfated; gs, glycol-split; RO-H, reduced oxyheparin; PBS, phosphate-buffered saline; OdeS, O-desulfated; GalA, L-galacturonic acid; w, weight average molecular weight.
2 F. Levy-Adam, G. Abboud-Jarrous, M. Guerrini, D. Beccati, I. Vlodavsky, and N. Ilan, in preparation.
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