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O-|3-D- Xylopyranoside Preferentially Inhibits Growth of Transformed Cells1

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O-|3-D- Xylopyranoside Preferentially Inhibits Growth of Transformed Cells1 [CANCER RESEARCH 58. 1099-1104. March 15. 1998] Advances in Brief Heparan/Chondroitin/Dermatan Sulfate Primer 2-(6-Hydroxynaphthyl)-O-|3-D- Xylopyranoside Preferentially Inhibits Growth of Transformed Cells1 Katrin Mani,2 Birgitta Havsmark, Susanne Persson, Yuji Kaneda, Hisao Vaniamolo. Katsukiyo Sakurai, Satoko Ashikari, Hiroko Habuchi, Sakaru Suzuki, Koji Kimata, Anders Malmström, (.imilla Westergren-Thorsson, and Lars-Âke Fransson Department of Cell Biology and Molecular Biology, Lund University, S-221 00 Lund. Sweden ¡K.M.. B. H.. S. P., A. M., G. W-T.. L-À.F.]; Tokyo Research Institute, Seikanaku Corp.. 103 Tokyo. Japan [Y. K.. H. Y.. K. S.]: and Institute for Molecular Science of Medicine. Aichi Medical University. 480-11 Aichi. Japan ¡S.A.. H. H.. S. S.. K. KJ Abstract PGs substituted with HS often remain attached to the cell surface but are also distributed to basement membranes and pericellulur Xylose forms the direct carbohydrate-protein link in extra- or pericel- matrices. A number of investigations have shown that the HS chains lular proteoglycans (PGs) that are substituted with either chondroitin have many important biological functions, including the promotion of sulfate (CS)/dermatan sulfate (DS) and/or heparan sulfate (HS). Cell surface PGs carrying HS are important regulators of cell growth. Xylose cell proliferation, adhesion, and differentiation. These functions are coupled to an aromatic compound can enter cells and initiate either ( 'S/I)S exerted via interactions with extracellular matrix proteins (1, 2), synthesis or both HS and CS/DS synthesis, depending on the nature of the growth factors, and growth factor receptors (3-6). Certain heparin- aromatic adduci. Here, we show that 2-(6-hydroxynaphthyl)-0-ß-D-xy- like HS species, as well as DS, also inhibit growth when added to lopyranoside, which can prime both types of glycan chains, inhibits cultures of normal fibroblasts (7. 8). These GAGs, especially HS. are growlh of a sel of normal and Iransformed cells. Transformed cells are complex and contain a number of structural motifs. The structures preferenlially inhibiled, and al a concenlration of 0.15-0.20 HIMxyloside, involved in growth inhibition have not yet been determined. Iransformed cells are totally growth arrested, whereas normal cells are only <50', inhibited. No inhibition of growth is observed with Ihe sle- Cells can use ß-D-xylopyranosides with hydrophobic aglycones as reoisomeric 2-(6-hydroxynaphlhyI)-0-ß-L-xylopyranoside, which does noi primers for GAG synthesis in most types of cells, and at sufficiently prime glycosaminoglycan synlhesis at all; with the nonhydroxylated high xyloside concentration, endogenous PG synthesis is competi 2-naphlhyl-O-ß-D-xylopyranoside, which only primes CS/DS synlhesis tively inhibited (9-12). The type of GAG produced depends on the under Ihese conditions; or wilh /»-nilrophenyl-O-ß-o-xylopyranoside, structure of the aglycone. Xylosides containing two fused aromatic- which is known lo prime only CS/DS synlhesis. We conclude lhal growlh rings, such as naphthol, can prime both CS/DS and HS chains, inhibition is due lo priming of HS and/or CS/DS synthesis, which may whereas simpler xylosides prime only CS/DS chains (11, 13). These either lead to Ihe formation of specific antiproliferative glycans or glycan free GAG chains are usually secreted into the extracellular environ fragments or to inlerference wilh endogenous PG synthesis and turnover. ment but may also bind to cell surface receptors. Introduction Xylosides may thus be used for several purposes, including manip ulation of PG synthesis and regulation of various cellular activities, Xylose, an unusual sugar in the animal kingdom, occupies a key such as growth. To perform sequence analysis of GAG, we have position in a family of complex macromolecular glycoconjugates explored the use of xylosides with iodinatable aglycones (such as called PGs,3 which are made and secreted by almost all cells. During /7-hydroxyphenol), and we have recently devised a procedure for biosynthesis of PGs, serine residues in consensus sequences of the end-labeling and sequence analysis of CS/DS chains (14). In search core protein, such as (D/E)GSG(D/E), become substituted with the ing for iodinatable xylosides with potential for HS priming, we tested trisaccharide Galßl-3Galßl-4Xylßl-,where xylose forms the carbo Xyl-NapOH. Here, we report that this xyloside. which has the poten hydrate-protein connection. Then the alternate addition of uronic acid tial to prime both HS and CS/DS synthesis, can also inhibit cell and hexosamine generates long, linear GAG backbones. Two kinds of proliferation, preferentially in transformed cells or tumor-derived backbones can be made, either type I (-3-N-acetyl-D-galactosamine- cells. /3-1-4-D-glucuronic acid-ß-l-)„or type II (-4-/V-acetyl-D-glucosa- mine-a-l-4-D-glucuronic acid-ß-l-)„.Inboth cases, further modifica Materials and Methods tions, such as /V-deacetylation/W-sulfation of /V-acetyl-D-glucosamine, C-5 epimerization of D-glucuronic acid (to L-iduronic acid), and Cells, Enzymes, and Column Media. Human embryonic lung fibroblasts various types of O-sulfation, are required to generate the mature and umbilical vein endothelial cells were obtained as described (8. 15). Human GAG: CS/DS, with type I backbones; or HS/heparin. with type II lung carcinoma cells (A 549). normal and SV4()-transtormed mouse 3T3 backbones. fibroblasts. and transformed endothelial cells (ECV 304) were obtained from the American Type Culture Collection (Rockville. MD). Monolayer cultures were maintained on plastic in Eagle's MEM (Life Technologies. Ltd.. Ren Received 11/6/97; accepted 1/29/98. frewshire. United Kingdom) except for umbilical vein endothelial cells, which The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with were grown in medium 199 (Sigma Chemical Co.). The media were supple 18 U.S.C. Section 1734 solely to indicate (his fact. mented with 109Õ-PCS (In Vitro AB. Stockholm. Sweden) or 2()7r human 1This work was supported by the Swedish Medical Research Council, the Swedish serum (for umbilical vein endothelial cells). 2 mM i.-glutamine (ICN Bio- Cancer Fund, the G. A. E. Nilsson Foundation, and the J. A. Persson Foundation. : To whom requests for reprints should be addressed, at Department of Cell and chemicals), penicillin (KM) units/ml), and streptomycin (I(K) ¿tg/ml). Cells Molecular Biology. P.O. Box 94. S-221 00 Lund. Sweden. Phone: 4646 222 8573; Fax: were regularly checked tor mycoplasma using GEN-PROBE Rapid detection 46462223128; E-mail: [email protected]. system (Skafte & Claesson. Molndal. Sweden). Chondroitin ABC lyase (EC 1The abbreviations used are: PG. proleoglycan; GAG. glycosaminoglycan: CS. chon 4.2.2.4) and HS lyase I (EC 4.2.2.8) were purchased from the Seikagaku Corp. droitin sulfate; DS. dermatan sulfate; HS. heparan sulfate; Xyl-NapOH. 2-(6-hy- (Tokyo. Japan). The following materials were obtained as follows: Nav^SO., droxynaphthyll-O-ß-D-xylopyranoside: Xyl-Nap. 2-naphthyl-O-ß-D-xylopyranoside; Xyl- PheNO,. /»-nitrophenyl-O-ß-D-xylopyranoside. (1310 Ci/mmol; Amersham International. Amersham. United Kingdom): DE 1099 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. XYLOSIDE INHIBITION OF GROWTH a 100 i I 1a. 50 i 0.05 0.1 0.15 0.2 0.05 0.1 0.15 XylNapOH (mM) XylNapOH (mM) Fig. I. Effect of Xyl-NapOH on growih of human lung fibroblasts (al. human lung carci noma cells (/>). mouse 3T3 fibroblasts (c). SV40- transformed mouse 3T3 fibroblasts (</). human umbilical vein endothelial cells (c). and trans formed human umbilical vein endolhelial cells (/). Cell number was determined after 72. 96. or 120 h of culturing and expressed as the percent age of growth in the absence of xyloside. Only results obtained after 96 h are shown. Dala /«wjf.v,means («= 5); SE was smaller in range than the size of the symbols. Thick siiliil line. 0.05 0.1 0.15 0.05 0.1 0.15 0.2 XylNapOH (mM) XylNapOH (mM) 0.05 0.1 0.15 0.2 0.05 0.1 0.15 XylNapOH (mM) XylNapOH (mM) 53 DEAE-cellulose (Whatman): and octyl-Sepharose CL-4B and Superóse 6 loxy-2-naphthy!-2,3,4-tri-O-benzyl-O-ß-D-xylopyranoside (1.27 g; 45% yield). HR 10/30 (Pharmacia-LKB). Debenzoylation followed by hydrogenolysis gave Xyl-NapOH (380 mg: 63% Xylosides. Synthesis and physical characterization of Xyl-Nap has been yield). 'H-NMR (400 MHz. CD,OD) gave 6„(ppm) = 7.53-6.92 (m, 6H, described (13). The sample used here was a generous gift from Dr. M. aromatic-H). 4.83 (d, IH J¡2 = 7.3 Hz, H-l), 3.85 (dd, IH J^ 4 = 4.9 Hz. Wilstermann (Lund University). A simplified synthetic route was used that •/s«,,sa*= 'I-2 Hz- H-5 eq), 3.52-3.27 (m, 4H). Optical rotation was -25.8° comprised treatment of a mixture of 1,2,3.4-tetra-O-acetyl-ß-D-xylopyranoside (c = 0.1; CH,OH), and decomposition point was 240°C.The L-xyloside was and 2-naphthol (or 2-naphthothiol) in dichloromethane with boron trifluoride prepared by the same procedure and with approximately the same yield, except etherate followed by de-0-acetylation. The analyses of the products were in that the starting material was the ß-i.-xylopyranose derivative, treatment with accordance with published data (13). Xyl-NapOH was synthesized as follows. trimethylsilyl trifluoromethanesulfonate was performed at -78°C, washing was 2,3,4-Tri-O-benzyl-l-phenylcarbamoyl-O-ß-i>xylopyranose (prepared from with aqueous NaHCO3, and recrystallization was made from methanol.
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