journal of che~stry vol. no. of for and biology, inc. in … · inhibition of lectin-mediated...

THE JOURNAL OF BIOLOGICAL C H E ~ S T R Y 0 1994 by The American Society for Biochemistry and Moleeular Biology, Inc.

Vol. 269, No. 36, Issue of September 9, pp. 22797-22803, 1994 Printed in U.S.A.

Inhibition of Lectin-mediated Ovarian Tumor Cell Adhesion by Sugar Analogs*

(Received for publication, May 16, 1994, and in revised form, July 1, 1994)

Barbara Woynarowska, David M. Skrincosky, Angela Haag, Moheswar Sharma, Khushi MattaS, and Ralph J. BernackiO From the Deoartments of Exoerimental Theraveutics and $Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, Ne; York 14263

Adhesion of A-121 human ovarian carcinoma cells to extracellular matrix is partly mediated via interaction between galaptin, an endogenous P-galactoside-binding lectin present in extracellular matrix, and specific cell surface carbohydrate receptors identified as lysosomal associated membrane proteins, lamp1 and lamp-2. In this study, we report that adhesion of human ovarian carcinoma cells to polystyrene plates coated with polymerized human splenic galaptin can be inhibited by polyclonal antibodies raised against lamp1 and lamp2 molecules and by pretreatment of A-121 human ovarian carcinoma cells with glucosamine analogs: S-acetamido- 1,4,6-tri-O-acetyl-3-deoxy-3-fluoro-a-~-glucopyranose (3- F-GlcNAc) and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4- fluoro-a-D-glucopyranose (4-F-GlcNAc). A 48-h exposure of A-121 cells to individual sugar analogs, or to a combination of the two, resulted in a concentration-dependent inhibition of cellular attachment to polymerized galaptin. Both drugs inhibited glycoprotein biosynthesis as measured by cellular incorporation of labeled [‘Hlglu- cosamine and [‘Hlfucose with negligible effects on [‘Hlthymidine and [‘Hlleucine incorporation and cell growth. As a result of drug action on glycoprotein biosynthesis, an alteration in the structure of the galaptin receptor was noted by indirect immunofluorescence and Western blot analysis. Moreover, probing gels of cell extracts with anti-lamp antibodies or Datura stramonium lectin demonstrated significant changes in the reactivity and pattern of glycoprotein staining, suggesting an effect of sugar analogs on the glycosylation of various cellular receptor molecules. The greatest change was observed when tumor cells were exposed to a combination of the two sugar analogs. These studies suggest that specific endogenous lectins and their surface receptors play a role in tumor cell adhesion and perhaps metastasis and may serve as suitable targets for therapeutic exploitation.

Human ovarian carcinoma cell adhesion is a critical event in the progression and spread of ovarian tumors within the peritoneal cavity. Previous studies have demonstrated that human ovarian carcinoma A-121 cells preferentially attach to extracellular matrix (ECM)’ as compared with mesothelial cells, which

* These studies were supported in part by NCI, National Institutes of Health, Grants CA 42898, CA 35329, and CA 13038. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

tal Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Sts., 5 To whom correspondence should be addressed: Dept. of Experimen-

Buffalo, NY 14263. Tel.: 716-845-3058; Fax: 716-845-8857. ‘The abbreviations used are: ECM, extracellular matrix; 3-F-

GlcNAc, 2-acetamido-1,4,6-tri-O-acetyl-3-deoxy-3-fluoro-~-~-glucopyranose; 4-F-GlcNAc, 2-acetamido-l,3,6-tri-O-acetyl-4-deoxy-4-fluoro-cu-o- glucopyranose; lamp-1 and 2, lysosomal associated membrane protein 1

line structures in the peritoneal cavity (1). One of the identified factors promoting tumor cell adhesion is an endogenous p-galactoside-binding lectin (galaptin) (2). Galaptin belongs to a class of S-type lectins, which are soluble, low molecular weight proteins for which the carbohydrate binding activity is divalent cation-independent and usually thiol-dependent (3). Galaptin has been found to be present in tumor cells in peritoneal effu- sions from ovarian cancer patients (4) and in the human ovarian carcinoma cell line A-121 (2).

Basement membranes of a variety of tissues (5-7) and bovine corneal endothelial cell-derived ECM contain galaptin (2, 6). We have identified the galaptin-binding glycoproteins on the A-121 ovarian carcinoma cell surface as being lysosomal associated membrane proteins, lamp-1 and lamp-2 (8). Lamps are heavily glycosylated proteins with oligosaccharide chains abundant in complex poly-N-acetyllactosamines (9-111, which consist of (Galpl+4GlcNAcpl-t3), repeats. Galp1-3GlcNAc linkages, corresponding approximately to 20% of total carbohy- drates, have also been found on oligosaccharide chains of lamp molecules (12). The treatment of A-121 cell extracts with N- glycanase resulted in decreased recognition of lamps by galaptin, thus confirming the carbohydrate-mediated nature of this interaction (8).

Inhibition by lactose of A-121 cell adhesion to polymerized galaptin strongly indicated the specificity of binding to cell surface oligosaccharides containing N-acetyllactosamine. Structure-activity relationship studies revealed that although substitution of a hydroxyl group at position C2 of glucose with an N-acetyl group (to generate N-acetyllactosamine) increases the binding activity (13-16), modification at the positions C4 and C6 of galactose and position C3 of glucose results in decreased binding (15-18). The proposed model of galaptin-ligand binding indicates the necessity of a free C3 position of glucose for binding to occur (18).

The objective of the present study was to evaluate 3- and 4-fluoroglucosamine analogs as modulators of A-121 cell surface glycoconjugates and, as a result, as inhibitors ofA-121 cell attachment. These glucosamine analogs were synthesized as potential oligosaccharide chain modifiers andor chain terminators. Substitution of fluorine a t position C4 of glucosamine was envisioned to prevent elongation of polylactosamine chains, whereas modification of glucosamine at position C3 was designed to weaken the binding. Such structural alterations in lamp polylactosamine residues should result in a weaker interaction between lamps and galaptin and lead to at least partial inhibition of A-121 cell adhesion.

EXPERIMENTAL PROCEDURES Materials-RPMI 1640, DMEM (lacking glucose), fetal bovine serum,

glutamine, and 0.05% trypsin, 0.53 mM EDTA were purchased from Life

and 2; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate- buffered saline; BSA, bovine serum albumin; TBS, Tris-buffered saline.

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22798 Inhibition of Ovarian Tumor Cell Adhesion Technologies, Inc. [Methyl-3H]Thymidine (specific activity 20 Ci/mmol), ~-[6-~H]glucosamine (specific activity 40.4 Ci/mmol), ~-[4,5-~H]leucine (specific activity 60.0 Ci/mmol), and ~-[5,6-~Hlfucose (specific activity 48.4 Ci/mmol) were obtained from DuPont NEN. Rainbow protein standards and ECL Western blotting detection kit were purchased from Amersham Corp. Datura stramonium biotin-conjugates were from Boehringer Mannheim. Rhodamine-conjugated donkey anti-rabbit IgG was from Accurate Chemical and Scientific Corp. (Westbury, N Y ) , X- Omat AR x-ray film was purchased from Kodak. Immulon 1 microenzyme-linked immunosorbent assay plates were obtained from Dynatech Laboratories (Chentilly, VA). Avidin-peroxidase, rabbit IgG, mouse anti- rabbit peroxidase IgG, fluorescein isothiocyanate-lectin conjugates and all others chemicals were from Sigma. Fluorica reference particles were purchased from Pandex Laboratories, Mundelein, IL. Previously characterized rabbit lamp-1 and lamp-2 polyclonal antibodies (9) were gen- erously provided by Dr. Minoru Fukuda (La Jolla Cancer Research Foundation).

A-121 Cell Culture-Human ovarian A-121 carcinoma cells have been characterized previously (19). Cultures were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 2 mM glutamine, and 15 mM HEPES. Cells were passaged every 4th day by harvesting cells by trypsinization and seeding 5 x lo5 cells in 20 ml of fresh medium.

Preparation of Polymerized Galaptin-Human splenic galaptin was isolated by afinity chromatography on lactose-Sepharose and by ion- exchange chromatography on DEAE-Sephacel as described previously (8). Galaptin was eluted from DEAE-Sephacel column with 10 mM Tris, 200 mM NaCl, pH 7.3, and 10 mg galaptin were readsorbed to a 1-ml bed of DEAE-Sephacel pretreated overnight with 100 mM iodoacetamide in TBS (Tris-buffered saline: 10 mM Tris, 20 mu NaCI, pH 7.3) for 1 h in the dark to alkylate galaptin. Alkylation was performed to eliminate the thiol requirement for retention of carbohydrate binding activity. Alkyl- ated galaptin was eluted with 8 mM sodium phosphate, 200 mM NaCI, pH 7.3, diluted to 1 mg/ml in the eluting buffer and polymerized with glutaraldehyde. Glutaraldehyde was slowly added to alkylated galaptin to a final concentration of 0.1%, and the reaction mixture was stirred for 15-24 h a t 4 "C in the dark in the presence of 0.1 M lactose to prevent polymerization in the carbohydrate binding site. After extensive dialy- sis against TBS, polymerized galaptin was repurified prior to use by affinity chromatography on lactose-Sepharose, followed by ion-exchange chromatography on DEAE-Sephacel and stored at -20 "C.

A-121 Cell Adhesion to Polymerized Galaptin-Polymerized galaptin (35 pg/ml in 10 mM Tris, 20 mM NaCl, pH 7.3) was added to wells (50 pl/well) of Immulon 1 microenzyme linked-immunosorbent assay plates and allowed to adsorb for 24 h at 4 "C. Nonadsorbed lectin was aspi- rated, wells were washed twice with PBS (phosphate-buffered saline: 2.7 mM KCI, 1.5 mM KH,PO,, 137 mM NaCl, 6.5 mM Na,HPO,, pH 7.3) and used later for adhesion experiments.

Adhesion experiments were performed as described previously (8). Briefly, 5 x lo5 cells were seeded onto Petri dishes in 5 ml of growth medium and allowed to attach overnight. Cell medium was exchanged with 3 ml of fresh medium containing drugs at the indicated concentrations, and tumor cell cultures were incubated further for 24 h. To radiolabel the cells, t3H1thymidine was added (1 pCi/ml) for an additional 24 h. Cells were detached with 0.53 mM EDTA in PBS, washed with PBS, and resuspended at lo5 cells/ml in DMEM containing 15 m~ HEPES and 1% BSA. Cell suspensions (100 pl) were added to polymerized galaptin-coated wells, and the percent adherent cells was esti- mated as described earlier (8).

The effect of anti-lamp antibodies on A-121 cell adhesion to polymerized galaptin was determined as follows. [3HlThymidine-labeled A-121 cells (3.3 x lo4) were suspended in 300 pl of RPMI 1640 containing 15 mM HEPES and 1% BSA. Samples were treated with 30 pl of polyclonal anti-lamp-1 IgG (78 pg of protein) or 30 pl of polyclonal anti-lamp-2 antibodies (93 pg of protein). Protein A (15 pg) was then added, and after a 15-min incubation 100-pl aliquots containing 10' cells were added to galaptin-coated plates. The percentage of adherent cells was determined after 30 min at 37 "C as described earlier. As controls, cell samples were treated with medium alone, medium plus protein A, rabbit IgG (90 pg), or rabbit IgG plus protein A.

Preparation ofA-121 Cell Extracts, SDS-Polyacrylamide Gel Electro- phoresis, and Western Blotting-Cultures of control and drug-treated A-121 cells were washed with PBS and detached with 0.53 mM EDTA. Cell pellets were lysed by incubation for 30 min on ice in 10 mM Tris, 500 mM NaCl, 2 mM EDTA, pH 7.3, containing 5 mM MgCl,, 1.5% Triton X-100, 50 pg/ml DNase I, 50 pg/ml RNase, 1 mM phenylmethylsulfonyl fluoride, and 10 pg/ml each of pepstatin A, aprotinin, and leupeptin. The cell lysate was sonicated and centrifuged at 100,000 x g for 1.5 h a t 4 "C.

The resulting cell extract was separated on 7% minigels by SDS-polyacrylamide gel electrophoresis (20) and electroblotted onto nitrocellulose membranes.

Nitrocellulose blots were probed with rabbit lamp-1 and lamp-2 polyclonal antibodies as described earlier (8). When probed with D. stramonium agglutinin biotin conjugates, an additional 1-h incubation with avidin horseradish peroxidase conjugate (1:20,000 in TBS, 2% BSA) was performed. Bound antibodies or lectin were detected using an enhanced chemiluminescence reaction (ECL Western blotting detection, Amer- sham) according to the manufacturer's protocol. Blots were exposed to X-Omat AR x-ray films, and the obtained autoradiograms were scanned using a Computing Densitometer (Molecular Dynamics).

Incorporation of Macromolecule Precursors-A-121 cells (5 x 10' cells/well/ml) were incubated in DMEM flacking glucose) for 6 h in the absence or presence of 3-F-GlcNAc a t given concentrations. I3H1Glu- cosamine (2 pCi/ml), r3H1thymidine (1 pCi/ml), or [3Hlleucine (4 pCi/ml) was added for the final hour of drug treatment, and the radioactivity incorporated into trichloroacetic acid precipitates was determined as described (21). [3HlFucose (20 pCi/ml) incorporation was measured after a 72-h exposure to drug in complete growth medium. Inhibition of incorporation of [3Hlglucosamine (1 pCi/ml), [3Hlgalactose (1 pCi/ml), and [3H]fucose (8 pCi/ml) by 4-F-GlcNAc was measured after a 48-h exposure to drug with a 6-h labeling pulse. Results are given as a percent control, expressed as c p d p g protein.

Indirect Immunofluorescence-Cells were grown in complete growth medium on glass coverslips in the absence and the presence of drug for 48 h. Monolayers of viable cells were blocked in PBS containing 0.4% BSAfor 15 min at 4 "C. Cells were treated with lamp-1 rabbit polyclonal antibodies or normal rabbit serum diluted 1:lOO in PBS, 0.4% BSA for 45 min at 4 "C then washed in PBS and PBS, 0.4% BSA. Bound antibodies were detected by incubating cells with rhodamine-conjugated donkey anti-rabbit IgG diluted 1:500 in PBS, 0.4% BSA for 45 min a t 4 "C. Cells were washed in PBS and fixed for 30 min in 2% paraformaldehyde in PBS; the coverslips then were mounted in Aqua-Poly Mount onto microscopic slides. Biotin-conjugated polymerized galaptin was used at a concentration of 10 pg/ml, and bound lectin was detected by incubation with avidin-Texas Red conjugates diluted 1500 as described above. Fluorescence micrographs were prepared using a Nikon micro- scope equipped with a Nikon FX-35WA camera and appropriate objec- tives and filter modules.

Protein Assay-Protein was determined by the modified Lowry procedure, according to Schacterle and Pollack (22).

Sugar Analogs-2-Acetamido-1,4,6-tri-O-acetyl-3-deoxy-3-fluoro-u- o-glucopyranose (3-F-GlcNAc) and 2-acetamido-1,3,6-tri-U-acetyl-4-deoxy-4-fluoro-u-~-glucopyranose (4-F-GlcNAc) were synthesized according to the methods described earlier (23, 24).

RESULTS

As we reported earlier, adhesion of human ovarian carcinoma A-121 cells to ECM is partially mediated by an interaction between galaptin and cell surface lamp-1 and lamp-2 (8). Heavily glycosylated lamp molecules contain long polylactosamine chains consisting of N-acetyllactosamine repeats, natural ligands for galaptin.

To confirm the specificity of galaptin-lamp interactions, lamp-1 and lamp-2 rabbit polyclonal antibodies were used to inhibit the adhesion ofA-121 cells to polymerized galaptin. The lamp-1 and lamp-2 antibodies were raised in rabbits using multiple antigen immunization (ll), thus the majority of im- munoglobulin was IgG (25). Our previous study showed that polymerized galaptin bound to the Fc fragment of rabbit IgG heavy chain. Therefore, protein A was used to mask the Fc region, preventing binding between the Fc fragment oligosaccharides and polymerized galaptin. As shown in Fig. 1, preincubation ofA-121 cells with lamp antibodies for 15 min followed by incubation with protein A resulted in approximately 50% inhibition of cell attachment to polymerized galaptin-coated wells. Preincubation ofA-121 cells with protein A, IgG alone, or the combination of both did not have any effect on A-121 cell adhesion. Similarly, anti-lamp antibodies alone did not show an inhibitory effect due to direct binding to polymerized galaptin through the Fc region (data not shown).


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I . .. . ~ . n n ’ Tumor Cell Adhesion 22799 lnhsbltron of uuarsan

v) 120 [ t I

120 I

Control IgG Prot A IgG Lamp-1 Lamp-2 I I I I

+ Prot A

FIG. 1. Inhibition of human ovarian carcinoma A-121 cell adhesion to polymerized galaptin by lamp-1 and lamp-2 antibodies. [3H]Thymidine-labeled A-121 cells (3.3 x lo4 cells) were suspended in 300 p1 of RPMI 1640, 15 mM HEPES, 1% BSA and treated

was added to selected samples and incubated for an additional 15 min. with lamp-1 and lamp-2 antibodies or rabbit IgG for 15 min. Protein A

Aliquots of 100 p1 (lo4 cells) were then added to wells adsorbed with polymerized galaptin, and after 15 min the percent of attached cells was determined as described under ”Experimental Procedures.”

It was reported that the stability and half-life of lamp molecules depend directly on the length of polylactosamine chains (Ga1/31+4GlcNAc/31+3), (12). Therefore, a substitution at the C4 position of GlcNAc molecule should result in chain termi- nation. Additionally, the binding specificity of galaptin is dependent on the hydroxyl group at the C3 position of glucosamine (15-18). Based on these observations, two sugar analogs, 3-fluoro- and 4-fluoroglucosamine, have been synthesized as potential carbohydrate chain modifiers andor chain terminators of polylactosaminyl side chains of target glycoproteins such as lamps.

To test the effect of the 3-F-GlcNAc on macromolecular biosynthesis, A-121 cells were treated with sugar analog at a wide range of concentrations for 6 h; radiolabeled precursors were added for the last hour of incubation. As shown in Fig. 2A the 3-F-GlcNAc preferentially inhibited the incorporation of labeled glucosamine and fucose into the cells, suggesting that this compound interferes with glycosylation pathways. Inhibi- tion of glucosamine incorporation was stronger and appeared at a lower drug concentration than inhibition of fucose incorporation. In contrast, little inhibition of DNA or protein synthesis was observed under the same conditions as measured by incorporation of labeled thymidine and leucine, respectively (Fig. 2A) . The exposure ofA-121 cells to 0.5 mM 4-F-GlcNAc for 48 h resulted in 50% inhibition of glucosamine incorporation to trichloroacetic acid precipitates and a smaller, 20%, inhibition of galactose and fucose incorporation (Fig. 2B).

Incubation of A-121 cells with drugs for 48 h resulted in decreased tumor cell adhesion to polymerized galaptin over the time of experiment (Fig. 3). The 3-F-GlcNAc at 0.5 mM caused 45% inhibition, whereas the more potent, 4-fluor0 analog (4-F- GleNAc) at 0.25 mM inhibited adhesion by 55%. The level of inhibition reached 80% when both drugs (0.2 m~ each) were given in combination (Fig. 4). This effect was proportional to the drug concentration; a decrease in concentration to 0.1 m~ resulted in decreased inhibition (from 80 to 50%) of A-121 cell adhesion.

The changes in oligosaccharide structure on the cell surface after sugar analog treatment were reflected by decreases in lectin binding. The binding level of concanavalin A, wheat germ agglutinin, Bandeiraea simplicifolia, and Phaseolus vulgaris decreased with increased drug concentration (data not shown). The binding of P. vulgaris (specific for -GlcNAc/31-.6 Man) was affected to the greatest degree.

0.01 0.1 0.5 1.0

3-F-GlcNAc [mM]

140 I I L I

120 a: ‘z 100

8 80 U 0 z W

[r W

t 60

o 40

a 20

0 0.1 0.5

4-F-GlcNAc [mM] FIG. 2. Macromolecular incorporation of labeled precursors

into A-121 ovarian carcinoma cells. Panel A, A-121 cells (5 x lo4 celldwelVm1) were incubated in DMEM (lacking glucose) for 6 h in the absence or presence of 3-F-GlcNAc at given concentrations. [3HlGlu- cosamine (2 pCi/ml; solid bars), [3Hlthymidine (1 pCi/ml; dotted bars) and [3Hlleucine (4 pCi/ml; open bars) were added for the final hour of drug treatment, and the radioactivity incorporated into trichloroacetic acid precipitates was determined as described under “Experimental Procedures.” [3HlFucose (20 pCi/ml; hatched bars) was measured after a 72-h exposure to drug in complete growth medium. The incorporation into control cells +. S.D. was at the level of 251 f 56, 3,099 e 1,100, 780 f 223, and 517 61 c p d p g of protein for [3Hlleucine, [3HJthymidine, [3Hlglucosamine, and [3Hlfucose, respectively. Panel B , the radioactivity incorporated into trichloroacetic acid precipitates was measured after a 48-h exposure to 4-F-GlcNAc at given concentrations and 6-h labeling period with 1 pCi/ml 13Hlglucosamine (solid bars), 1 pCi/ml r3Hlgalac- tose (crossed bars), and 8 pCi/ml [3Hlfucose (hatched bars). The incorporation into control cells S.D. was at the level of 4,175 2 711, 8,757 f 2,386 and 427 81 cpdwel l for [3H]glucosamine, [3H]galactose, and [3Hlfucose, respectively.

The large amount of high affinity binding sites for I? vulgaris on A-121 cell surface are correlated with the elevated levels of 0-N-acetylglucosaminyl transferase V, an enzyme responsible for the formation of branched oligosaccharide structures in glycoproteins (26). These tri- and tetraantenary branched structures often carry poly-N-acetyllactosamine sequences. Among glycoproteins, lamp molecules, A-121 cell surface ligands for galaptin, are the main carriers of lactosamine residues. To de- termine whether drug treatment caused any changes in polylactosamine chains in lamp molecules, cell extracts from control and drug-treated cells were obtained and probed with lamp polyclonal rabbit antibodies. Dot-blot analysis indicated that A-121 cell extract contained more lamp-1 than lamp-2 molecules. Treatment with 2.5 mM 3-fluor0 analog resulted in almost a 50% decrease in lamp-1 reactivity (Fig. 5 A ) , whereas reactivity of lamp-2 was less affected (Fig. 5B).

An analysis of cell extracts subjected to 7% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes was performed with lamp-1 antibodies and D. stramonium agglutinin lectin, which specifically binds Galp(l-4)GlcNAc structures. Lamp-1 polyclonal antibody re-


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22800 Inhibition of Ovarian Tumor Cell Adhesion

5 t I

0 15 30 45 60 75

TIME [rnin]

FIG. 3. Inhibition of A-121 ovarian carcinoma cell adhesion to polymerized galaptin-coated wells after treatment of cells with sugar analogs. A-121 cells (1 x 105/ml) were incubated for 48 h without (0) and with 3-F-GlcNAc (0.05 mM, 0; 0.5 mM, 4 )and 4-F-GlcNAc (0.05 mh1, n; 0.2 mM, A). [”]Thymidine was added for last 16 h of incubation. Cells were removed from monolayers with 0.53 mM EDTA, and lo4 cells resuspended in DMEM, 20 mM HEPES, 10 mg/ml BSA were added to the wells coated with polymerized galaptin. The percent of adherent cells was measured at the indicated time intervals as described under “Experimental Procedures.” The asterisks point to statistically significant differences between control and drug-treated samples: p < 0.01, t test.

15 30 45 60 Time [min]

FIG. 4. Effect of sugar analogs on A-121 adhesion to polymerized galaptin. Details of the experimental procedure are as described in Fig. 3. Control, open bars; 0.2 mM 3-F-GlcNAc, hatched bars; 0.2 mM 4-F-GlcNAc, dotted bars; both drugs in combination, solid bars. The asterisks point to statistically significant differences between control and samples treated with the drugs in combination: *, p < 0.01, t test, and between values obtained for drugs given separately and in combination: **, p < 0.01, t test.

acted with a protein in the control cell extract corresponding to the mature form of lamp-1 with an apparent molecular mass of 116 kDa. Very weak reactivity was associated with a lower molecular mass protein (66-69 ma). Treatment of A-121 cells with 0.2 mM 3-F-GlcNAc resulted in a %fold amplification of the lower molecular mass protein band, suggesting increased content of a shorter, more immature species (Fig. 6, lanes 1 and2).

When D. stramonium agglutinin was used for probing the nitrocellulose membranes, an additional protein band($, rich in Galpl+lGlcNAC, with a molecular mass of 95-97 kDa, was detected. The treatment of cells with 4-F-GlcNAc caused an almost total disappearance of the staining in this area (Fig. 6, lanes 3 and 4) .

To probe further the changes in galaptin ligand(s) on A-121 cell surface, control and 0.5 mM 3-F-GlcNAc-treated cells were subjected tcj indirect immunofluorescence using anti-lamp antibodies as described under “Experimental Procedures.” Both lamp-1 and lamp-2 were detected on cell surfaces in control and sugar analog-treated cells (Fig. 7,A and B). Although in control cells lamp-1 was detected on cellular extensions, the lamp-2 binding was not detected in that area (data not shown). Im-

L v) Z W 0 W

I- z 4 w IT

t

v) Z W 0 W

I-

k

z 5 W U

3.50

A: LAMP-I

0.00 0.25 0.50 0.75 1.00

CELL EXTRACT [pg protein]

0.00 0.25 0.50 0.75

CELL EXTRACT [pg protein]

FIG. 5. Lamp-1 (punel A) and lamp-2 (punel B ) expression in A-121 cells after sugar analog treatments. A-121 cells were incubated without (closed circles) or with 2.5 mM 3-F-GlcNAc (open circles) and 0.5 mM 4-F-GlcNAc (closed triangles) for 48 h. The cell extract was obtained as described under “Experimental Procedures.” Indicated amounts of protein extract from control and drug-treated cells were spotted onto nitrocellulose membrane using a dot blotter, blocked with TBS, 2% BSA and probed with anti-lamp-1 antibodies. The developed dot blots were scanned using Image Scan and quantified as described under “Experimental Procedures.”

1 2 3 4

-116kD - 97

- 66

FIG. 6. Western blot analysis of protein extracts from A-121 cells. Equal amounts of protein extracts (10 pg/lane) from control cells (lanes 1 and 3 ) , 0.2 mM 3-F-GlcNAc (lane Z), and cells treated with 0.2 mM 4-F-GlcNAc (lane 4 ) were separated on 7% minigels by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membrane, and probed with a 1:400 dilution of lamp-1 rabbit polyclonal antibodies (lanes 1 and 2) or with 5 pg/ml biotin-conjugated D. stramonium agglutinin (lanes 3 and 4) . Blots were washed, treated with appropriate secondary antibodies, and processed as described under “Ex- perimental Procedures.”

munofluorescence analysis revealed that drug treatment caused significant alteration in cell morphology, leading to reduction in numbers of cellular extensions. The residual cellular extensions appear to show less intense staining for lamp-1.

The structural and morphological changes caused by drug treatment to cell surface lamp molecules were reflected by decreased binding of polymerized galaptin to A-121. polymerized galaptin bound efficiently to the surface ofcontrol cells (Fig. 8A).


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Inhibition of Ovarian

lamp-1. Control cells ( p n r ~ e l AI and 0.5 m\I 3-F-GlcNAc 48-h-treated FIG. 7. Indirect immunofluorescence microscopy of A-121 cell

cells (panel B ) . Monolayers of viable cells grown on coverslips were incubated with a 1:lOO dilution of lamp-1 polyclonal antibodies. Cells were washed and treated with a 1:150 dilution of rhodamine B isothiocyanate donkey anti-rabbit IgG, fixed in 2 6 paraformaldehyde, and mounted onto slides with Aqua-Poly Mount. Note the absence of cellular extensions following sugar analog treatment. Magnification, x 40.

Treatment with 0.2 mM 4-F-GlcNAc caused a strong reduction in binding (Fig. 8 0 . When both drugs were used in combination only a limited amount of polymerized galaptin molecules bound to cell surfaces (Fig. 8D). A 48-h incubation of A-121 cells with 0.5 mM 3-F-GlcNAc resulted only in a slight decrease in galaptin binding (Fig. 8B). These results confirmed previous observations regarding the apparent synergistic activity of these sugar analogs, which led to decreased adhesion of drug-treated human ovarian carcinoma cells to galaptin-coated wells.

DISCUSSION The spread of human ovarian cancer is frequently limited to

the peritoneal cavity (27). The progression of ovarian cancer involves the shedding of solid tumor cells into the peritoneal cavity with subsequent attachment and growth of tumor cells on structures throughout the peritoneum (28). Therefore, iden- tification of the adhesion mechanisms underlying these events with the development of agents capable of interfering with these processes would be of great significance.

Adhesion of human ovarian carcinoma A-121 cells is at least partially mediated through carbohydrate-lectin interactions. Galaptin, a P-galactoside-binding lectin found in ECM, inter- acts with A-121 ovarian carcinoma cell surface lamp molecules. Galaptin present on the surface ofA-121 cells may also interact with other glycosylated components of ECM, such as laminin and fibronectin. Ozeki et al. (29) suggested that tissue fibronectin may function as an endogenous ligand for P-galactoside-

lhmor Cell Adhesion 22801

binding lectin as well as serve as a ligand for integrins. These authors implied that the lectin plays a role in cell adhesion by interacting with ECM components including fibronectin and laminin through protein-carbohydrate interactions (29). This type of carbohydrate-mediated interaction can a t least partially explain the preferential adhesion ofA-121 to ECM rather than to mesothelial cells, as noted in previous studies (1).

The incubation ofA-121 cells with polyclonal lamp antibodies in the presence of protein A resulted in significant inhibition of adhesion to polymerized galaptin. The presence of protein Adid not affect the binding of lamp antibodies. The use of protein A was necessary to eliminate direct binding of lamp antibodies to polymerized galaptin through the Fc region. Because of this direct binding, no inhibitory effect on A-121 cell adhesion by lamp antibodies alone was observed (data not shown). How- ever, in the studies reported by Amos and Lotan (301, the adhesion of F9 murine embryonal carcinoma cells to laminin and fibronectin was not affected by anti-lamp-1 and anti-lamp-2 antibodies. Although the differentiation of F9 cells led to increased glycosylation of lamps and their more abundant presence on the cell surface, adhesion to laminin and fibronectin by differentiated cells was decreased when compared with undif- ferentiated cells. I t is very likely that heavily glycosylated polylactosamine chains on the cell surface masked cell surface receptors for laminin and fibronectin leading to decreased adhesion. Moreover, under those reported experimental conditions, cell attachment was mediated to a greater extent by protein-protein than lectin-oligosaccharide interactions. In this study, we employed polymerized galaptin-coated wells as a model substrate to evaluate tumor cell adhesion. Polymerized galaptin adsorbed to plastic-mediated adhesion of lymphoblas- toid (31) and A-121 ovarian cells more efficiently than unpo- lymerized galaptin dimers probably because of the presence of more carbohydrate binding sites accessible for ligand binding.

To modulate the structure of tumor cell surface glycoproteins, 3-flUOrO- and 4-fluoroglucosamine analogs have been evaluated as potential oligosaccharide chain terminators and/or modifiers. Substitution of a hydroxyl group with fluorine at C3 of glucosamine, based on binding specificity studies for galaptin, was expected to alter the interaction between galaptin and its ligand. Moreover, if 3-F-GlcNAc is incorporated into the polylactosamine chain, fluorine a t C3 should prevent formation of the Fucal+3 linkage. On the other hand, substitution of fluorine at C4 should prevent elongation of poly-N- acetyllactosamine chains and lead to structurally altered lamps having shorter half-lives. Both anticipated effects of sugar analogs should result in decreased carbohydrate-mediated cell attachment.

Treatment ofA-121 ovarian tumor cells with 3-F-GlcNAc and 4-F-GlcNAc led to a concentration-dependent inhibition of cellular attachment to polymerized galaptin-coated wells (Fig. 31, with the greatest effect observed when both drugs were used in combination (Fig. 4). The inhibition of attachment was a consequence of structural modification of cell surface oligosaccharides, since drug treatment selectively affected the biosynthesis of glycoconjugates (Fig. 2) and lectin binding. Western blots of protein extracts from control and drug-treated cells showed a difference in reactivity with anti-lamp antibodies and D. stramonium agglutinin lectin. Probing with anti-lamp antibodies revealed an additional band (67 kDa) that probably corre- sponds to an immature, not fully glycosylated species of lamp. On the other hand, probing with D. stramonium agglutinin showed the presence of an additional glycoprotein of unknown structure and function (95-97 kDa), rich in Galpl-+4GlcNAc structure, which was also affected by glucosamine analog treatment. Since D. stramonium agglutinin specifically recognizes


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22802 Inhibition of Ovarian lbmor Cell Adhesion

with biotin-conjugated polymerized galaptin (10 pg/ml). Cells were washed and treated with a 1:500 dilution of avidin-Texas Red conjugates, fixed FIG. 8. Binding of biotin-conjugated polymerized galaptin to A-121 cells. Monolayers of viable cells grown on cowrslips were incubated

in 2% paraformaldehyde, and mounted onto slides with Aqua-Poly Mount. Panel A, control cells; panel B, 0.5 mM 3-F-GlcNAc for 48 h; panel C , 0.2 m M 4-F-GlcNAc for 48 h; and panel D, both drugs in combination. Magnification, x 40.

Galpl+4GlcNAc, the results indicate a decrease in the amount of this structure in 4-F-GlcNAc-treated cells. These findings strongly suggest that incorporation of this modified sugar into polylactosamine chains prevents further elongation and fuco- sylation. As a consequence, the binding of polymerized galaptin to A-121 cell surface was reduced because of a smaller amount of available ligand. After treatment with both drugs in combination, there was almost complete reduction in the binding of polymerized galaptin to A-121 cell surface, correlating well with the observed inhibition of adhesion (Fig. 3).

Increased amounts of poly-N-acetyllactosamine chains in lamp-1 and lamp-2 led to prolonged half-lives of lamp molecules in differentiated HL-60 cells. Based on these observations, Lee et al. (12) suggested that more stable lamp molecules might facilitate the fusion of lysosomes with cell membrane. Since the presence of lamp molecules on the cell surface is necessary for participation in tumor cell adhesion, the reduction in their amount and/or changes in polylactosamine structure on the cell surface should decrease tumor cell adhesion mediated by lectin-lamp interaction. As reported by Huang et al. (32), modu- lation of oligosaccharide chain structure by a galactosamine analog led to altered cellular adhesion. These authors showed an inhibition of synthesis of [3H]glucosamine-labeled mucins in human colon cancer cells by benzyl-a-GalNAc. Treatment of HM7 cells, high mucin variants of the colon cancer cell line, with benzyl-a-GalNAc decreased the level of peripheral mucin- associated carbohydrate antigens such as sialyl Le" and sialyl Le" and increased lectin binding to T and Tn antigens. These changes in the structure of mucin oligosaccharides were accom- panied by a decrease in cell binding to E-selectin, thus affecting

carbohydrate-mediated adhesion of human colon cancer cells. Although lamps are lysosomal proteins, they have been

shown to be present on the cell surfaces of several different tumor cell types (30,33-35). At either an intra- or extracellular location, they are believed to protect cellular components in the microenviroment from degradation by hydrolytic enzymes. In several cellular systems, increased expression of lamps on cell surfaces or increased amounts of polylactosamines attached to cell surface lamps has been associated with an enhanced metastatic potential of these cells (36-38). We also demonstrated that A-121 cells express lamp-1 and lamp-2 on their surface (8) and degrade ECM by releasing lysosomal hydrolytic enzymes (l), whereas a nontumorigenic and noninvasive counterpart, human ovarian carcinoma A-1 cell line, expresses significantly less highly branched lamp molecules on its cell surfaces and does not degrade ECM in vitro (39,40).

Moreover, in several cellular systems, (41-43) increased levels of P-galactoside-binding lectins have been associated with an increased ability of tumor cells to aggregate, adhere, and metastasize homotypically to the lung (44, 45). Inhibition of these activities by antibodies against lectins and galactose-rich glycoproteins, including lamp, further supports the involve- ment of P-galactoside-binding lectin in tumor cell recognition, adhesion, and metastasis (46-49). An elegant study by Sawada and co-workers (50) directly correlated the cell surface expression of lamp and lamp-borne sialyl-Le" structures with the E-selectin-mediated adhesion to endothelial cells. Using a se- ries of human colonic cell lines with different metastatic potential, the authors showed a strong relation between E-selectin mediated adhesion and metastatic potential, thus pointing to


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Inhibition of Ovarian Tumor Cell Adhesion 22803

another potential target for chemotherapy (50). In conclusion, we have demonstrated that modification of

human ovarian tumor cell surfaces by treatment of cells with 3- and 4-fluoro-substituted sugar analogs results in inhibition of galaptin-mediated cellular adhesion. Interference with the carbohydrate-mediated interactions such as homotypic aggrega- tion and adhesion may lead to reduced tumor growth in situ and decreased metastatic potential. In the case of ovarian tumors, which express anchorage-dependent growth, adhesion factors may be exploitable targets, and an inhibition of adhesion may be useful as a supplementary therapy along with surgery and/or chemotherapy.

cussions and many helpful suggestions during the preparation of this Acknowledgments-We thank Dr. Howard Allen for stimulating dis-

manuscript and Dr. Minoru Fukuda for kindly providing antibodies to lamp-1 and lamp-2.

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B Woynarowska, D M Skrincosky, A Haag, M Sharma, K Matta and R J BernackiInhibition of lectin-mediated ovarian tumor cell adhesion by sugar analogs.

1994, 269:22797-22803.J. Biol. Chem.

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