Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro*

We previously reported that the xyloside 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH- and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate.

We previously reported that the xyloside 2-(6-hydroxynaphthyl) ␤-D-xylopyranoside (XylNapOH, Fig. 1), in contrast to 2-naphthyl ␤-D-xylopyranoside (XylNap, Fig. 1) and most other xylosides, specifically reduces tumor growth both in vitro and in vivo (16 -18). The mechanism of action has not been elucidated; however, the xyloside-primed GAGs have been suggested to be involved, possibly as a result of the formation of GAGs with different composition depending on xyloside and cell type. Here, we have isolated XylNapOH-and XylNapprimed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and studied the relationship between GAG structure and its effect on cell growth.
Isolation of XylNapOH-and XylNap-primed GAGs-HCC70 cells and CCD-1095Sk cells (passages 5-25) cultured in T75 flasks (Thermo Scientific) to ϳ70% confluence were preincubated in DME/F-12 medium supplemented with 10 g/ml insulin, 25 g/ml transferrin (all from Sigma), 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10 ng/ml EGF (Corning) for 24 h. The cells were then incubated in 15 ml of fresh medium supplemented with 100 M XylNapOH or XylNap. For radiolabeling, the medium was additionally supplemented with 5 Ci/ml [ 35 S]sulfate (PerkinElmer Life Sciences). After 24, 48, or 72 h of incubation, the cell media were collected and subjected to ion exchange chromatography, hydrophobic interaction chromatography, and precipitation as previously described (19). The precipitate was dissolved in deionized H 2 O, freeze-dried, and resuspended in deionized H 2 O before purification of the XylNapOH-and XylNapprimed GAGs on a Superose 12 HR 10/30 column (GE Healthcare) connected to a Thermo Scientific UltiMate 3000 Quaternary Analytical system. The mobile phase consisted of 70% 60 mM NH 4 OAc, pH 5.6, and 30% MeCN in an isocratic mode at a flow rate of 0.7 ml/min. The XylNapOH-and XylNap-primed GAGs were detected using a FLD-3400RS fluorescence detector (excitation ϭ 229 nm and emission ϭ 372 nm for Xyl-NapOH and excitation ϭ 229 nm and emission ϭ 342 nm for XylNap). The fluorescent fractions were collected and pooled, freeze-dried, and quantified using the 1,9-dimethyl-methylene blue method (20) using CS A from bovine trachea (Sigma) and HS (Iduron) as standards.
Isolation of PG-GAGs-The procedure was the same as for the isolation of the XylNapOH-and XylNap-primed GAGs, with the following exceptions. The medium used was supplemented with 5 Ci/ml [ 35 S]sulfate only; after 48 h of incubation, the media were subjected only to ion exchange chromatography before precipitation, and the PG-GAG fractions from the HPLC purification were collected based on radioactivity instead of fluorescence. Radioactivity was measured using a liquid scintillation counter (PerkinElmer Life Sciences).
Cell Growth Assay Using the Crystal Violet Method-Confluent HCC70 cells and CCD-1095Sk cells (passages 10 -25) were dissociated using TrypLE TM Express Enzyme (Thermo Scientific) and seeded in 96-well microculture plates (Greiner Bio-One) at plating densities set to obtain an approximate 2.5-fold increase in cell number after 96 h (1000 -5000 cells/well). After 24 h of plating, the cells were allowed to grow in DME/F-12 medium supplemented with 10 g/ml insulin, 25 g/ml transferrin, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 10 ng/ml EGF, and increasing concentrations of XylNapOH-or XylNap-primed GAGs from HCC70 cells (1, 2.5, 5, 7.5, and 15 g/ml) or CCD-1095Sk cells (2.5, 5, 10, 20, and 30 g/ml), or CS A from bovine trachea, CS B from porcine intestinal mucosa, heparin from porcine intestinal mucosa (all from Sigma), or CS C from shark cartilage (a gift from Dick Heinegård) (2.5, 5, 10, 20, and 30 g/ml). Untreated cells, blanks only containing medium, and controls with xylosides as references were included. After 24 h or 96 h, the cell density was measured using the crystal violet method as previously described (21). Concurrently with the initiation of each experiment, a plate containing cells at day 0 was fixed and stored at room temperature in Hanks' balanced salt solution, pH 7.4, until staining. After staining, the amount of bound dye was measured by absorbance at 595 nm in a microplate reader. The relative cell number (in % of untreated cells (UT)) was calculated according to Equation 1, where Abs is absorbance (in absorbance units   (Sigma) in 150 l of 0.1 M NaOAc, pH 5.0, for 16 h. After treatment, the samples were boiled for 10 min and subsequently centrifuged at 10,000 ϫ g for 10 min before supernatants were dried by centrifugal evaporation. The GAGs were purified on a Superdex Peptide HR 10/30 column (GE Healthcare), run in 0.2 M NH 4 HCO 3 at a flow rate of 0.5 ml/min. The radioactive fractions eluting at a retention time corresponding to that of untreated XylNapOH-primed GAGs were collected and pooled, freeze-dried, and quantified using the 1,9-dimethylmethylene blue method before being used in the cell growth assay (at concentrations of 2.5, 5, and 7.5 g/ml). For molecular weight estimation, ϳ4 g of XylNapOH-and XylNap-primed GAGs were treated with 20 milliunits of xylanase in 65 l of the above described buffer for 16 h. After treatment, the samples were worked up as described above. Subsequently, the GAGs were dissolved in deionized H 2 O, and the free aglycons were removed by centrifugal filtration using filters with a cut-off of 3 kDa (VWR Collection) before size-exclusion chromatography n a Superose 6 column (GE Healthcare) run in 0.2 M NH 4 HCO 3 at a flow rate of 0.3 ml/min. The elution profiles were compared with those of heparin standards (Iduron and gift from Dilafor).

Structure of Cytotoxic Xyloside-primed Glycosaminoglycans
Determination of Apoptosis of Cells Treated with XylNapOHprimed GAGs from HCC70 Cells-Confluent HCC70 cells and CCD-1095Sk cells (passages 10 -25) were dissociated using TrypLE TM Express Enzyme and seeded in 4-well glass chamber slides at densities of 1 ϫ 10 5 cells/well. After 24 h of plating, the cells were preincubated with DME/F-12 medium supplemented with 10 g/ml insulin, 25 g/ml transferrin, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10 ng/ml EGF for 24 h before treatment with XylNapOHprimed GAGs (2.5, 5, and 7.5 g/ml) from HCC70 cells. Untreated cells were included as a control. After 24 h, apoptotic cells were identified using a TUNEL assay (BioVision) performed according to the manufacturer's instructions. Imaging was performed using a Carl Zeiss AxioObserver inverted fluorescence microscope equipped with an EC Plan-NEOFLUAR 20ϫ/0.5 objective. The images were captured using an AxioCam MRm camera and the AxioVision Rel 4. 8 mM CaCl 2 , and 2.5 milliunits of each enzyme at 37°C for 16 h. CS A, CS B, CS C, and heparin were included as controls. Identification and quantification was performed by comparing the HPLC-separated 2-aminoacridone-labeled enzyme-generated disaccharides with 2-aminoacridone-labeled HPLC-separated disaccharide standards (Iduron) as previously described (22). The HS/CS/DS ratios of the Xyl-NapOH-and XylNap-primed GAGs and the PG-GAGs were calculated as the relative proportions (% of total) of HS, IdoUA-containing disaccharides in CS/DS (CS/DS IdoUA ), and GlcUA-containing disaccharides in CS/DS (CS/DS Gl -cUA) according to Equations 2-4, respectively, where m is mass (in ng) after degradation with the indicated enzymes. Equation 3 was generated based on the cleavage sites of chondroitinase B and chondroitinase AC-I and -II (23). Enzymatic Degradation of XylNapOH-and XylNap-primed GAGs for Cell Growth Assay-Approximately 15 g of XylNapOH-and XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells were degraded using chondroitinase ABC or heparinase I-III. Chondroitinase ABC degradations were performed using 100 milliunits of enzyme, and heparinase I-III degradations were performed using 25 milliunits of heparinase I and 50 milliunits of each heparinase II and III, both in 250 l of above described buffers at 37°C for 16 h. After degradation, the samples were worked up as described above followed by purification of the non-degraded GAGs on a Superdex Peptide HR 10/30 column, run in 0.2 M NH 4 HCO 3 at a flow rate of 0.5 ml/min. Fluorescent fractions eluting at a retention time corresponding to that of untreated XylNapOH-and XylNap-primed GAGs were collected and pooled, freeze-dried, and quantified using the 1,9-dimethylmethylene blue method before being used in the cell growth assay (at concentrations of 2.5, 5, and 7.5 g/ml).
Statistical Analysis-All statistical calculations and curve fitting to data were performed using GraphPad Prism 6.

XylNapOH-primed GAGs from HCC70 Cells Reduce Growth of HCC70 Cells and CCD-1095Sk Cells-XylNapOH-and
XylNap-primed GAGs were isolated from HCC70 cells and CCD-1095Sk cells after treatment with 100 M XylNapOH or XylNap for 48 h. Little or no xyloside-primed GAGs were isolated from the cell extracts, and therefore, only the xyloside-primed GAGs from the culture media were investi-gated. Very little PG was detected in the culture media after xyloside treatment. The amount of recovered xylosideprimed GAGs depended on the number of cell passages and differed between the cell lines; the amount recovered from the HCC70 cells increased with the number of passages, whereas the amount recovered from the CCD-1095Sk cells decreased with the number of passages. In general, treatment with XylNap resulted in smaller amounts of recovered xyloside-primed GAGs than treatment with XylNapOH. The amounts of XylNapOH-and XylNap-primed GAGs recovered were in the range of 2.5-8 g/10 6 HCC70 cells and 10 -30 g/10 6 CCD-1095Sk cells.
To investigate the effect of the xyloside-primed GAGs on cell growth, HCC70 cells and CCD-1095Sk cells were treated for 96 h with increasing concentrations of XylNapOH-or XylNapprimed GAGs isolated from HCC70 cells or CCD-1095Sk cells. The growth of both HCC70 cells ( Fig. 2A) and CCD-1095Sk cells (Fig. 2E) was reduced by treatment with XylNapOHprimed GAGs from HCC70 cells. The effect was concentrationdependent and corresponded to a mean IC 50 value of 5.9 Ϯ 2 g/ml in HCC70 cells and 7.8 Ϯ 0.2 g/ml in CCD-1095Sk cells. At concentrations above, on average, 7.5 g/ml and 10 g/ml, the number of HCC70 cells and CCD1095Sk cells, respectively, was reduced below the initial number of cells, suggesting a cytotoxic effect. XylNap-primed GAGs derived from HCC70 cells had little effect on the growth of HCC70 cells (ϳ15% reduction at 15 g/ml of XylNap-primed GAGs from HCC70 cells; Fig. 2C) and none on the growth of CCD-1095Sk cells (Fig. 2G).
Neither XylNapOH-nor XylNap-primed GAGs derived from CCD-1095Sk cells had any effect on the growth of either of the cell lines despite the fact that the xyloside-primed GAGs were administered at higher concentrations ( Fig. 2, B, D, F, and H). The results were consistent across experiments and independent of the number of cell passages before isolation of the xyloside-primed GAGs.
The incubation time with xyloside before GAG isolation is a parameter that potentially could influence the composition of the xyloside-primed GAGs. Therefore, we compared the effect of XylNapOH-primed GAGs isolated from HCC70 cells after 24, 48, and 72 h of incubation on HCC70 cell growth and found it to be similar irrespective of incubation time before isolation (Fig. 3A). This suggests that the composition of the XylNapOHprimed GAGs is constant over time or that the effect on cell growth is insensitive to changes in GAG composition. Alternatively, the xyloside part of the xyloside-primed GAGs could play a role for the effect.
The GAG Part of XylNapOH-primed GAGs from HCC70 Cells Is Responsible for the Reduction in Cell Growth-To determine whether the GAG part or the xyloside part of the XylNapOH-primed GAGs or a combination of both was responsible for the reduction in cell growth, we repeated the cell growth assay using GAGs that lacked the xyloside part. We used an endo-1,4-␤-xylanase from T. longibrachiatum that has an endo-␤-xylosidase activity to cleave off the aglycon from the XylNapOH-primed GAGs.
The aglycon, 2,6-dihydroxynaphthlene, of the XylNapOHprimed GAGs is fluorescent but not the GAGs themselves. A comparison of the fluorescence chromatogram of the xylanasetreated XylNapOH-primed GAGs to those of the untreated XylNapOH-primed GAGs and of the aglycon showed that xylanase treatment resulted in a complete shift in retention time of the XylNapOH-primed GAGs to that of the aglycon (Fig. 3B), implying that the cleavage was efficient and occurred specifically between the xylose and the aglycon. In addition, the chromatograms of untreated and xylanase-treated [ 35 S]sulfate-labeled XylNapOH-primed GAGs remained unchanged (Fig.  3C), indicating that the GAG chains were unaffected by the xylanase treatment.
Treatment of HCC70 cells with untreated or xylanasetreated XylNapOH-primed GAGs from HCC70 cells resulted in similar effects on cell growth (Fig. 3D), implying that the GAG part rather than the xyloside part (the aglycon) was responsible for the reduction in cell growth. In comparison, the commercially available GAGs, CS A, CS B, CS C, and heparin, together representing the majority of disaccharide units present in CS/DS and HS (supplemental Tables S1 and S2), had no significant effect on growth on any of the cell lines when administered at the same concentrations as the xyloside-primed GAGs (Fig. 3, E and F), indicating that GAGs in general do not have a growth-reducing effect on HCC70 cells and CCD-1095Sk cells.
The XylNapOH-primed GAGs from HCC70 Cells Induce Apoptosis in HCC70 Cells and CCD-1095Sk Cells-Because the growth-reducing effect of the XylNapOH-primed GAGs appeared to be mediated by a cytotoxic effect, we investigated whether apoptosis was involved. Apoptotic cells, identified using the TUNEL technique, were detected in HCC70 cells and CCD-1095Sk cells treated for 24 h with XylNapOH-primed GAGs from HCC70 cells, whereas they were not detected in untreated cells (Fig. 4A). Because the TUNEL experiments were performed after 24 h of treatment, we performed a cell growth assay after 24 h of treatment. The effect of the XylNapOHprimed GAGs derived from HCC70 cells on cell growth was observed also after 24 h and was similar to that after 96 h (Fig. 4B).
XylNapOH-and XylNap-primed GAGs from HCC70 Cells or CCD-1095Sk Cells Are 22-44 Disaccharide Units in Size-As an initial step in investigating the structure of the XylNapOHand XylNap-primed GAGs from the two cell lines, their molecular weights were estimated using size-exclusion chromatography HPLC. To prevent interactions between the xyloside-primed GAGs and the column, with peak broadening as a result, [ 35 S]sulfate-labeled xyloside-primed GAGs where the aglycons had been removed by xylanase were used. The peak maxima of the xyloside-primed GAGs on a Superose 6 column were compared with those of heparin with known molecular weights run on the same column (Fig. 5). Although some variability in the peak maxima of the different xyloside-primed GAGs was observed, the molecular weights were estimated to be, on average, 10,000 -20,000, which corresponds to ϳ22-44 disaccharide units in size (determined using a molecular weight of 459 disaccharide). The molecular weights correspond to IC 50 values of 0.3-0.8 M for the XylNapOH-primed GAGs from HCC70 cells in HCC70 cells and CCD-1095Sk cells. PG-GAGs are commonly in the range of 25-100 kDa in size (24); thus, the XylNapOH-and XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells were somewhat smaller than PG-GAGs. The similarity in size of the XylNapOH-and XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells indicates that size is not a determinant for the effect of the XylNapOH-primed GAGs from HCC70 cells. This was further supported by the fact that the CS/DS and HS from HCC70 cells primed on either XylNapOH or XylNap were of similar size as non-degraded XylNapOH-and XylNap-primed GAGs from HCC70 cells (as determined by comparing the sizes of heparinase I-III-or chondroitinase ABC-degraded XylNapOH-and XylNap-primed GAGs from HCC70 cells with those of the non-degraded Xyl-NapOH-and XylNap-primed GAGs from HCC70 cells; data not shown).
The Disaccharide Composition of the XylNapOH-and Xyl-Nap-primed GAGs Is Similar when the GAGs Are Derived from the Same Cell Line but Differs when Derived from Different Cell Lines-To gain further insight into the GAG structures, disaccharide fingerprinting of the XylNapOH-and XylNap-primed GAGs from the two cell lines was performed. The xylosideprimed GAGs were degraded using chondroitinase ABC, chondroitinase AC-I and -II, chondroitinase B, or heparinase I-III followed by 2-aminoacridone labeling of the disaccharides and separation of these on HPLC (Fig. 6A). The disaccharides were qualitatively and quantitatively characterized by comparing with disaccharide standards. Both the chondroitinase ABCand heparinase I-III-generated disaccharides varied considerably in composition between the xyloside-primed GAGs isolated from HCC70 cells and CCD-1095Sk cells (Fig. 6, B-E). Interestingly, the disaccharide composition of the xylosideprimed GAGs derived from the same cell line was similar irrespective of whether they were primed on XylNapOH or XylNap. Likewise, the disaccharide composition of the Xyl-NapOH-primed GAGs isolated from HCC70 cells after different incubation times was similar irrespective of incubation time (supplemental Tables S1 and S2). The major difference in CS/DS disaccharides between the xyloside-primed GAGs derived from HCC70 cells and CCD-1095Sk cells was in the ⌬UA-GalNAc,4S (A unit)/⌬UA-GalNAc,6S (C unit) ratio, which was shifted between the two cell lines: ϳ15%/ϳ82% in the xyloside-primed GAGs from HCC70 cells and ϳ56%/ϳ38% in the xyloside-primed GAGs from CCD-1095Sk cells (Fig. 6B). In addition, ⌬UA-GalNAc,4S,6S (E unit) was present in the xyloside-primed GAGs from the HCC70 cells (ϳ2.8%) but only in trace amounts in those from CCD-1095Sk cells. Instead, ⌬UA,2S-GalNAc,4S (B unit) (ϳ1.7%) and ⌬UA,2S-GalNAc,6S (D unit) (ϳ3.8%) were present in the xyloside-primed GAGs from the CCD-1095Sk cells (Fig. 6B).
Based on the chondroitinase AC-I and -II degradations and chondroitinase B degradations, we concluded that the disaccharide units of the XylNapOH-and XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells contained both GlcUA and IdoUA (Fig. 6, C and D). For example, the A units and E units of XylNapOH-and XylNap-primed GAGs from HCC70 cells were a mix of GlcUA and IdoUA, as they were detected after chondroitinase AC-I and -II degradation and after chondroitinase B degradation. The B unit of XylNapOH-and XylNap-primed GAGs from CCD-1095Sk cells was detected only after chondroitinase B degradation, whereas the D unit was detected only after chondroitinase AC-I and -II degradation.
The HS/CS/DS Ratios of the XylNapOH-and XylNap-primed GAGs Differ between the Different Cell Lines but Is also Influenced by the Xyloside-The HS/CS/DS ratios of the studied XylNapOH-and XylNap-primed GAGs (Fig. 7) were calculated based on the disaccharide data according to Equations 2-4 under "Experimental Procedures." The proportion of IdoUAcontaining disaccharides in CS/DS (CS/DS IdoUA ) calculated using Equation 3 includes the IdoUA-containing disaccharides present both in blocks and as single IdoUA-containing disaccharides, whereas the proportion of IdoUA-containing disaccharides generated by chondroitinase B degradation includes those present in blocks only.
Similar to the disaccharide composition, differences in the HS/CS/DS ratios of the xyloside-primed GAGs derived from the two cell lines were observed (Fig. 7). The xyloside-primed GAGs from HCC70 cells were composed of a higher proportion of HS (ϳ20 and 35% for XylNapOH-and XylNap-primed GAGs, respectively) than those from CCD-1095Sk cells, where HS was almost absent (ϳ1.4%). Instead, the xyloside-primed GAGs from CCD-1095Sk were composed of a higher proportion of CS/DS IdoUA (ϳ37%) than the xyloside-primed GAGs from HCC70 cells (ϳ8.9%). Analogously, the proportion of IdoUA-containing disaccharides generated after chondroitinase B degradation of xyloside-primed GAGs from CCD-1095Sk cells (ϳ17%) was higher than that generated after chondroitinase B degradation of xyloside-primed GAGs from HCC70 cells (ϳ1.4%).  Unlike for the disaccharide composition, variability in the HS/CS/DS ratios of the XylNapOH-and XylNap-primed GAGs derived from HCC70 cells was detected. The proportion of HS in the XylNapOH-primed GAGs from HCC70 cells increased with incubation time before isolation, from ϳ7.4% after 24 h to ϳ20% after 48 h and to ϳ25% after 72 h. In the XylNap-primed GAGs isolated from HCC70 cells after 48 h the proportion of HS was ϳ35%. Thus, the HS/CS/DS ratio of xyloside-primed GAGs, at least in the HCC70 cells, appears to be influenced not only by the cell type, but also by the xyloside, as previously observed (10,16).

The Composition of the Xyloside-primed GAGs Does Not Mirror That of the PG-GAGs Derived from the Same Cell Line, but
There Are Similarities-To investigate if the xyloside-primed GAGs structurally resembled the endogenous PG-GAGs derived from the same cell line, the PG-GAGs secreted into the culture media of untreated HCC70 cells and CCD-1095Sk cells were studied. The amounts of PG-GAGs recovered after 48 h were 0.7-0.9 g/10 6 HCC70 cells and 0.9 -1.5 g/10 6 CCD-1095Sk cells, which was considerably lower than the amounts recovered after xyloside treatment. The disaccharide composition of both the CS/DS and the HS of the xyloside-primed GAGs from HCC70 cells was similar to that of the PG-GAGs isolated from HCC70 cells, whereas the HS/CS/DS ratios differed (supplemental Tables S1 and S2). The main difference was in the proportion of HS, which was lower in the xyloside-primed GAGs (ϳ20 and 35% for XylNapOH-and XylNap-primed GAGs, respectively) than in the PG-GAGs (ϳ56%). In addition, the proportion of CS/DS IdoUA in the xyloside-primed GAGs (ϳ8.9%) was slightly higher than that in the PG-GAGs (ϳ5.0%). Analogously, the proportion of IdoUAcontaining disaccharides generated after chondroitinase B degradation of the xyloside-primed GAGs (ϳ1.4%) was slightly higher than that generated after chondroitinase B degradation of the PG-GAGs (ϳ0.4%), possibly explaining why the E unit was not observed in the PG-GAGs after chondroitinase B degradation.
The xyloside-primed GAGs from CCD-1095Sk cells differed from the PG-GAGs from CCD-1095Sk cells both in disaccharide composition and in HS/CS/DS ratios (supplemental Tables S1 and S2). The xyloside-primed GAGs were composed of a higher proportion of C units (ϳ38%) and to a lower proportion of A units (ϳ56%) than the PG-GAGs from CCD-1095Sk cells (ϳ14 and ϳ81%, respectively). The disaccharides generated after chondroitinase AC-I and -II degradation were the main source of this difference, as the disaccharides generated after chondroitinase B degradation were similar. In addition, fewer HS disaccharides were represented in xyloside-primed HS from CCD-1095Sk cells than in the corresponding PG-HS, possibly as a result of a lower proportion of HS in the xyloside-primed GAGs (ϳ1.4%) than in the PG-GAGs (ϳ8.5%). In addition, the proportion of CS/DS IdoUA of the xyloside-primed GAGs (ϳ37%) was lower than that of the PG-GAGs (ϳ46%). Analogously, the proportion of IdoUA-containing disaccharides generated after chondroitinase B degradation of the xylosideprimed GAGs (ϳ17%) was lower than that generated after chondroitinase B degradation of the PG-GAGs (ϳ36%).
Taken together, these results indicate that the overall GAG composition of the xyloside-primed GAGs does not mirror that of the PG-GAGs of their corresponding cell line. However, some similarities were observed, in particular the disaccharide composition of the GAGs derived from HCC70 cells.

CS/DS from HCC70 Cells Primed on Either XylNapOH or XylNap Is Cytotoxic but Can Be Inhibited by XylNap-primed HS-
To determine whether CS/DS or HS was responsible for the cytotoxic effect of the XylNapOH-primed GAGs from HCC70 cells, XylNapOH-primed GAGs from HCC70 cells were treated with either heparinase I-III or chondroitinase ABC before treating HCC70 cells. Heparinase I-III treatment of the GAGs, which investigated the effect of the CS/DS, did not alter the cytotoxic effect of the XylNapOH-primed GAGs, whereas chondroitinase ABC treatment of the GAGs, which investigated the effect of HS, resulted in a complete loss of cytotoxic effect (Fig. 8A). This suggests that the XylNapOH-primed CS/DS was responsible for the cytotoxic effect. Because the disaccharide composition of XylNapOH-and XylNap-primed GAGs from HCC70 cells was similar, we performed the same experiment using XylNap-primed GAGs. Interestingly, heparinase I-III-treated XylNap-primed GAGs from HCC70 cells had a similar effect on HCC70 cells as the XylNapOH-GAGs from HCC70 cells (Fig. 8B). The XylNapOH-and XylNap-primed GAGs from CCD-1095Sk cells were included as controls, showing that neither chondroitinase ABC treatment nor heparinase I-III treatment of the xyloside-primed GAGs from CCD-1095Sk cells affected the growth of the cells (data not shown). Taken together, this implies that xyloside-primed CS/DS GAGs from HCC70 cells are cytotoxic irrespective of whether they are primed on XylNapOH or XylNap. In addition, the HS primed on XylNap appeared to inhibit the effect of the CS/DS.
To confirm that the XylNap-primed HS from HCC70 cells had an inhibitory effect on the cytotoxic CS/DS, HCC70 cells were treated with a combination of chondroitinase ABCtreated XylNap-primed GAGs and heparinase I-III-treated XylNapOH-primed GAGs, both from HCC70 cells. Increasing concentrations of the two resulted in complete inhibition of the cytotoxic effect of the heparinase I-III-treated XylNapOHprimed GAGs (Fig. 8C). Likewise, a combination of a fixed concentration of the heparinase I-III-treated XylNapOH-primed GAGs (7.5 g/ml) and increasing concentrations of the chondroitinase ABC-degraded XylNap-treated GAGs (0.5, 1, and 2 g/ml) resulted in a concentration-dependent inhibition of the cytotoxic effect of the heparinase I-III-treated XylNapOHprimed GAGs (Fig. 8D), thus confirming that the XylNapprimed HS had an inhibitory effect on the cytotoxic CS/DS.

Discussion
We have previously reported a tumor-specific growthinhibiting effect of the xyloside XylNapOH and suggested that the XylNapOH-primed GAGs could be responsible for this effect. Furthermore, we have observed differences in HS/CS/DS composition between GAGs primed on XylNapOH and GAGs primed on other xylosides, such as XylNap, that lack effect on cell growth. Here, we have characterized the structure-function relationship of XylNapOH-and XylNap-primed GAGs derived from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk. We demonstrated that XylNapOH-primed GAGs isolated from HCC70 cells reduced growth of HCC70 cells and CCD-1095Sk cells, with IC 50 values  Tables S1 and S2). The data are the means of two independent experiments.
in the low micromolar range. The growth-reducing effect was a cytotoxic effect, mediated by apoptosis. Importantly, we demonstrated that the GAG part rather than the xyloside part of the XylNapOH-primed GAGs was responsible for the cytotoxic effect. Furthermore, the effect was specific for the XylNapOHprimed GAGs from HCC70 cells, as neither XylNap-primed GAGs from HCC70 cells nor XylNapOH-or XylNap-primed GAGs from CCD-1095Sk cells or commercially available GAGs showed any clear effect on cell growth.
Both HS and CS/DS GAGs have previously been shown to affect growth of cancer cells either after direct administration or as a result of enzymatic treatment of cells. For example, HS fragments generated after heparinase I treatment or heparinase III treatment of tumor cells have been shown to either promote or inhibit tumor growth in vivo, depending on their composition (25). Similarly, treatment of melanoma cells with chondroitinase AC or chondroitinase B has been reported to inhibit growth and migration in vitro (26), and other studies have showed inhibition as well as promotion of cell growth by HS and CS/DS (27)(28)(29). Here, we demonstrated that the CS/DS was responsible for the cytotoxic effect of the XylNapOHprimed GAGs from HCC70 cells, whereas the XylNapOHprimed HS did not have any effect on cell growth. Furthermore, the XylNap-primed CS/DS from HCC70 cells had a similar effect, whereas the XylNap-primed HS inhibited the cytotoxic effect of the XylNapOH-or XylNap-primed CS/DS in a concentration-dependent manner, explaining why no cytotoxic effect of the non-degraded XylNap-primed GAGs was observed. Neither the CS/DS nor the HS derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on cell growth, further supporting the notion that the cytotoxic effect was associated with the carcinoma cells.
The fact that HS and CS/DS appear to affect different cells differently indicates that besides possible differences in susceptibility to various GAGs, the composition of the GAGs is important for the biological effect. Thus, we characterized the disaccharide composition of the XylNapOH-and XylNap-primed GAGs. The data revealed considerable differences in disaccharide composition between the xyloside-primed GAGs from HCC70 cells and CCD-1095Sk cells. Interestingly, we found that the disaccharide composition of the xyloside-primed GAGs derived from the same cell line was similar irrespective of whether they were primed on XylNapOH or XylNap. This indicates that the disaccharide composition is specific and cell type-dependent rather than xyloside-dependent. In addition, the results suggest the disaccharide composition as a possible determinant for the biological effect of the GAGs. Interestingly, the disaccharide composition of the xylo- side-primed GAGs from HCC70 resembled that of the corresponding PG-GAGs.
The XylNapOH-and XylNap-primed CS/DS derived from HCC70 cells were primarily composed of three disaccharide units: C units, A units, and E units, whereas those derived from CCD-1095Sk cells were primarily composed of A units, C units, D units, and B units. Thus, the prevalence of the E unit, D unit, and B unit distinguished the two. Interestingly, CS E, primarily composed of E units, is involved in some of the few identified functional CS/DS sequences. For example, CS E has been reported to interact with Wnt signaling (5), and L-and P-selectin (6) and to enhance CD44 cleavage (30), all of which could influence tumor progression. However, prevalence of the E unit is unlikely a single determinant for cytotoxicity, not the least because the commercially available CS B contained the E unit (supplemental Table S1), but did not have any effect on cell growth. Thus, the sequential order of the disaccharides, in addition to the disaccharide composition, is likely to be important for the biological effects.
Variations in the HS/CS/DS ratio of xyloside-primed GAGs depending on aglycon have been extensively studied over the years (10,16,31,32). The amount of HS has been of particular interest, as xylosides were initially thought to only induce synthesis of CS. We found that the HS/CS/DS ratios differed between the xyloside-primed GAGs derived from the different cell lines and that the proportion of HS was influenced by the xyloside, which was evident in the HCC70 cells, where the proportion of HS was substantial. This supports previous results showing that the HS/CS/DS ratio is both cell type-dependent and xyloside-dependent.
Although the CS/DS from HCC70 cells primed on both XylNapOH and XylNap appeared structurally and functionally similar, the HS had, despite a similar disaccharide composition, different effects depending on whether it was primed on XylNapOH or XylNap. The XylNap-primed HS inhibited the cytotoxic effect of the XylNapOH-and XylNap-primed CS/DS from HCC70 cells, whereas the XylNapOH-primed HS was inactive. This did not appear to be a result of different proportions of HS in the XylNapOH-and XylNap-primed GAGs (ϳ20 and ϳ35%, respectively), as the proportion of HS primed on XylNapOH increased with increasing incubation time without having any impact on the cytotoxic effect. In addition, the cytotoxic effect of the XylNapOH-primed GAGs remained the same upon HS removal. We speculate that the difference in effect could be due to the sequential order of the disaccharide units; however, this remains to be elucidated.
IdoUA-containing GAGs are associated with an increased structural flexibility compared with equivalent GlcUA-containing GAGs, thereby altering their interaction properties with various biomolecules (33). CS/DS IdoUA was present in the xyloside-primed GAGs from both HCC70 cells and CCD-1095Sk cells; thus, the presence of CS/DS IdoUA does not appear to be a single determinant for effect by the GAGs. However, the proportion could potentially have an impact on the effect, as it differed between the GAGs from HCC70 cells (8.9%) and CCD-1095Sk cells (37%). Interestingly, there was also a considerable difference in the distribution of IdoUA-containing disaccharides between the xyloside-primed GAGs derived from the dif-ferent cell lines (ϳ20% versus ϳ50% of the CS/DS IdoUA was present in blocks in the xyloside-primed GAGs from HCC70 cell and CCD-1095Sk cells, respectively), another factor that potentially could influence the biological effect.
The data presented here provide new perspectives on xyloside-primed GAGs and their properties. In addition, to our knowledge, this is the first study that demonstrates a cytotoxic effect on cancer cells and normal cells by CS/DS and that this effect can be inhibited by HS.
Author Contributions-A. P. and K. M. designed and coordinated the study. A. P. performed the experiments and analyzed the data. A. P. and E. T. performed the disaccharide fingerprint experiments. A. P., E. T., G. W.-T., A. M., U. E., and K. M. interpreted and reviewed the results. AP wrote the paper, which was reviewed and approved by all of the authors.