Effects of Thomsen-Friedenreich Antigen-specific Peptide P-30 on β-Galactoside-mediated Homotypic Aggregation and Adhesion to the Endothelium of MDA-MB-435 Human Breast Carcinoma Cells

Cell Lines and Cultures.

The MDA-MB-435 human breast carcinoma cell line, originally isolated from the pleural effusion of a patient with breast carcinoma, was kindly provided by Dr. Janet E. Price, M. D. Anderson Cancer Center, Houston, TX. This cell line was selected for our study because it was found to be highly metastatic in nude mice from mammary fat-pad tumors as well as on i.v. inoculation in vivo (14 , 15) and exhibited superior aggregation and survivability in vitro compared with the other lines tested (16) . Tumor cells were maintained in 5% CO₂/95% air at 37°C in a humidified incubator in tissue culture flasks as a monolayer culture using RPMI 1640 supplemented with l-glutamine, 10% fetal bovine serum, sodium pyruvate, and nonessential amino acids.

HUVECs pooled from multiple isolates were purchased from Cascade Biologicals, Inc. (Portland, OR). The cultures were free of HIV-1, Hepatitis B and C viruses, Mycoplasma, bacteria, yeast, and fungi. The cells were positive for the DiI-acetylated low density lipoprotein uptake and expression of von Willebrand factor and CD31 but not for the α-actin expression. The HUVECs were maintained on plastic as a monolayer culture in a humidified incubator in 5% CO₂/95% air at 37°C. The basal Medium 200 (Cascade Biologicals), supplemented with low serum growth supplement containing fetal bovine serum (2% v/v final concentration), hydrocortisone, human fibroblast growth factor, heparin, and human epidermal growth factor, was used. The cells at population doublings of approximately 8–12 were used for the adhesion experiments.

Peptide Synthesis and Purification.

T antigen-binding peptide P-30 (HGRFILPWWYAFSPS) and control peptide (RRLLFYKYVYKRYRAGKQRG) were chemically synthesized on the Applied Biosystems peptide synthesizer 431A using N-(9-fluorenyl)methoxycarbonyl-based chemistry and purified to homogeneity on a C-18 reverse-phase high-performance liquid chromatography column (ISCO Corp.).

Antibodies.

A rabbit polyclonal anti-galectin-1 antiserum was a generous gift from Dr. Douglas W. N. Cooper (University of California, San Francisco, CA). A rat monoclonal anti-galectin-3 (anti-Mac-2) antibody (17) was used as described previously (18) . Rabbit anti-galectin-4 serum was raised using the COOH-terminal domain of rat intestinal galectin-4 as immunogen as described previously (19) . Cy5-conjugated goat antirabbit IgG was purchased from Jackson Immuno Research Laboratories (West Grove, PA). Goat Texas Red-conjugated antirat antibody was purchased from Molecular Probes (Eugene, OR).

Cytochemical Analysis of T Antigen.

The cytochemical analysis of T antigen was performed using PNA lectin-horseradish peroxidase conjugate, and subsequent color reaction was performed with diaminobenzidine tetrahydrochloride. The direct binding of T antigen-specific PNA lectin to MDA-MB-435 human breast carcinoma cells was performed as described previously (13) with one minor modification. After dissociation of cells from the plastic and before fixing them with 2% formaldehyde-PBS solution and placing on a microscope slide, the cells were allowed to aggregate for 30 min in serum-free RPMI 1640 at 37°C.

Cell Aggregation Assay.

A homotypic aggregation assay of MDA-MB-435 cells was performed as previously described (4 , 20) . The only modification was made for the samples prepared for the cytological analysis of T antigen. In these experiments, cancer cells were allowed to aggregate for 30 min instead of 1 h to avoid formation of excessively large multicellular aggregates.

Analysis of Galectins Expression in MDA-MB-435 Cells.

The metabolic [³⁵S]methionine/cysteine labeling of galectins followed by affinity purification on lactosyl-Sepharose and separation by SDS-PAGE was performed exactly as described previously (21) . Densitometry of SDS-PAGE of the purified galectins was used to assess the relative amounts of each galectin. On the basis of the absolute yield of lactosyl-Sepharose purified galectins and the estimated volume of the confluent monolayer of MDA-MB-435 cells, the approximate molar concentrations of galectins 1, 3, and 4 were calculated as described previously (21) .

Adhesion to the Endothelium.

HUVECs were grown to confluence directly on microscope slides using the four-well Lab-Tec II chamber slide system (NalgeNunc, Naperville, IL). Twenty-four h before the adhesion experiment, the endothelial cell cultures were switched to quiescence medium (Medium 200 without low serum growth supplement), and MDA-MB-435 human breast carcinoma cells were prelabeled with 5 μg/ml solution of DiI (Molecular Probes) in serum-free RPMI 1640 for 60 min at 37°C. Immediately before the experiment, cancer cells were dissociated from plastic using a nonenzymatic cell dissociation reagent (Sigma, St. Louis, MO), and pipetted to produce a single-cell suspension. DiI-labeled breast carcinoma cells [5 × 10⁴ cells per chamber in 2.5 ml of serum-free medium supplemented with various concentrations of P-30 (0 to 0.1 mg/ml) or control peptide] were added to the monolayer of the endothelial cells. The chambers were sealed with adhesive tape while ensuring that no air bubbles were trapped. The cells were allowed to adhere for 1 h at 37°C, after which the chambers were inverted and left upside down for 30 min to allow sedimentation of nonadhered cells. At the end of the incubation, the medium was drained while chambers were still upside-down. Samples were gently rinsed with PBS, fixed for 30 min in 2% formaldehyde solution in PBS, mounted under cover glass, and examined by fluorescent microscopy. Four random fields in each well were photographed at ×250, and the total number of adhered cells in every field was counted. The assay was performed in quadruplicate for each concentration of the peptides tested.

Laser Scanning Confocal Microscopy.

The samples for laser scanning confocal microscopy were prepared exactly as described above in “Adhesion to the Endothelium,” except that the cancer cells used in these experiments were not prelabeled with DiI, and samples were fixed (but not permeabilized) in 2% formaldehyde solution in PBS for 24 h. The antibodies against galectins-1, -3, and -4 were used as described previously (20) . The goat Texas-Red-conjugated antirat antibody and Cy5-conjugated goat antirabbit IgG were used as secondary antibodies at a dilution of 1:100. The laser scanning confocal microscopy was performed with a Bio-Rad MRC 600 confocal system. The RHS and YHS blocks were used to detect fluorescence emitted by Cy5 and Texas Red respectively. The Z stacks were prepared by obtaining serial sections with 0.5-μm increments and analyzed in orthogonal projections (Y-Z and X-Z sections) using the MetaMorph Imaging System software (Universal Imaging, Hallis, NH).

Involvement of T Antigen in Homotypic Aggregation of MDA-MB-435 Human Breast Carcinoma Cells.

Multicellular aggregate formation is an important feature of metastatic cancer cells directly correlating with their increased survival potential in vitro (20) and metastatic propensity in vivo (22) . The cancer-associated T antigen has been implicated in tumor cell adhesion through carbohydrate-lectin interactions (6 , 23) . We previously reported the expression of large quantities of T antigen on the surface of MDA-MB-435 cells that was confirmed by the binding of T antigen-specific PNA lectin (13) . In this study, we investigated the role of T antigen in homotypic aggregation of the MDA-MB-435 breast cancer cells. Tumor cells collected from subconfluent (70–80%) cultures were allowed to form multicellular aggregates as described in “Materials and Methods.” The direct binding of T antigen-specific PNA lectin, conjugated to horseradish peroxidase followed by color reaction with diaminobenzidine tetrahydrochloride, was used to visualize T antigen. The cytochemical analysis of the samples that contained multicellular aggregates revealed significant accumulation of T antigen at the sites of cell contacts (Fig. 1A ⇓ ), which suggested participation of T antigen in homotypic aggregation of MDA-MB-435 breast carcinoma cells. Consistent with this is the fact that the addition of different concentrations of synthetic T antigen-specific peptide, P-30 (HGRFILPWWYAFSPS), inhibited homotypic aggregation of MDA-MB-435 cells in a dose-dependent manner (Fig. 1B ⇓ ). A maximal inhibitory effect (>70%) was achieved at a peptide concentration of 0.1 mg/ml. The control peptide (RRLLFYKYVYKRYRAGKQRG), which does not interact with T antigen (13) , failed to inhibit homotypic aggregation of MDA-MB-435 cells (Fig. 1, C–E ⇓ ). These findings, as well as the previously reported ability of P-30 to inhibit asialofetuin-mediated aggregation of mouse melanoma cells (13) , suggest that the effect of P-30 on homotypic aggregation of MDA-MB-435 cells is T antigen-specific.

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Fig. 1.

A, direct binding of T antigen-specific PNA to the MDA-MB-435 human breast carcinoma cells. Arrows, accumulation of T antigen at the sites of the cell-cell contact. B, dose-dependent inhibition of spontaneous homotypic aggregation of the MDA-MB-435 human breast carcinoma cells by T antigen-specific peptide P-30. C–E, inhibition of spontaneous homotypic aggregation of MDA-MB-435 human breast carcinoma cells by 0.1 mg/ml of synthetic P-30 (E) but not by the same concentration of the control peptide (D) compared with the control (C).

Expression of β-Galactoside-specific Lectins (Galectins) on MDA-MB-435 Cells.

Because T antigen (Galβ1 → 3GalNAc) has β-galactose as a terminal sugar, it is likely that T antigen-mediated interactions may involve the participation of β-galactoside-specific lectins (galectins). Thus, we studied the expression profile of the galectins, namely galectin-1, galectin-3, and galectin-4 in MDA-MB-435 cells. Metabolic [³⁵S]methionine/cysteine labeling followed by affinity purification on lactosyl-Sepharose and separation by SDS-PAGE was used to isolate galectins and characterize their expression in this cell line. The results of these experiments (Fig. 2 ⇓ ) identified galectin-1 as a major β-galactoside-specific lectin expressed in MDA-MB-435 breast carcinoma cells. The estimated molar concentrations of galectins-1, -3, and -4 in MDA-MB-435 cells were in the range of 1–3 μm, 100–500 nm, and 10–50 nm, respectively. The presence of the additional minor band corresponding to a molecular weight of M_r ∼40,000 (Fig. 2 ⇓ ) suggests the weak expression of another, yet unidentified, soluble lactose-binding lectin (Gal-X). Human galectin-3 displays a 20-fold higher specific activity in binding to the Galβ1→3GalNAc disaccharide than galectin-1 (24) . Thus, galectin-3 is most likely to interact with T antigen. Previously reported inhibition by T antigen-specific P-30 of asialofetuin-mediated aggregation of B16-F1 cells (13) , known to be galectin-3-dependent (25) , is also supportive of this interaction. The analysis of β-galactoside-binding lectins in 11 other human breast carcinoma cell lines established from pleural or ascitic effusions revealed similar galectin expression profiles, 4 which suggests that galectin-1 and galectin-3 overexpression is a phenomenon frequently occurring in metastatic breast cancer.

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Fig. 2.

[³⁵S]Methionine/cysteine-labeled expression profile of β-galactoside-specific lectins (galectins) in MDA-MB-435 human breast carcinoma cells. On the left, the position of the molecular weight markers (46, 29, and 14, for M_r 46,000, 29,000, and 14,000, respectively).There is an abundant expression of galectin-1 and galectin-3 compared with a very weak expression of galectin-4. The additional weak band corresponding to the molecular weight of approximately M_r 40,000 suggests a low level of expression of an unidentified soluble β-galactoside-specific lectin (Gal-X).

Adhesion of MDA-MB-435 Breast Carcinoma Cells to the Endothelium.

Both galectin-1 and galectin-3 were found to be expressed on the endothelium of various origins in different species including humans (26) . Galectin-1 was also shown to participate in murine RAW117-H10 large-cell lymphoma cell adhesion to liver microvessel endothelial cells (26) , and galectin-3 was suggested to be, at least in part, responsible for the preferential adhesion of prostate cancer cells to human bone marrow endothelial cells (27) . Thus, it was of interest to analyze whetherβ -galactoside-specific lectins participate in adhesion of the MDA-MB-435 cells to the endothelium. Confocal laser microscopy revealed the clustering of both galectin-1 and galectin-3 to the sites of contact between MDA-MB-435 cells and human umbilical endothelial cells (Fig. 3, A and B ⇓ ) indicative of their involvement in the interaction between cancer and endothelial cells. We could not observe, however, any sign of galectin-4 participation in this process, which was consistent with the data on its low level of expression in MDA-MB-435 cells.

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Fig. 3.

Involvement of the galectin-1 (A) and galectin-3 (B) in the adhesion of the MDA-MB-435 human breast carcinoma cells to the monolayer of human umbilical endothelial cells as revealed by laser confocal microscopy. The X-Z sections shown were obtained as described in “Materials and Methods” using a ×60 lens. The images were pseudocolored (green, galectin-1; red, galectin-3). Arrows, clustering toward the cell contacts of galectin-1 on cancer cells (A) and of galectin-3 on endothelial cells (B). The superimposed fluorescent photomicrographs of DiI-labeled MDA-MB-435 breast cancer cells adhered to the monolayer of HUVEC cells (C–E). There is an inhibition of the adhesion by 0.1 mg/ml of the T antigen-specific synthetic P-30 (E) but not by the same concentration of the control peptide (D) compared with the control (C).

Interestingly, galectin-1 and galectin-3 reacted differently on tumor and endothelial cells. A strong galectin-1 signal accumulated at the sites of tumor-endothelial cell contact predominantly on the cancer cells (Fig. 3A ⇓ ), which suggested the involvement of one or more of its cognate ligands on the endothelium. Galectin-3, in contrast, although also being strongly expressed on the tumor cells, clearly demonstrated signal accumulation toward the sites of the cell contact on HUVEC (Fig. 3B ⇓ ) possibly interacting with T antigen or other putative ligands on cancer cells. We hypothesized that if galectin-3 on the endothelial cells interacts with T antigen on MDA-MB-435 cells, then T antigen-specific P-30 should inhibit this interaction as it did in the case of homotypic aggregation. Thus, we performed experiments in which cancer cells were allowed to adhere to a monolayer of endothelial cells in the presence of P-30 (0.1 mg/ml final concentration) or a control peptide of identical concentration. The results of these experiments (Fig. 3, C–E ⇓ ) showed that the control peptide did not effect the adhesion of the MDA-MB-435 cells to the endothelial cells (Fig. 3, C and D ⇓ ), whereas T antigen-specific P-30 significantly (2-fold) inhibited it (Fig. 3E ⇓ ). When adhesion experiments were performed with different concentrations of P-30, we found the peptide’s effect to be dose-dependent with the maximal inhibition achieved up to 50% (Fig. 4 ⇓ ). These data demonstrated that adhesion of the MDA-MB-435 human breast carcinoma cells to the endothelial cells was, at least in part, mediated by T antigen. We observed the same inhibitory effects of the P-30 peptide on both adhesion to the endothelium and spontaneous homotypic aggregation of DU-145 human prostate carcinoma cells (data not shown), which suggests that similar molecular mechanisms of adhesion could be involved in different human malignancies.

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Fig. 4.

Dose-dependent inhibition of the adhesion of MDA-MB-435 human breast carcinoma cells to the endothelium by synthetic T antigen-specific peptide P-30. The maximum inhibitory effect (about 50%) was achieved at 0.1 mg/ml concentration of the peptide. Representative results from one of three similar experiments are presented as mean of quadruplicate determinations. Bars, SD.

Multicellular aggregate formation and adhesion of tumor cells to the endothelium are crucial events during early stages of cancer metastasis. Taken together, our data indicate thatβ -galactoside and particularly T antigen-mediated cell-cell interactions are important components of these events. To the best of our knowledge, this is the first observation that directly shows the accumulation of galectin-1 and galectin-3 at sites of contact between cancer and endothelial cells, which is indicative of their active participation in the adhesion of tumor cells to the endothelium.

Strikingly different behavior of these twoβ -galactoside-specific lectins reflects the complexity of the adhesion process. The accumulation of galectin-1 at the sites of cell-cell contacts predominantly on cancer cells and galectin-3 on endothelial cells suggests that several of their cognate ligands may be simultaneously involved here on both tumor and endothelial cells. Inhibition of tumor cell adhesion by the T antigen-specific P-30 peptide, however, highlights an active role for this cell surface carbohydrate structure in cancer-endothelial cell interactions. Recent observations of Al-Mehdi et al. (7) indicate that hematogenous metastases arise from the endothelium-attached tumor cells, which makes them particularly vulnerable to intravascular drugs capable of disrupting cancer-endothelial cell interactions. The ability of a short synthetic peptide to effectively interfere with this line of intercellular communication may also be of functional significance for the development of new antiadhesive therapies of cancer metastasis.

Two other types of compounds that also targetβ -galactoside-mediated adhesion have already been proven to be effective inhibitors of cancer metastases in vivo (28 , 29) . Specifically, synthetic analogues of naturally occurring conjugates of carbohydrates and amino acids (glycoamines) were shown to inhibit up to 75% both the incidence and number of MDA-MB-435 human breast cancer metastases in nude mice experiments (28) . Modified citrus pectin, as reported by Pienta et al. (29) , was also demonstrated to be an effective inhibitor of B16-F1 murine melanoma lung colonization as well as MAT-LyLu Dunning rat prostate cancer metastasis. Both synthetic glycoamines and modified citrus pectin act through the interaction with β-galactoside-specific lectins, specifically galectin-3, presumably by mimicking corresponding glycoepitopes of the cell surface glycomacromolecules or circulating glycoproteins (30) . It is reasonable to hypothesize that the development of molecules directed against appropriate carbohydrate structures may likewise lead to the development of new effective antiadhesive therapies of cancer metastases.

This suggests new approaches to the concept of antiadhesive therapy of cancer (reviewed in Ref. 31 ), originally developed by early pioneering works of Dr. R. Kerbel and colleagues (32, 33, 34) and Dr. A. Raz and colleagues[ Meromsky et al. ( 4 and Inohara and Raz (25)] . Traditional approaches to such therapy would be to generate appropriate sugar-specific antibodies. The difficulties of raising highly specific antibodies against carbohydrate moieties, as well as of the large-scale production of such antibodies, are well known, however. The development of carbohydrate-specific synthetic peptides using combinatorial bacteriophage display libraries could be a valid complimentary approach.