Tumor attenuation by 2(6-hydroxynaphthyl)-ß-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

Katrin Mani1,2, Mattias Belting2, Ulf Ellervik3, Niklas Falk3, Gabriel Svensson2, Staffan Sandgren2, Fang Cheng2 and Lars-Åke Fransson2

2 Department of Cell and Molecular Biology, Biomedical Centre, Lund University, SE-221 84 Lund, Sweden, and 3 Organic and Bioorganic Chemistry, Centre for Chemistry and Chemical Engineering, Lund University, POB 124, SE-221 00, Lund, Sweden

Received on May 27, 2003; revised on October 26, 2003; accepted on November 17, 2003


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 Abstract
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 Results
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We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide–dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70–97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.

Key words: heparan sulfate / nitric oxide / tumor growth / xyloside


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Xylose (Xyl) is a unique component of proteoglycans (PGs) and growth of tumor cells is dependent on PG expression (Esko et al., 1988Go). Xylose is the first sugar added to the nascent PG core protein when the posttranslational assembly of the glycosaminoglycan (GAG) side chains is initiated (for review, see Fransson et al., 2000Go). During transport through the secretory pathway the GAG chains are polymerized on the common linkage region GlcUA-Gal-Gal-Xyl-protein. This saccharide serves as primer for either chondroitin sulfate (CS) type or heparan sulfate (HS) type GAG chain synthesis.

Biosynthesis of GAG chains can also take place independently of core protein synthesis by using xylosides as primers. Xylosides with hydrophobic aglycons that can penetrate cell membranes will initiate synthesis by serving as acceptors in the first galactosylation step. In most cases, priming of CS dominates and synthesis of free HS chains is low or undetectable. Increased yields of HS can be obtained when the aglycons of the xylosides comprise aromatic, polycyclic structures, such as naphthol derivatives (for references see Belting et al., 2002Go).

We have previously reported that the naphthol-based and both HS- and CS-priming glycoside 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) inhibits growth of transformed or tumor-derived cells to a greater extent than that of normal cells (Mani et al., 1998Go). The underlying mechanism has not been clarified. However, priming of HS synthesis appeared to be a necessary but insufficient requirement. A unique and important step in HS biosynthesis is the regional deacetylation of GlcNAc moieties in the (GlcUA-GlcNAc)n backbone to GlcNH2 followed by N-sulfation, yielding GlcNSO3. However, a few glucosamine residues may remain N-unsubstituted (for review, see references in Fransson, 2003Go). We have shown (Ding et al., 2001aGo; Mani et al., 2000Go) that during intracellular recycling of the HS PG Gpc-1, HS chains are cleaved at these GlcNH2 residues by endogenously produced nitric oxide (NO)–generating products that contain reducing terminal anhydromannose (anMan). Some of the degradation products also appear in the nucleus (Belting et al., 2003Go). Growth inhibition by Xyl-2-Nap-6-OH may depend on the nature of the aglycon or may be due to xyloside hydrolysis generating toxic products or to HS priming and nuclear targeting of antiproliferative products generated by NO-dependent deaminative cleavage.

To explore these possibilities, we studied the antiproliferative effect of different synthetic analogs of Xyl-2-Nap-6-OH on normal, mutated, or transformed cell lines in vitro. We have also examined their ability to initiate HS biosynthesis and whether xyloside-primed HS chains are degraded by NO and transported to the nucleus. Finally, we tested the ability of Xyl-2-Nap-6-OH to inhibit tumor formation in vivo.


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Effect of Xyl-2-Nap-6-OH (compound 1) on cell growth in vitro
As summarized in Table I, the xyloside Xyl-2-Nap-6-OH (1) has a moderate or minimal inhibitory effect on normal fibroblasts, such as human fibroblasts from lung (HFL-1 s) (ED50 0.25 mM); endothelial cells, such as human umbilical vein endothelial cells (HUVECs) (ED50>0.50 mM); and epithelial cells, such as Chinese hamster ovary (CHO-K1) (ED50 0.20 mM). In contrast, tumor-derived or transformed cells, that is, A549 (ED50 0.13 mM), 3T3-SV40 (ED50 0.03 mM), T24 (ED50 0.05 mM), and HepG2 (ED50 0.07 mM) were growth inhibited at lower concentrations of xyloside. In the case of 3T3-A31 cells, the inhibitory effect was obtained at an intermediary concentration (ED50 0.17 mM).


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Table I. Effect of Xyl-2-Nap-6-OH, xyloside 1, on cell proliferation

 
Effect of different xyloside analogs on cell growth in vitro
A comparison between different xyloside analogs (Table II) indicated that Xyl-2-Nap (2) and Xyl-2-Nap-6-OMe (3) were both essentially inactive on the cell lines tested (HFL-1, A549, and T24). Hence, a free hydroxyl opposite the Xyl position appeared to be necessary for bioactivity. To determine whether the 2,6-orientation was obligatory, the effect of the isomeric Xyl-1-Nap-5-OH (4) was examined. This xyloside had a similar and moderate effect on HFL-1 (ED50 0.27 mM) and T24 cells (ED50 0.20 mM). However, on A549 cells xyloside 4 was almost as effective as 1 (ED50 0.15 mM and 0.13 mM, respectively). Another 2-xylosylated dihydroxynaphthol but with the free hydroxyl in a different position (Xyl-2-Nap-7-OH, compound 5) was also tested. This xyloside proved to be essentially inactive on all cell lines tested. Finally, a xyloside with a single aromatic ring in the aglycon but with the free hydroxyl opposite the Xyl (Xyl-1-Phe-4-OH, compound 6) was also inactive on T24 cells.


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Table II. Effect of aglycon ring modification on the ability of xylosides to affect cell growth

 
The effect of the various aglycons that were part of xylosides 1–5 was also tested on cells (Table II). All of the free aglycons appeared to be quite toxic, even those that were part of an essentially inactive xyloside (2, 3, and 5). Even the cell number in confluent cultures was markedly reduced (data not shown).

Antiproliferative activity in vitro and GAG priming
The antiproliferative effect of xyloside 1 (Xyl-2-Nap-6-OH) was tested on various CHO cell lines with normal or depressed production of GAG (Table III). Wild-type CHO-K1 cells had an ED50 of 0.20 mM, whereas CHO cells with a defect in galactosyltransferase I (pgsB-618), which precludes all GAG priming by xyloside (for references see Belting et al., 2002Go), were completely insensitive. CHO cells, which can produce CS but not HS (pgsD-677), were also completely insensitive, indicating that HS production is required for the generation of antiproliferative activity.


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Table III. Effect of Xyl-2-Nap-6-OH, xyloside 1, on proliferation of wild-type, GAG-deficient, and HS-deficient CHO cells

 
The type of GAG chains primed by the different xylosides at a concentration of 0.1 mM were examined. Both the GAG chains secreted into the medium and those in the cells were isolated and separated on Superose 6. The chromatograms obtained after treatment with Xyl-2-Nap (2) are shown in Figure 1. All the other xylosides tested yielded similar patterns (data not shown) and the results are summarized in Table IV. In normal cells (HFL-1), the xylosides generated a large pool of free GAG chains that were secreted into the medium (see bar in Figure 1A, solid line). Cells treated with xylosides 2, 3, and 4 secreted exclusively CS whereas xyloside 1 also primed HS (1–4 in Table IV).



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Fig. 1. GAG priming by 2-naphthyl-O-ß-D-xylopyranoside in (A, B) HFL-1 and (C, D) T24 cells. Polyanionic macromolecules from the culture medium (A, C) and the cells extracts (B, D) of confluent cells in 9-cm2 dishes in the presence of 0.1 mM xyloside (solid line; dashed line, results obtained in the absence of xyloside) were isolated as described in Materials and methods and subjected to gel exclusion chromatography on Superose 6. The cell culture material pooled as indicated by bars in A, C was alkali-resistant (data not shown). The HS content was determined as described in Materials and methods and is shown in Table IV together with results obtained with the other xylosides that were tested. In the cell extracts three pools (I–III) were obtained. Pool I contained both PG material (alkali-sensitive) and long GAG chains (alkali-resistant; data not shown). The HS content of the GAG component remaining in pool I after alkali treatment and that in pools II (when present) and III was determined as described in Materials and methods.

 

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Table IV. Effect of aglycon ring modification on heparan sulfate priminga

 
Much less GAG was generally recovered from the extracts of HFL-1 cells than from the medium (Figure 1B, solid line). Cell-associated, free GAG chains included one pool of large-size material (present in pool I) and one pool eluting in the same position as the secreted GAG chains (pool II). The relative contents of HS in the large and small GAG pools generated by the different xylosides are shown in Table IV. No HS was detected in large-size material from cells treated with xyloside 1. However, the smaller material (pool II) contained HS (42% of total GAG). When xylosides 2, 3, and 4 were used, the HS content of the large-size GAG pool ranged from 33–40% and that of the smaller-size from 35–47%. Hence, both the potentially bioactive xyloside 1 and the three closely related but inactive ones were able to prime HS synthesis.

In cultures of tumor-derived T24 cells, which were sensitive to growth inhibition by xyloside 1, the various xylosides generated less secreted GAG chains (see bar in Figure 1C) than in HFL-1 cells (Figure 1A). The relative amount of HS in the GAG pool secreted by T24 cells ranged from 12–49% (1–4 in Table IV) with the lowest yield from cells treated with the bioactive xyloside (1).

In the cell extract of T24 cells not treated with xyloside, there are both PGs eluting in fractions 25–35 and endogenously generated HS oligosaccharides eluting in fractions 45–55 (see dashed line in Figure 1D). The HS degradation products result from extensive turnover of the endogenous, recycling HS PG Gpc-1 (Ding et al., 2001aGo, 2002Go; Cheng et al., 2002Go). In the present experiments GAGs from pool II and GAG oligosaccharides (pool III) were analyzed. When cells were treated with the bioactive xyloside 1, no HS was recovered from pool II. When cells were treated with the inactive xylosides 2, 3, and 4, this GAG pool contained 40–47% HS (Table IV). The HS-content of the oligosaccharide (pool III) ranged between 30% and 57% for xylosides 1–4, with the lowest yield from 1 (Table IV). Overall, fewer HS chains were obtained from T24 cells than from HFL-1 cells. This could be due to diminished HS production or increased HS degradation in the sensitive T24 cells.

Nuclear localization of xyloside-primed HS
We have recently demonstrated that during Gpc-1 recycling, HS is degraded both enzymatically by heparanase and nonenzymatically by NO, in the latter case generating oligosaccharides terminating with anMan (Ding et al., 2001aGo, 2002Go; Cheng et al., 2002Go). To identify the cellular localization of xyloside 1–primed HS degradation products, we used confocal immunofluorescence microscopy. A polyclonal antiserum against human Gpc-1 (Ding et al., 2001aGo) and a monoclonal antibody against an epitope comprising the anMan residues of deaminatively cleaved HS (Ding et al., 2002Go) were used.

In untreated subconfluent HFL-1 cells, there was ongoing deaminative cleavage of Gpc-1 HS chains resulting in partial colocalization between Gpc-1- and anMan-containing degradation products (yellow in Figure 2A). Treatment of HFL-1 cells with xyloside 1, which did not markedly inhibit growth (Table I), appeared to interfere with the NO-dependent degradation process (less green in Figure 2B). In contrast, T24 cells, which did not contain anMan-positive degradation products (Figure 2C) and were growth inhibited by xyloside 1 (Table I), accumulated anMan-containing degradation products in the nucleus after treatment with xyloside 1 (green in Figure 2D). Formation and nuclear targeting of such degradation products was markedly inhibited by quenching of NO/nitrite with sulfamate (Figure 2E), confirming that the products were generated by deaminative cleavage. When T24 cells were exposed to the inactive xyloside 2, some anMan-containing products were generated, but they were mainly at paranuclear sites, colocalizing with Gpc-1 (Figure 2F).



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Fig. 2. Effect of xylosides on the formation of deaminatively generated HS degradation products. HFL-1 (AB) or T24 cells (CF) were either left untreated (A, C) or were treated with 0.1 mM Xyl-2-Nap-6-OH, xyloside 1 (B, D, and E) or with 0.1 mM Xyl-2-Nap, xyloside 2 (F) for 24 h. In E, cells were also exposed to 10 mm ammonium sultamate. 6pc-7 (red) was detected using polyclonal antiserum and HJ degradation products by using a mAb directed against an Mcn-containing HS oligosaccharides (green). Only the merged images are shown. Scale bar, 20 µm.

 
It should be added that the anMan-containing epitope makes up a tetrasaccharide motif and that free anMan is not detected by the monoclonal antibody. Hence, in untreated T24 cells (Figure 2C) anMan-containing oligosaccharides may have been generated but further degraded into monosaccharides. HS chains primed by either xyloside 1 or 2 may have inhibited this last step, resulting in the appearance of anMan-containing oligosaccharides (extranuclear yellow in Figure 2D and 2F).

Antiproliferative activity in vitro and HS degradation
To further examine whether degradation and processing of xyloside-primed HS was involved in the generation of growth inhibition, we interfered with the enzymatic and nonenzymatic degradation steps. By inhibiting heparanase with suramin and NO-dependent deaminative cleavage by addition of sulfamate (Ding et al., 2001aGo) we tested whether the growth inhibition exerted by Xyl-2-Nap-6-OH (xyloside 1) could be counteracted.

As shown in Figure 3, xyloside 1 inhibited growth of T24 cells in a concentration-dependent manner with an ED50 of 0.05 mM. Prevention of deaminative cleavage alone had no major effect on growth up to a concentration of 6 mM sulfamate (~90% of control growth). However, simultaneous treatment with this agent and xyloside 1 counteracted the growth-inhibitory effect of the xyloside. Hence, 6 mM sulfamate restored growth from ~45% to 75% of control in cells exposed to 0.05 mM Xyl-2-Nap-6-OH and from ~30% to 65% in cells exposed to 0.1 mM xyloside. Suramin, which per se is moderately growth-inhibitory to T24 cells (~80% of control growth at 0.2 mM) did not affect the antiproliferative activity of xyloside 1 (data not shown), indicating that cleavage by heparanase was not required for growth inhibition.



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Fig. 3. Effect of sulfamate on growth inhibition exerted by 2-(6-hydroxynaphthyl)-O-ß-D-xyloside. Growth of T24 cells in the presence of Xyl-2-Nap-6-OH or ammonium sulfamate alone and in the presence of combinations of xyloside and ammonium sulfamate was determined as described in the Materials and methods. Cell number was determined after 96 h of culturing and expressed as the percentage of growth in the absence of drugs.

 
Effect of NO depletion on GAG priming by xyloside 1 in vitro
If growth inhibition by Xyl-2-Nap-6-OH-primed HS required generation of NO to achieve partial degradation of HS at the GlcNH2 residues, then more xyloside-primed HS chains should be obtained from NO-depleted cells. In this case we used sulfamate to inhibit deaminative cleavage, aminoguanidine to inhibit NO synthase, and neocuproine to inhibit release of NO from nitrosothiols (Ding et al., 2001aGo, 2002Go). As shown in Figure 4, when T24 cells were treated with xyloside 1 during NO depletion, the yield of HS increased. In the secreted xyloside-primed GAG pool (pool II in Figures 4A and 4C, solid lines) the proportion increased almost four-fold. In the cell-associated low-molecular-weight pool (pool III in Figures 4B and 4D, solid lines), the proportion of HS increased more than twofold.



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Fig. 4. Effects of NO depletion on 2-(6-hydroxynaphthyl)-O-ß-D-xyloside-primed HS production in T24 cells. Cells grown to confluence in 25-cm2 dishes were preincubated for 24 h in the arginine-poor medium 199 with no additional substances (A, B) or in the presence of 10 mM ammonium sulfamate, 10 mM aminoguanidine, and 0.1 mM neocuproine (C, D). Thereafter, 50 µCi/ml 35SO4 was added, and cultures were incubated for an additional 24 h with 0.1 mM Xyl-2-Nap-6-OH (A, B) or 10 mM ammonium sulfamate, 10 M aminoguanidine, 0.1 mM neocuproine, and 0.1 mM Xyl-2-Nap-6-OH (C, D). Polyanionic macromolecules from the culture medium (A, C) and the cell extracts (B, D, solid line; dashed lines, results obtained in the absence of xyloside) were isolated as described in Materials and methods and subjected to gel exclusion chromatography on Superose 6. Material was pooled as indicated, and the HS content in pools obtained from xyloside-, and xyloside- and drug-treated cells was determined as described in Materials and methods.

 
Potentiation of xyloside-induced growth inhibition by {alpha}-difluoromethylornithine (DFMO)
Cellular proliferation also depends on an adequate supply of polyamines (putrescine, spermidine, and spermine) and inhibitors of polyamine synthesis, such as DFMO, have been used to treat cancer. DFMO increases the GlcNH2 content of HS in Gpc-1 (Ding et al., 2001bGo) and stimulates polyamine uptake (for a comprehensive review, see references in Thomas and Thomas, 2001Go). We have shown that Gpc-1 is a carrier during polyamine uptake (Belting et al., 1999Go, 2003Go; Ding et al., 2001aGo) and that tumor attenuation can be achieved by combined treatment using DFMO to inhibit polyamine synthesis and naphthol-based xylosides to interfere with HS PG synthesis (Belting et al., 2002Go). We therefore tested whether the antiproliferative effect of xyloside 1 was potentiated by pretreatment of cells with DFMO. We compared HFL-1 and A549 cells because the latter required a relatively high concentration of xyloside 1 for inhibition (ED50 0.13 mM). As shown in the notes to Table I the ED50 for HFL-1 cells was unchanged, whereas A549 cells were growth-inhibited at a fourfold lower xyloside concentration (ED50 0.03 mM), when cells were made dependent on polyamine uptake.

Antitumor effect of xyloside 1 in SCID mice
To study potential toxicity, bioavailability and antiproliferative activity of Xyl-2-Nap-6-OH in vivo, a subcutaneous tumor model was established in female severe combined immunodeficiency SCIDnodCA mice. Human bladder carcinoma T24 cells formed subcutaneous tumors that were clearly palpable after 10–14 days of incubation. Initial screening for xyloside toxicity, as described in Materials and methods, showed no adverse effects in treated mice, that is, no toxic symptoms or loss in body weight were observed. No deaths occurred in the course of the experiments. In the first set of tumor growth experiments, mice received xyloside 1 in the drinking water (1.7 mM) and/or by daily injections subcutaneously (0.5 ml, 1.7 mM) for a period of 19 days. As shown in Figure 5A, tumor load (T24 cells) was substantially reduced after both peroral (~70% growth inhibition as compared with control) and subcutaneous (~96% growth inhibition) administration. Combined administration resulted in ~97% reduction in average tumor mass, and in one individual no tumor was found. In another set of experiments, xyloside 1 was injected intraperitoneally (Figure 5B), showing 80% reduction in average tumor mass as compared with control (n = 8, p<0.001). In the experiments described, xyloside treatment was initiated concomitant with inoculation of T24 cells. To test the effect of xyloside on preformed tumors, mice were inoculated with T24 cells and left untreated for a period of 3 weeks and then treated with xyloside for another 3 weeks (Figure 5C). Mice that received xyloside subcutaneously or intraperitoneally had an average tumor load of ~40–50 mg compared to ~135 mg in control animals, which corresponds to ~70% growth inhibition of established tumors.



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Fig. 5. Inhibition of in vivo tumor growth by Xyl-2-Nap-6-OH (xyloside 1). Scatter plots showing the end tumor mass in mice that were injected with human bladder carcinoma T24 cells. In (A) and (B), xylosides were administered perorally (1.7 mM ad libitum) and/or subcutaneous (0.5 ml, 1.7 mM) concomitant with tumor cell inoculation, whereas in (C) animals with preformed tumors received xyloside treatment via intraperitoneal (0.5 ml, 1.7 mM) or subcutaneous (0.5 ml, 1.7 mM) injections. Bar indicates average tumor load (n = 4–8 for each group).

 

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The growth inhibition exerted by xyloside 1 could be due to cleavage of the xyloside by glycosidase, generating a toxic aglycon, or to inhibition of HS PG synthesis or to generation of antiproliferative xyloside-primed HS chains or oligosaccharides. Xyloside cleavage is unlikely because xylosides 1 and 5 differ only with respect to the position of the free OH group and ought to be equally cleavable, generating toxic aglycons (Table II). Yet xyloside 5 was completely inactive. Furthermore, HS-deficient cells, which have no known glycosidase defect, were insensitive to xyloside 1.

The latter result also shows that priming of HS synthesis by xyloside is necessary for growth inhibition. Xylosides can also compete with GAG assembly onto PG core proteins. However, much higher concentrations of xylosides are usually required to disturb PG synthesis (Moses et al., 1999Go).

HS degradation products were detected in the nuclei of T24 cells after treatment with the antiproliferative xyloside 1. The products were generated via NO-dependent cleavage at GlcNH2 residues. This cleavage appears to be a prerequisite for antiproliferative activity because NO depletion counteracted the effect of the xyloside. The selective growth-inhibitory effect of xyloside 1 on transformed cells was sensitive to alterations in the hydroxyl substitution pattern of the naphthol rings. Both the HS component and the aglycon may be necessary for nuclear targeting and ensuing growth inhibition. The selective sensitivity of transformed or tumor-derived cells may partly be due to their increased capacity to generate NO (Doi et al., 1996Go; Thomsen and Miles, 1998Go) as well as to their greater dependence on supply of polyamines (Belting et al., 1999Go; Thomas and Thomas, 1998). Synergistic effects between xylosides, DFMO, and NO-donating or NO-releasing compounds may thus be anticipated.

Animal experiments indicated that xyloside 1, administered in various ways was adsorbed and made available to tumor cells located subcutaneously. Treatment with xyloside 1 reduced the average tumor load by 70–97% in mice receiving the xyloside concomitant with tumor cell inoculation as well as in mice with preformed tumors. Because virtually all cells in the body have the capacity to synthesize GAG on xylosides, the lack of obvious toxic effects in vivo suggests that nontumor cells are unable to transform HS-primed xyloside 1 into antiproliferative compounds in significant amounts. Side effects via inhibition of PG synthesis in cartilage and connective tissues are unlikely, because xyloside concentrations greater than 2 mM are required for their inhibition (Moses et al., 1999Go).

Recent studies in another laboratory have shown that exogenously supplied HS oligosaccharides generated by HS lyase (also known as heparinase-III) inhibit tumor growth in vivo (Liu et al., 2002Go). Our approach may have generated similar HS oligosaccharides in situ using low-molecular-weight xylosides that easily penetrate the cell membranes. To further investigate this approach, other hydroxylation variants of the present naphthol-based xyloside lead compound, as well as radioactively labeled xylosides, are currently being synthesized.


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Cells, enzymes, chemicals, and columns
HUVECs were the same as used previously (Mani et al., 1998Go). The human bladder carcinoma cell line T24 and the transformed human cell line HepG2 were obtained from Prof. Inge Olsson and Prof. Åke Nilsson, respectively, Department of Medicine, Lund University. HFL-1 and lung carcinoma cells (A549), normal and SV40-transformed mouse 3T3 fibroblasts, wild-type CHO-K1 cells and CHO cells deficient in total PG (pgsB-618) or HS biosynthesis (pgsD-677) were obtained from ATCC (Rockville, MD). Regular cell culture media, L-glutamine, penicillin-streptomycin, trypsin, and donor calf serum were obtained from Life Technologies (Grand Island, NY). Dulbecco's modified Eagles medium (DMEM), medium 199, and Ham's F-12 medium were purchased from Sigma (St. Louis, MO), DFMO from ILEX Oncology (San Antonio, TX), suramin (Germanine) from Bayer (Germany), and ammonium sulfamate from BDH (U.K.). Na235SO4 (1310 Ci/mmol) was obtained from Amersham International (Little Chalfont, U.K.). Epidermal growth factor was purchased from Genzyme (Cambridge, MA) and crystal violet from Merck (Darmstadt, Germany). The prepacked columns (Superose 6 HR 10/30, PD-10, and Mono Q HR 5/5) and Dextran T-500 were from Pharmacia-LKB (Uppsala, Sweden) and DE-53 DEAE-cellulose was from Whatman. GAG lyases and other reagents were the same as those used previously (Mani et al., 1998Go, 2000Go).

Synthesis of xylosides
Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker DRX-400 (400 MHz) instrument and fast atom bombardment mass spectrometry spectra with a JEOL SX-120 mass spectrometer. Chemical shifts are given in ppm downfield from the signal of SiMe4, with reference to internal CHD2OD. Reactions were monitored with thin-layer chromatography glass plates coated with silica gel Merck 60 F254 and visualized using either UV light or a solution of orcinol (400 mg/L) in 10% aqueous H2SO4. Flash chromatography was performed using Grace Amicon silica gel (35–70 µm). Solid phase extraction (SPE) was performed using IST Isolute SPE column 500 mg PH. 2-Naphthyl-ß-D-xylopyranoside (Xyl-2-Nap, compound 2) and its thioglycoside analog, 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside (Xyl-2-Nap-6-OH, compound 1) and p-hydroxyphenyl-ß-D-xylopyranoside used for in vitro studies were the same preparations as used previously (Mani et al., 1998Go).

The large-scale preparation of 1 (10 g) used for animal experiments was made according to the procedure in Scheme 1.



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Scheme 1.
 
2,6-Dihydroxynaphthalene (25 g, 0.156 mol) was dissolved in dioxane (150 ml, filtered through Al2O3) and heated to 100°C for 30 min. To the hot solution was added pyridine (11 ml) and benzoyl chloride (50 ml). The mixture was refluxed for 4 h and then poured out on ice water (3.3 L). The crystals were filtered off and dried under vacuum to give a mixture of the mono- and dibenzoylated products (56 g, 39% monobenzoylated product). The crystals were dissolved in CH2Cl2 (1500 ml, MS4A) together with 1,2,3,4-tetra-O-acetyl-ß-D-xylopyranose (35 g, 0.110 mol) and stirred at room temperature for 20 min. BF3{bullet}OEt2 (40 ml, 0.33 mol) was added, and the mixture was stirred for 45 min and then washed with saturated aqueous NaHCO3 and water. The organic phase was dried (Na2SO4) and concentrated. The residue was chromatographed (SiO2, heptane-EtOAc, 10:1->pure EtOAc) followed by recrystallization from diethylether-EtOAc-heptane to give crystals (21 g). This material was dissolved in CH2Cl2 (100 ml, MS4A) and MeOH (1.00 L, MS4A) and stirred at room temperature for 15 min. NaOMe-MeOH (1 M, 35 ml) was added and the mixture was stirred for 1 h, and then acetic acid (120 ml) was added and the mixture was concentrated. The residue was chromatographed (SiO2, CH2Cl2-MeOH 20:1->pure MeOH) and recrystallized to give pure 1.

The small-scale preparation of compounds 3–5 were made by the procedure shown in Scheme 2.



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Scheme 2.
 
2-(6-Methoxynaphthyl)-ß-D-xylopyranoside (Xyl-2-Nap-6-OMe, 3). To a solution of 2,3,4-tri-O-acetyl-{alpha}-D-xylopyranosyl trichloroacetimidate (Mori et al., 1990Go; 50.0 mg, 0.119 mmol) and 6-methoxy-2-naphthol (12.9 mg, 0.0667 mmol) in MeCN (1 ml) was added BF3{bullet}OEt2 (0.005 ml, 0.04 mmol) at room temperature. The solution was stirred for 30 min, and then NaOMe-MeOH (1 M, 0.2 ml) was added. A milky slurry was formed, and after 10 min H2O (3 ml) was added. The mixture was applied onto an SPE column and washed with H2O (5 ml). The column was then eluted with MeOH (5 ml) to give 3 (15.8 mg, 70%). 1H NMR (CD3OD): {delta} 7.74–7.66 (m, 2H), 7.41–7.36 (m, 1H), 7.28–7.23 (m, 1H), 7.22–7.18 (m, 1H), 7.15–7.09 (m, 1H), 4.99 (d, J = 7.2 Hz, 1H), 3.99 (dd, J = 11.3, 5.3 Hz, 1H), 3.90 (s, 3H), 3.68–3.59 (m, 1H), 3.54–3.41 (m, 3H); HRMS (FAB+): calculated for C16H18NaO6+ (M + Na+): m/z 329.1001. Found: m/z 329.1000.

1-(5-Hydroxynaphthyl)-ß-D-xylopyranoside (Xyl-1-Nap-5-OH, 4) was synthesized as compound 3 using 5-benzoyloxy-1-naphthol (Buess et al., 1965Go). Chromatography (SiO2, CH2Cl2-MeOH, 8:1) gave 4 (3.9 mg, 24%). 1H NMR (CD3OD): {delta} 7.85 (m, 2H), 7.30 (m, 1H), 7.25 (m, 1H), 7.11 (m, 1H), 6.83 (m, 1H), 5.04 (d, J = 7.5 Hz, 1H), 3.66–3.59 (m, 2H), 3.51–3.44 (m, 1H), 3.39 (dd, J = 11.4, 10.1 Hz, 1H); HRMS (FAB+): calculated for C15H16NaO6+ (M + Na+): m/z 315.0845. Found: m/z 315.0854.

2-(7-Hydroxynaphthyl)-ß-D-xylopyranoside (Xyl-2-Nap-7-OH, 5) was synthesized as compound 3 using 7-benzoyloxy-2-naphthol. Chromatography (SiO2, CH2Cl2-MeOH, 8:1) gave 5 (6.9 mg, 42%). 1H NMR (CD3OD): {delta} 7.64 (m, 2H), 7.20 (m, 1H), 7.03 (m, 2H), 6.94 (m, 1H), 4.98 (d, J = 7.2 Hz, 1H), 3.96 (dd, J = 11.4, 5.2 Hz, 1H), 3.60 (m, 1H), 3.52–3.39 (m, 3H); HRMS (FAB+): calculated for C15H16NaO6+ (M + Na+): m/z 315.0845. Found: m/z 315.0844.

Cell culture, radiolabeling, and extraction procedures
Cells were cultured as monolayers in DMEM supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) in an incubator with humidified atmosphere and 5% CO2 at 37°C. Confluent cells were preincubated for 1 h in low-sulfate, MgCl2 labeling medium supplemented with 2 mM glutamine. The preincubation medium was replaced by fresh medium containing 50 mCi/ml 35SO4 and different xylosides. Dilutions were made from 20 mM stock solutions in Me2SO/water (1:1, v/v). Low-arginine medium (medium 199) was used for preincubation in the presence of NO synthase inhibitors/nitrite quenchers. After preincubation for 24 h, 35SO4 and xylosides were added and cultures were incubated for an additional 24 h. Drugs used were suramin (0.2 mM), ammonium sulfamate (10 mM), aminoguanidine (10 mM), and neocuproine (0.01 mM). After the incubation period, culture medium was collected and pooled with two washings of ice-cold phosphate buffered saline (0.137 M NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.5). Cells were extracted with 0.1–0.2 ml/cm2 dish of 0.15 M NaCl, 10 mM ethylenediamine tetra-acetic acid, 2% (v/v) Triton X-100, 10 mM KH2PO4, pH 7.5, 5 µg/ml ovalbumin containing 1 mM diisopropylphosphorofluoridate on a slow shaker at 4°C for 10 min.

Isolation of xyloside-primed radiolabeled GAG
The procedures have been described in detail previously (Fransson et al., 1992Go). 35SO4-labeled polyanionic macromolecules were isolated from the culture medium by ion exchange chromatography on DEAE-cellulose at 4°C. Samples were mixed with 1.3 volumes of 7 M urea, 10 mM Tris, pH 7.5, 0.1% Triton X-100, 10 mM N-ethyl maleimide and passed over a 1-ml column of DE-53 equilibrated with 6 M urea, 0.5 M NaOAc, pH 5.8, 5 µg/ml ovalbumin, 0.1% Triton X-100. After sample application, the columns were washed successively with 10-ml portions of (a) equilibration buffer; (b) 6 M urea, 10 mM Tris, pH 8.0, 5 µg/ml ovalbumin, 0.1% Triton X-100; and (c) 50 mM Tris pH 7.5. Bound material was eluted with 5x1 ml 4 M guanidine-HCl, 50 mM NaOAc, pH 5.8, 5 µg/ml ovalbumin, 0.2% Triton X-100. Radioactive fractions were pooled and precipitated with 5 volumes of 95% ethanol overnight at –20°C using 100 µg dextran as carrier. After centrifugation in a Beckman JS-7.5 at 4000 rpm and 4°C for 45 min, material was dissolved in 4 M guanidine-HCl, 50 mM NaOAc, pH 5.8, 0.2% Triton X-100 and subjected to gel permeation FPLC on Superose 6 at a flow rate of 0.4 ml/min in the same buffer. Radioactivity was determined in a ß-counter, and fractions were pooled.

Cell-associated 35SO4-labeled PGs and GAGs were isolated by passage over PD-10 columns. The PD-10 columns were equilibrated with distilled water. The samples in a total volume of 2.5 ml were passed through a 9.1-ml column. The procedure was tested with HS lyase digests of radiolabeled HS and free radiosulfate. Products larger than disaccharide were eluted with 3.5 ml distilled water and free radiosulfate eluted later. Samples were freeze-dried and dissolved in 4 M guanidine-HCl, 50 mM NaOAc, pH 5.8, 0.2% Triton X-100, then subjected to gel permeation FPLC on Superose 6.

Degradation procedures
GAG chains were released from PGs by treatment with 0.5 M NaOH, 0.1 M NaBH4 at room temperature overnight. Samples were neutralized with HOAc. CS chains were degraded by treatment with chondroitin ABC lyase (see references in Mani et al., 1998Go). HS chains were degraded by using the pH 1.5-HNO2 method (Shively and Conrad, 1976Go). The samples were freeze-dried and redissolved for analysis by gel permeation chromatography on PD-10 or Superose 6 to determine the proportions of total HS.

In vitro growth assay
The procedure has been described elsewhere (Mani et al., 1998Go). Cells were seeded into 96-well microculture plates at 3000 cells/well in DMEM supplemented with insulin (10 ng/ml), transferrin (25 ng/ml), and 10% fetal calf serum. After 4 h of plating, the cells were placed in serum-free Ham's F-12 medium supplemented with insulin (10 ng/ml) and transferrin (25 ng/ml) for an additional 24 h. Cells were then allowed to proliferate supported by 10 ng/ml of epidermal growth factor in the presence of 0.01, 0.025, 0.05, 0.1, and 0.2 mM of aglycon or xyloside. In some experiments cells were pretreated with DFMO to up-regulate spermine uptake (Belting et al., 1999Go, 2002Go, 2003Go). Cells were then exposed to xyloside in the continued presence of 5 mM DFMO and 1 µM spermine. Controls without growth factor as well as solvent controls were included. The total exposure time was 96 h, and the growth rate was determined by counting cells after different time intervals. Cells were fixed in 1% glutaraldehyde dissolved in Hanks balanced salt solution (NaCl 80 g/L, KCl 4 g/L, glucose 10 g/L, KH2PO4 600 mg/L, NaHPO4 475 mg/L) for 15 min, then cell nuclei were stained with 0.1% crystal violet. After washing and cell lysis for 24 h in Triton X-100, the amount of bound dye was measured at A600 in a microplate photometer (Titertek multiscan). The inhibitory effect of the compounds is expressed as ED50 (mM) scored after 96 h of exposure.

Confocal laser immunofluorescence microscopy
The various procedures including seeding of cells, fixation, use of priming and secondary antibodies, generation of images and data processing were the same as used previously (Cheng et al., 2002Go; Ding et al., 2002Go; Mani et al., 2003Go).

Animal experiments and tumor formation in vivo
Female SCIDnodCA mice (7–8 weeks old) were kept under pathogen-free conditions in the animal barrier facility at the Biomedical Center, Lund University, according to the Swedish guidelines for humane treatment of laboratory animals. The experimental setup was approved by the ethical committee for animal research in Malmö/Lund, Sweden. For toxicity studies, mice (n = 8, average weight 20 g) received a 1.7 mM xyloside 1 solution,that is, a saturated solution, either ad libitum via the drinking water or by daily injections SC (0.5 ml) or IP (0.5 ml) for a total period of up to 2 weeks. Controls received drinking water with no additives or daily injections with sterile water. Food and water intake and changes in body weight were monitored. For assessment of xyloside 1 antitumor activity, human bladder carcinoma T24 cells (1x106 cells in 200 µl phosphate buffered saline) were injected SC in the dorsal region of 7–8-week-old mice (n = 4–8). The xyloside was administered as indicated in Results, either concurrent with or 3 weeks postinjection. In the latter case, the animals were randomly divided into control and treatment groups with no significant differences in tumor volume. The animals were sacrificed following 2–3 weeks of treatment, and tumor mass was recorded.


    Acknowledgements
 
The technical assistance of Ms. Birgitta Havsmark and Susanne Jonsson is greatly appreciated. This work was supported by grants from the Swedish Science Council (VR-M and VR-NT), the Cancer Fund, XYLOGEN AB, the Tegger Foundation, the Knut and Alice Wallenberg Foundation, the G.A.E. Nilsson Foundation, Polysackaridforskning i Uppsala AB, and the J. A. Persson Foundation. It is dedicated to the memory of Prof. Göran Magnusson who, as a supervisor to N.F., initiated the xyloside synthesis project.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: katrin.mani{at}medkem.lu.se


    Abbreviations
 
CHO, Chinese hamster ovary; CS, chondroitin sulfate; DFMO, difluoromethylornithine; DMEM, Dulbecco's modified Eagles medium; GAG, glycosaminoglycan; HFL-1, human fibroblasts from lung; HS, heparan sulfate; HUVEC, human umbilical vein endothelial cell; NO, nitric oxide; PG, proteoglycan; SPE, solid phase extraction; NMR, nuclear magnetic resonance; SCID, severe combined immunodeficiency


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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