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Experimental Therapeutics |
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, San Diego, California, 92103-0687 [M. M. F.], and Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, 92093-0687 [J. R. B., L. W., J. D. E.]
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ABSTRACT |
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-activated endothelial cells. Altering glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of wild-type mice over a 3-h period after i.v. injection. No significant difference in biodistribution was noted after the injection of AcGnG-NM-treated tumor cells into P-selectin deficient mice, although the extent of lung seeding was reduced compared with that in wild-type mice. In vitro, we demonstrate that normal mouse platelets, but not P-selectin-deficient platelets, bound to control tumor cells and protected them from leukocyte-mediated cytolysis. Conversely, treatment of tumor cells with disaccharide markedly reduced the ability of normal platelets to protect them from cytolysis. Finally, in an experimental metastasis model, we show that treatment of tumor cells with the disaccharide markedly reduced their lung colonization potential after injection into severe combined immunodeficient mice. These findings suggest that this compound may represent a novel class of chemotherapeutic agents for prevention and treatment of metastatic disease. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2,3Galß1,4(Fuc1,3)GlcNAc]3
and sLea [Sia2,3Galß1,3(Fuc1,4)GlcNAc] on cell-surface glycoconjugates endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Studies of human tumors and mice bearing genetic alterations in one or more selectins underscore the importance of these interactions in hematogenous spread of cancer cells (6, 7, 8, 9, 10)
. The carbohydrate ligands for the selectins are predominantly O-linked glycoprotein mucins and glycolipids that display sLeX or sLea in clustered arrangements (11, 12, 13) . Several aggressive solid tumors display significant reactivity to anti-sLeX mAbs and to E-and P-selectins. These include a relatively large proportion of tumors from the lung, colon, and breast (4 , 7 , 13, 14, 15, 16, 17) . Adhesion interactions involving sLeX constitute important early steps in the pathophysiology of metastasis (9 , 10 , 18, 19, 20, 21) possibly by stabilizing "neoplastic emboli " via P-selectins on platelets or L-selectin on leukocytes, or by facilitating adhesion to, and possible extravasation thorough, the endothelium. The importance of these interactions derives from studies in patients postresection from colon, lung, gastric, and other carcinomas that show that survival correlates inversely with tumor expression of sLeX (22, 23, 24) .
With this information in mind, we have focused on the development of small molecule inhibitors that might block the expression of Lewis carbohydrate antigens on cells. Per-O-acetylated disaccharides (acetylated forms of Galß1,4GlcNAcß-O-naphthalenemethanol (AcGGn-NM) or GlcNAcß1,3Galß-O-naphthalenemethanol (AcGnG-NM)) are taken up by cells, deacetylated, and acted on as substrates by relevant glycosyltransferases located in the Golgi. Assembly of oligosaccharides on the disaccharides takes place, resulting in the diversion of glycan biosynthesis from endogenous glycoconjugates (25, 26, 27)
. The result is a concomitant reduction of sLeX expression on the cell surface. The monosaccharide GalNAc-O-benzyl behaves in a similar fashion, altering the expression of O-linked chains on mucins of colon and leukemia cell lines in vitro and altering cell adhesion to platelets and endothelia (28, 29, 30)
. However, much higher concentrations of the monosaccharide are needed to achieve a similar level of inhibition as the disaccharide (1–5 mM versus 10–50 µM, respectively). This report examines the ability of the more potent disaccharide, AcGnG-NM, to inhibit adhesion of adenocarcinoma cells to both immobilized recombinant selectins and to selectins on activated human platelets and endothelia. We show that inhibiting tumor cell glycosylation in this way leads to decreased interactions with selectins, increased susceptibility to leukocyte-mediated lysis, and reduction in organ colonization in an experimental model of metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MEM medium (LS180), F12 (A549), F12/DMEM (A427), or RPMI 1640 (HAL-8). All of the media (Life Technologies, Inc.) were supplemented with 10% (v/v) FBS (HyClone Laboratories), L-glutamine (0.3 g/L), streptomycin sulfate (100 µg/ml), and penicillin (100 units/ml). Cells were passaged every 4–6 days using ATV trypsin solution (Life Technologies, Inc.). HMVECs were grown in EBM-2 media (Clonetics) supplemented with 10% FBS, subcultured using a solution of 0.025% trypsin/0.01% EDTA, and harvested on first or second passage for adhesion assays. All of the cell lines were maintained at 37°C in a humidified incubator under an atmosphere of 5% CO2 and 95% air.
Peracetylated forms of GlcNAcß1,3Galß-O-naphthalenemethanol (AcGnG-NM) and Galß1,3Galß-O-naphthalenemethanol (AcGG-NM) were prepared as described previously (27)
. The compounds were dissolved in DMSO and were added to growth medium to achieve the concentrations indicated in the figures. The supplemented medium was then exchanged for the medium in established cultures of cells to avoid lysis caused by adding concentrated DMSO directly to the plates. The final concentration of DMSO was adjusted to 
0.5% (v/v). After the specified number of days, the cells were harvested with 2 mM EDTA in PBS (20 min) and were used for experiments.
Cell Sorting.
To detect the presence or absence of the relevant carbohydrate determinants, cells were stained with CSLEX-1 (anti-sLeX, 5 µg/ml; Becton-Dickinson) or CA-19–9 (anti-Lea, 14.5 µg/ml; Chemicon) and analyzed by flow cytometry (FACScan Becton-Dickinson, Franklin Lakes, NJ). Approximately 5 x 105 cells were incubated for 1 h at 4°C in 100 µl PBS/1% BSA containing CSLEX-1 or CA-19–9, followed by phycoerythrin-conjugated rabbit antimouse IgG (2 µg/ml). As a negative control, cells were treated with nonspecific mouse isotype-matched antibody (0.5 µg/ml; Sigma) for 1 h at 4°C in 100 µl PBS/1% BSA followed by phycoerythrin-conjugated rabbit antimouse IgG (2 µg/ml).
Cell Adhesion to Immobilized Selectins.
We coated 96-well plates overnight at 4°C with recombinant E- (4 µg/ml) or P-selectin (2 µg/ml; R & D Systems) and blocked with 1% BSA/PBS. LS180 cells were grown for 5 days with various amounts of acetylated disaccharide and were harvested and labeled with calcein AM (5 µM; Molecular Probes) in DMEM/1% FBS, and were allowed to settle at room temperature on selectin-coated wells (5 x 104 cells/well). Plates were then stirred at 75 rpm for 30 min (Orbit shaker; Lab-Line Instruments) followed by immersion upside-down in a vessel filled with HBSS (Sigma), which allowed nonadherent cells to fall under gravity (31)
. The wells were then washed by aspiration using HBSS. LS180 cells were less adherent to P-selectin and the immersion step was not necessary before washing. Controls included treating tumor cells for 1 h at 37°C with AUS (Calbiochem; 20 mU/1 x 106 cells) in 0.05 M HEPES buffer (pH 6.9), pretreating selectin-coated wells with anti-E- or anti-P-selectin mAbs (1 µg/well; PharMingen), or growing tumor cells in 50 µM of the inactive disaccharide primer, peracetylated Galß1,3Galß-O-NM. Fluorescence was measured using a 96-well fluorimeter (CytoFluor II), and the average of triplicate measurements was determined ± SE. Cell viability was judged to be >90% by trypan blue exclusion at the end of each experiment.
Adhesion of Tumor Cells to Activated Human Endothelial Cells.
HMVECs were added to 96-well plates (1 x 104 cells/well) in EBM-2 medium (Clonetics) and were allowed to grow to confluence over 2 days. The cells were activated with TNF- (20 ng/ml; R&D Systems) for 4 h at 37°C. Calcein-labeled tumor cells, harvested after growth for 5 days in various levels of AcGnG-NM, were added to HMVECs at 2.5 x 104 cells/well in 100 µl of DMEM and were allowed to settle for 20 min. The wells were washed twice with cold PBS, and the extent of binding was determined by fluorimetry. In some experiments, anti-E-selectin mAb (2 µg/well) was added before the addition of tumor cells, cells were treated with sialidase (20 mU/106 cells), or TNF- was omitted.
Adhesion of Activated Human Platelets to Tumor Cells.
Tumor cells were seeded into 6-well plates (5 x 104/well), and allowed to grow into colonies in the presence or absence of 50 µM AcGnG-NM. After 3 days, platelets were isolated from 15 ml of normal human blood collected into 20% (v/v) Acid-Citrate-Dextrose (ACD) anticoagulant. A platelet-rich plasma was prepared by centrifugation and repeated washing using 5 mM HEPES (pH 6.8), 145 mM NaCl, 4 mM KCl, 0.5 mM sodium phosphate, 5.5 mM glucose, 0.5% BSA, and 25 nM prostaglandin E1 (Sigma). The platelets were then labeled with calcein AM (5 µM), and counted with a hemocytometer. Wells containing tumor cells were washed with HBSS, and 3 x 106 platelets were added in 1 ml of HBSS, followed by activation with human thrombin (0.8 IU/well; Sigma). The plates were rocked for 10 min, and the wells were washed twice with HBSS, fixed with 5% formalin in HBSS, and analyzed by fluorescence microscopy. Controls included pretreatment of tumor cells with AUS [20 mU/well in 0.05 N HEPES (pH 6.9), 1 mM CaCl2, and 1 mM MgCl2], pretreatment of tumor cells with O-sialoglycoproteinase (2.4 µg/ml; Cedarlane) before the addition of platelets, addition of anti-P-selectin mAb (10 µg/ml in HBSS) to the platelet suspension before thrombin activation and addition to tumor cells, or omission of thrombin.
Platelets bound to tumor cells were visualized by fluorescence microscopy (NikonDiaphot) with a digital camera (Nikon) linked to an Apple Macintosh computer running Adobe Photoshop. The fluorescence image showing the platelets was superimposed on a phase-contrast picture of the cells, and the number of attached platelets was quantified. A "Platelet Association Index" was generated for each well by dividing the number of tumor-associated platelets by the area occupied by tumor cells.
Biodistribution Studies in Mice.
LS180 cells were grown for 3 days in the presence or absence of 50 µM AcGnG-NM. Methyl-[3H]thymidine (10 µCi/ml; NEN Life Sciences Products) was added to the medium, and cells were incubated for another 3 days. The cells were then harvested with EDTA, resuspended in sterile 0.9% saline, and injected (1 x 105 cells/100 µl) into the lateral tail vein of anesthetized (inhaled methoxyflurane, Janssen Pharmaceuticals) 6–8 week-old wild-type C57BL/6 mice or P-selectin-deficient mice bred on the same background (Jackson Laboratory; Ref. 32
). On awakening, mice were observed for 3 h, anesthetized, bled (
200 µl each), sacrificed via cervical dislocation, and dissected for collection of lungs, liver, kidney/adrenals, spleen, and brain. The organs were digested at 55°C overnight with proteinase K (0.15 µg/ml; Boehringer Mannheim) in 2 ml of PBS containing 1% SDS, and homogenized by repeated passage through an 18-gauge needle. The amount of radioactive DNA in the blood and the organ extracts was then determined using an Easy DNA kit (Invitrogen) and liquid scintillation spectrometry. The total counts in blood were estimated by assuming a total blood volume of 2 ml/mouse. Proper attention has been given to experimental ethical considerations toward animals as prescribed by the Animal Subjects Program at the University of California.
Tumor Formation.
LS180 cells were grown in the presence or absence of 50 µM AcGnG-NM for 6 days, harvested with EDTA, and resuspended in sterile PBS. Approximately 3 x 105 tumor cells in 150 µl of PBS were injected into the lateral tail vein of anesthetized 7-week-old immunodeficient mice (Fox Chase SCID; Charles River, MA). The mice were then maintained in microbe-free housing with free access to standard laboratory chow and water, and inspected regularly for any signs of distress. After 4 weeks, mice were euthanized by CO2 asphyxiation under anesthesia, and lungs, livers, brains, kidneys/adrenals, and spleens were fixed in Bouin’s solution (Sigma) for 6 h, followed by transfer to 70% ethanol. Lungs for each animal were inspected under a dissecting microscope for the total number of surface tumors. Histological sections (H&E) were examined for tumor foci, and representative photomicrographs were taken. The other organs were also reviewed histologically for any tumor foci.
Cytolytic Assays.
LS180 cells were grown to near-confluence in the presence or absence of 50 µM AcGnG-NM for 6 days, harvested using 2 mM EDTA/PBS, washed, and resuspended in 2 ml RPMI 1640 containing 10% FBS and 15 µCi Na251CrO4 (435 mCi/mg, DuPont NEN). After 2 h, at 37°C, the cells were then washed twice with medium and placed into a conical 96-well plate (1500 cells/well). Effector cells were prepared from normal C57BL/6 mouse spleens by mincing the tissue and sieving the cells over a fine screen. Red cells were lysed by resuspension in 0.83% NH4Cl in PBS, and the enriched leukocytes were resuspended in RPMI medium containing 10% FBS and 200 units/ml of recombinant human IL-2 (Life Technologies, Inc.). After 3 days of culture, the leukocytes were added to the wells containing tumor cells. Some wells also received platelets (104 platelets/tumor cell) isolated from pooled whole blood (2–4 mice/group) prepared by the same procedures described above for the isolation of human platelets (33)
. After 3 h at 37°C, the amount of 51Cr released was measured by centrifuging the plate at 1500 rpm and taking an aliquot of the supernatant. Spontaneous release (Rspont) of radioactivity was measured by incubating target cells in RPMI medium only. Maximum release (Rmax) was measured after complete lysis of targets in 2% SDS. Specific lysis was determined according to the equation: % specific lysis = (Rexp - Rspon) x 100/(Rmax - Rspon), where Rexp = counts released in the presence of effector cells. In some experiments, labeled tumor cells were added to whole human blood (1.6 ml mixed with 0.4 ml of Acid-Citrate-Dextrose anticoagulant) and incubated with stirring at 37°C for 3 h.
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RESULTS |
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. Several disaccharides related to mucin-like oligosaccharides that carry sLeX determinants were shown to be effective as primers, with peracetylated GlcNAcß1,3Galß-O-NM (AcGnG-NM) exhibiting the highest potency (27)
. Priming of oligosaccharides in this way inhibited the expression of sLeX by HL-60 and U937 cell lines. In this study, we examined the effect of this disaccharide on selectin-binding and tumor-forming properties of LS180 human colon adenocarcinoma cells. These cells were chosen because they express carbohydrate ligands known to bind to E- and P-selectins, and they form lung tumors in an experimental murine model of hematogenous metastasis (9
, 10 , 13
, 16
, 34)
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6 h, indicating that no permanent damage to the cells had occurred. Inhibiting the expression of sLeX in this way reduced the ability of LS180 cells to adhere to recombinant E- and P-selectin immobilized on plastic dishes (Fig. 3)
. Adhesion to P-selectin was more sensitive to the inhibitor than was adhesion to E-selectin under these conditions. The inhibitory effect of AcGnG-NM was specific because the incubation of cells with acetylated Galß1,3Galß-O-NM (AcGG-NM) had no effect on expression of sLeX (data not shown) or adhesion to either selectin conjugate (Fig. 3)
. The maximum extent of inhibition approached the values obtained when the cells were pretreated with sialidase or blocking antibody to the corresponding selectin.
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-activated HMVECs (Fig. 4)
. In this system, adhesion was mostly dependent on E-selectin expression because blocking antibody or absence of TNF- stimulation dramatically lowered the extent of adhesion. AcGnG-NM inhibited adhesion with a dose response similar to that observed using immobilized receptor (Fig. 3)
. The maximum extent of inhibition was similar to that obtained after treatment of the tumor cells with sialidase, which destroys sLeX, or by using a blocking antibody to E-selectin.
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. The extent of inhibition was comparable with that achieved by treating tumor cells with sialidase and O-sialoglycoproteinase, which requires clustered oligosaccharide chains for cleavage of the underlying protein core (16
, 35)
. The extent of inhibition was not as great as that achieved by blocking antibody or by omitting thrombin activation, suggesting that the disaccharide did not fully suppress expression of sLeX or, alternatively, that non-Sia-containing ligands for P-selectin exist. Similar effects were observed in studies of two lung adenocarcinoma cell lines, A549 and A427 (Figs. 5, B and C
, respectively). These cells also express selectin ligands and sLeX determinants,4
but they varied in parallel in their response to sialidase and AcGnG-NM treatment.
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60% of recovered counts remained in the lung, 20% in the liver, 15% in the blood, and lesser amounts in other organs (Fig. 6A)
. Inhibitor-treated cells exhibited a different biodistribution after injection. Seeding of the lungs was substantially reduced and accompanied by a corresponding increase in counts recovered in the blood compartment, without significant differences in seeding of other tissues. When treated and untreated cells were injected into P-selectin-deficient mice, no difference was observed in the distribution of the cells (Fig. 6B)
, although the extent of seeding was reduced compared with wild-type mice. Thus altering either P-selectin or its carbohydrate ligand had similar effects, suggesting that the interaction of the tumor cell glycans with host cellular elements expressing P-selectin affected the fate of the cells.
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. Numerous foci were present on lungs from animals that had been given injections of untreated cells, whereas foci were less numerous in animals receiving disaccharide-treated cells (P < 0.0002, Student’s t test; Fig. 7C
). A similar trend was noted on examination of foci in histological sections (P < 0.02). No foci were found in other organs by surface and histological surveys. A human metastatic lung adenocarcinoma cell line (HAL8) was also examined. These cells behaved similarly, although the absolute number of tumor foci was much lower (data not shown).
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. The extent of cell lysis was proportional to the E:T ratio. Adding platelets to the incubation significantly reduced cytolysis, although some lysis was noted at very high E:T values outside the range of values that would occur in a typical experimental metastasis assay performed in mice (indicated by the broken vertical lines in Fig. 8
). P-selectin-deficient platelets showed a marked reduction in their ability to protect tumor cells over the same range. Platelet protection was also markedly reduced after treatment of the tumor cells with AcGnG-NM. Exposure of disaccharide-treated and untreated LS180 cells to whole human blood gave comparable results (Fig. 8
, inset), although the overall effect and extent of lysis was significantly greater. Together, these findings demonstrate that P-selectin on platelets binding to sLeX determinants on tumor cells provides protection against leukocyte-mediated cytolysis. Moreover, they show that treatment with AcGnG-NM inhibits platelet-mediated protection of the cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The treatment of tumor cells with AcGnG-NM has a particularly important effect on metastatic potential as a result of altered platelet adhesion. As shown in Fig. 8
, selectin-mediated platelet adhesion endows tumor cells with significant protection from immune-mediated cytolysis, which may explain the higher tumorigenicity of untreated cells compared with cells treated with the disaccharide inhibitor (Fig. 7)
. Because these experiments were performed in SCID mice, humoral factors and T-cell mediated responses should not be involved, but elements of innate immunity (e.g., innate cytotoxic responses, natural killer cells, and so forth) may play a role (38)
. The apparent protection of tumor cells by platelets critically depends on P-selectin-carbohydrate interactions because P-selectin deficiency dampens the effect both in vivo and in vitro. On injection of treated cells, sLeX begins to reappear on the cell surface with a t1/2 of 6 h. This suggests that cytolysis occurs relatively rapidly and that interfering with platelet adhesion to tumor cells soon after their release into the circulation will render the cells more sensitive to killing. Other inhibitory agents, such as heparin or mucin fragments, also transiently block selectin-dependent adhesion and block tumor formation (10)
. These agents are rapidly cleared from the circulation but nevertheless have profound effects on ultimate colonization of the lungs by metastatic cells. Thus, antimetastatic agents that target selectin-carbohydrate interactions need only to act in a narrow time frame to be effective.
A final consideration is the ability of selectins to participate in "arresting" newly circulating tumor cells in organ capillary beds. Entrapment of emboli may have important consequences on the eventual uptake and growth of tumor "seeds " into large metastatic tumor foci. AcGnG-NM-treated LS180 cells showed a limited ability to eventually grow as tumors in the lungs of immunodeficient mice harboring the cells for a 4-week period after tail-vein delivery (Fig. 7)
. Whereas alterations in tumor sLeX may have an as-yet-unexplained effect on tumor growth (including apoptosis), an initial inhibition of selectin-mediated capillary arrest may be critical to the survival of metastases. Additional evidence supporting this view includes: (a) treatment of adenocarcinoma cells with AcGnG-NM (up to 50 µM) has minimal effects on LS180 growth in culture; (b) experimental mice were not maintained on pharmacological AcGnG-NM after tumor cell injection, indicating that the effects are rapid and independent of continuous inhibition; and (c) whereas tumors that grew in experimental mice were markedly fewer in number, tumor size in the two groups was approximately the same. A recent study highlights the importance of an early period of intravascular tumor residence and proliferation before extravasation and uptake (39)
. The probability of securing a prolonged intravascular period of arrest should increase after selectin-mediated formation of platelet-tumor emboli and direct contact of tumor cells with endothelial selectins. Inhibition of sLeX-mediated adhesion would be expected to decrease these parameters. A recent study demonstrates that the inhibition of ß3 integrins also interferes with hematogenous metastasis in a platelet-dependent fashion, consistent with this idea (40)
. These findings imply a potential role for AcGnG-NM or related compounds as possible antimetastasis agents for treating human cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by University of California Tobacco-Related Disease Research Program (TRDRP) Fellowship Award 8FT-0118 and a Pulmonary Fellowship Award from Glaxo-Wellcome (to M. M. F.), and NIH Grants HL23594 and CA46462 (to J. D. E.). J. R. B. was supported in part through a University of California Biotechnology Research and Education Grant. 
2 To whom requests for reprints should be addressed, at Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0687. Phone: (858) 822-1100; Fax: (858) 534-5611; E-mail: jesko{at}ucsd.edu
3 The abbreviations used are: sLeX, sialyl Lewis X; sLea, sialyl Lewis a; AcGnG-NM, per-O-acetylated GlcNAcß1,3Galß-O-naphthalenemethanol; AcGG-NM, per-O-acetylated Galß1,3Galß-O-naphthalenemethanol; Gal, galactose; Fuc, fucose; GlcNAc,N-acetylglucosamine; Sia, sialic acid; AUS, Arthrobacter ureafaciens sialidase; FBS, fetal bovine serum; HMVEC, human microvascular endothelial cell; TNF, tumor necrosis factor; mAb, monoclonal antibody; SCID, severe combined immunodeficient.
4 J. R. Brown, M. Fuster, T. Whisenant, and J. D. Esko, in press.
Received 8/ 6/02. Accepted 3/25/03.
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L. Borsig, L. Wang, M. C. M. Cavalcante, L. Cardilo-Reis, P. L. Ferreira, P. A. S. Mourao, J. D. Esko, and M. S. G. Pavao Selectin Blocking Activity of a Fucosylated Chondroitin Sulfate Glycosaminoglycan from Sea Cucumber: EFFECT ON TUMOR METASTASIS AND NEUTROPHIL RECRUITMENT J. Biol. Chem., May 18, 2007; 282(20): 14984 - 14991. [Abstract] [Full Text] [PDF]
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 S. Mathieu, M. Prorok, A.-M. Benoliel, R. Uch, C. Langlet, P. Bongrand, R. Gerolami, and A. El-Battari Transgene Expression of {alpha}(1,2)-Fucosyltransferase-I (FUT1) in Tumor Cells Selectively Inhibits Sialyl-Lewis x Expression and Binding to E-Selectin without Affecting Synthesis of Sialyl-Lewis a or Binding to P-Selectin Am. J. Pathol., February 1, 2004; 164(2): 371 - 383. [Abstract] [Full Text] [PDF]
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, adhesion to E-selectin; 
, adhesion to P- selectin. The extent of adhesion was normalized to the value obtained for cells not treated with disaccharide. Samples treated with sialidase, anti-E- or anti-P selectin mAb, or 50 µM of the inactive disaccharide primer acetylated-Galß1,3Galß-O-NM gave values of 0.28–0.32, 0.05–0.33, and 0.95–1.1, respectively. Each experimental condition was done in quadruplicate, and the average values ± SEs are given.
, P-selectin-positive platelets were added; 
, P-selectin-negative platelets were added; 
, tumor cells were treated with peracetylated GnG-NM and mixed with P-selectin-positive platelets. Inset, the effect of whole human blood on the lysis of 51Cr loaded tumor cells with and without treatment with AcGnG-NM. The experiment was performed in triplicate, and average values ± SDs are shown.