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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Key words: heparan sulfate / nitric oxide / tumor growth / xyloside
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Introduction |
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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., 2002).
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., 1998). 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, 2003
). We have shown (Ding et al., 2001a
; Mani et al., 2000
) 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., 2003
). 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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Results |
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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., 2002), 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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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 1249% (14 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 2535 and endogenously generated HS oligosaccharides eluting in fractions 4555 (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., 2001a, 2002
; Cheng et al., 2002
). 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 4047% HS (Table IV). The HS-content of the oligosaccharide (pool III) ranged between 30% and 57% for xylosides 14, 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., 2001a, 2002
; Cheng et al., 2002
). To identify the cellular localization of xyloside 1primed HS degradation products, we used confocal immunofluorescence microscopy. A polyclonal antiserum against human Gpc-1 (Ding et al., 2001a
) and a monoclonal antibody against an epitope comprising the anMan residues of deaminatively cleaved HS (Ding et al., 2002
) 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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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., 2001a) 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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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 1014 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
4050 mg compared to
135 mg in control animals, which corresponds to
70% growth inhibition of established tumors.
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Discussion |
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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., 1999).
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., 1996; Thomsen and Miles, 1998
) as well as to their greater dependence on supply of polyamines (Belting et al., 1999
; 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 7097% 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., 1999).
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., 2002). 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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Materials and methods |
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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 (3570 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., 1998).
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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The small-scale preparation of compounds 35 were made by the procedure shown in Scheme 2.
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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., 1965). Chromatography (SiO2, CH2Cl2-MeOH, 8:1) gave 4 (3.9 mg, 24%). 1H NMR (CD3OD):
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), 3.663.59 (m, 2H), 3.513.44 (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): 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), 3.523.39 (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 0.10.2 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., 1992). 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., 1998). HS chains were degraded by using the pH 1.5-HNO2 method (Shively and Conrad, 1976
). 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., 1998). 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., 1999
, 2002
, 2003
). 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., 2002; Ding et al., 2002
; Mani et al., 2003
).
Animal experiments and tumor formation in vivo
Female SCIDnodCA mice (78 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 78-week-old mice (n = 48). 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 23 weeks of treatment, and tumor mass was recorded.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Belting, M., Borsig, L., Fuster, M.M., Brown, J.R., Persson, L., Fransson, L.-Å., and Esko, J.D. (2002) Tumor attenuation by combined heparan sulfate and polyamine depletion. Proc. Natl Acad. Sci. USA, 99, 371376.
Belting, M., Mani, K., Jönsson, M., Cheng, F., Sandgren, S., Jonsson, S., Ding, K., Delcros, J.-G. and Fransson, L.-Å. (2003) Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide. J. Biol. Chem., 278, 4718147189
Buess, C.M., Guidici, T., Kharasch, N., King, W., Lawson, D.D., and Saha, N.N. (1965) The synthesis of thyromimetic substances and potential inhibitors of thyroxine. J. Med. Chem., 8, 469474.[ISI][Medline]
Cheng, F., Mani, K., van den Born, J., Ding, K., Belting, M., and Fransson, L.-Å. (2002) Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated Gpc-1 takes place in caveolin-1-containing endosomes. J. Biol. Chem., 277, 4443144439.
Ding, K., Jönsson, M., Mani, K., Sandgren, S., Belting, M., and Fransson, L.-Å. (2001a) N-unsubstituted glucosamine in heparan sulfate of recycling Gpc-1 from suramin-treated and nitrite-deprived endothelial cells. Mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein. J. Biol. Chem., 276, 38853894.
Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-Å. (2001b) Modulations of Gpc-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites. J. Biol. Chem., 276, 4677946791.
Ding, K., Mani, K., Cheng, F., Belting, M., and Fransson, L.-Å. (2002) Copper-dependent autocleavage of Gpc-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols. J. Biol. Chem., 277, 3335333360.
Doi, K., Akaike, T., Horie, H., Noguchi, Y., Fujii, S., Beppu, T., Ogawa, M., and Maeda, H. (1996) Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth. Cancer, 77, 15981604.[CrossRef][ISI][Medline]
Esko, J.D., Rostand, K.S., and Weinke, J.L. (1988) Tumor formation dependent on proteoglycan biosynthesis. Science, 241, 10921096.[ISI][Medline]
Fransson, L.-Å. (2003) Glypicans. Int. J. Biochem. Cell Biol., 35, 125129.[CrossRef][ISI][Medline]
Fransson, L.-Å., Karlsson, P., and Schmidtchen, A. (1992) Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts. Biochim. Biophys. Acta, 1137, 287297.[ISI][Medline]
Fransson, L.-Å., Belting, M., Jönsson, M., Mani, K., Moses, J., and Oldberg, Å. (2000) Biosynthesis of decorin and glypican. Matrix Biol., 19, 367376.[CrossRef][ISI][Medline]
Liu, D., Shriver, Z., Venkataraman, G., El Shabrawi, Y., and Sasisekharan, R. (2002) Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis. Proc. Natl Acad. Sci. USA, 99, 568573.
Mani, K., Havsmark, B., Persson, S., Kaneda, Y., Yamamoto, H., Sakurai, K., Ashikari, S., Habuchi, H., Suzuki, S., Kimata, K., and others. (1998) Heparan/chondroitin/dermatan sulfate primer 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside preferentially inhibits growth of transformed cells. Cancer Res., 58, 10991104.[Abstract]
Mani, K., Jönsson, M., Edgren, G., Belting, M., and Fransson, L.-Å. (2000) A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of Gpc-1 in vascular endothelial cells. Glycobiology, 10, 577586.
Mani, K., Cheng, F., Havsmark, B., Jönsson, M., Belting, M., and Fransson, L.-A. (2003) Prion or amyloid-b-derived Cu(II)- or free Zn(II)-ions support S-nitroso-dependent autocleavage of Gpc-1 heparan sulfate. J. Biol. Chem., 278, 3895638965.
Mori, M., Ito, Y., and Ogawa, T. (1990) Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1-3)-beta-D-Manp-(1-4)-beta-D-Glcp-(1-1)-Cer and alpha-D-Manp-(1-3)-[beta-D-Xylp-(1-2)]-beta-D-Manp-(1-4)-beta-D-Glcp-(1-1) Cer. Carbohydr. Res., 195, 199224.[CrossRef][ISI][Medline]
Moses, J., Oldberg, Å., and Fransson, L.-Å. (1999) Initiation of galactosaminoglycan biosynthesis. Separate galactosylation and dephosphorylation pathways for phosphoxylosylated decorin protein and exogenous xyloside. Eur. J. Biochem., 260, 879884.
Shively, J.E. and Conrad, H.E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry, 15, 39323942.[ISI][Medline]
Thomas, T. and Thomas, T.J. (2001) Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci., 58, 244258.[ISI][Medline]
Thomsen, L.L. and Miles, D.W. (1998) Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev., 17, 107118.[CrossRef][ISI][Medline]
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