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Advances in Brief |
Cancer Research Center [V. V. G.] and Department of Biochemistry [V. V. G., S. L. D., T. P. Q.], University of Missouri, Columbia, Missouri 65211; La Jolla Institute for Allergy and Immunology [M. E. H.], San Diego, California 92121; and Sidney Kimmel Cancer Center and Metastat, Inc. [G. V. G.], San Diego, California 92121
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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There has been a tremendous surge in research to characterize the roles
of cell surface carbohydrate structures in cell-cell communication as
mediators of tumor cell proliferation, adhesion, and metastasis.
Alterations in cell surface carbohydrate structures of cancer cells are
postulated to effect normal cellular interactions and have been
shown to facilitate tumor cell colonization and metastasis (2
, 3
, 8)
. One such cancer-associated carbohydrate antigen, the T
antigen,3
has been the focus of much research into its role in tumor cell
adhesion and metastasis (8)
. The immunodominant portion of
the T antigen is the terminal Galß1 
3GalNAc carbohydrate moiety
(5)
. Cryptic, covalently or structurally masked and
nonimmunoreactive, T antigen is present on the surfaces of healthy
cells in most tissues. It is, however, exposed and immunoreactive on
most human carcinomas and T-cell lymphomas (8
, 10)
. T
antigen has been proposed to be involved in tumor cell adhesion and
tissue invasion. The existence of T antigen-mediated cell adhesion
between highly metastatic murine lymphoma cells and hepatocytes is
supportive of a role for this cell surface carbohydrate structure in
the metastatic process (6)
. Large quantities of T antigen
have been detected on the outer surface membranes of human breast
carcinomas, which makes it an attractive target for the development of
tumor diagnostic and therapeutic agents (10
, 11)
. In our
laboratory, several peptides that bind T antigen have been
affinity-selected from a 15-amino-acid-random-peptide bacteriophage
display library and characterized for their binding affinities and
specificities (12
, 13)
. One of the peptides, P-30, has
been shown to selectively bind several cancer cell lines that display T
antigen on their surfaces including MDA-MB-435 human breast carcinoma
cells. It was also found to efficiently inhibit asialofetuin-induced
homotypic aggregation of B16-F1 murine melanoma cells
(13)
. We hypothesized that if T antigen mediates
spontaneous homotypic aggregation of breast cancer cells, then a T
antigen-binding peptide may likewise inhibit this aggregation. In this
study, we demonstrate that T antigen accumulates at the sites of cell
contact in multicellular aggregates of MDA-MB-435 human breast
carcinoma cells, which suggests the involvement of T antigen in
spontaneous aggregation. Indicative of the participation of T antigen
in homotypic aggregation is the ability of T antigen-specific P-30 to
significantly (>70%) inhibit this aggregation in a dose-dependent
manner.
Cell type-specific carbohydrates facilitate cell-cell communication through selective interactions with carbohydrate-binding proteins, including cell surface lectins (1) . The early works of Dr. A. Raz and colleagues [Meromsky et al. (4) and Raz and Lotan (9) ] suggest an important role of soluble ß-galactoside-specific lectins (galectins) in cancer cell adhesion and metastasis. Because the terminal residue of T antigen is ß-galactose, one can reasonably suggest its possible interactions with members of the ß-galactoside binding lectin family. Therefore, we studied the expression profiles of galectins, namely galectin-1, galectin-3, and galectin-4, in MDA-MB-435 cells. Our data indicated the abundant expression of 35S-labeled galectin-1 and galectin-3 but not galectin-4 in these cells, which suggested a potential interplay of T antigen with galectins, most likely with galectin-3.
Both galectin-1 and galectin-3 appear to participate in the adhesion of the MDA-MB-435 cells to a monolayer of human endothelial cells as revealed by laser confocal microscopy. We observed the accumulation of the galectin-3 on endothelial cells at the sites of their contact with cancer cells, which would be supportive of possible interactions between T antigen and galectin-3. The T antigen-specific P-30 peptide was able to inhibit this adhesion by up to 50%.
The results presented in this paper demonstrate that ß-galactoside-mediated, in particular T antigen-mediated, cell-cell interactions are important components of both the spontaneous homotypic aggregation of the MDA-MB-435 human breast carcinoma cells and their adhesion to the endothelium. The ability of P-30 to inhibit T antigen-mediated tumor cell aggregation and adhesion highlights its potential functional significance for antiadhesive therapy of cancer metastasis.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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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 
-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 x 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 x250,
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).
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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), 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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3GalNAc) has ß-galactose as a
terminal sugar, it is likely that T antigen-mediated interactions may
involve the participation of ß-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 ß-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
Mr 
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 Galß13GalNAc
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 ß-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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) 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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), 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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Strikingly different behavior of these two ß-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 target ß-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 ß-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.
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Acknowledgments |
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FOOTNOTES |
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1 Supported by Cancer Research Center
(V. V. G.), grants from United States Army (DAMD-179717198) and
Department of Energy (ER-61661; to T. P. Q.), grant from Department
of Defense DAMD17–98-1-8320 (to S. L. D.), grants from American
Cancer Society RPG-97-104-01-CSM and California Breast Cancer Research
Program, University of California, Office of President 3 IB-0059 (to
M. E. H.). 
2 To whom requests for reprints should be
addressed, at Department of Biochemistry, University of Missouri, 117
Schweitzer Hall, Columbia, MO 65211. Phone: (573) 882-6099; Fax: (573)
884-4812; E-mail: QuinnT{at}missouri.edu
3 The abbreviations used are: T antigen,
Thomsen-Friedenreich antigen; HUVEC, human umbilical vein endothelial
cell; PNA, peanut agglutinin; DiI, 1,1'-dioctadecyl-3,3,
3',3'-tetramethylindocarbocyanine; Cy5,
N,N'-biscarboxypentyl-5,5'-disulfonatoindodicarbocyanine.
4 B. Lundin-Jensen, M. Jazayeri, A. Ponce, F-T.
Liu, P. Bryant, and M. E. Huflejt. Galectins in human
breast cancer cell lines established from various stages of the breast
disease, submitted for publication.
Received 11/16/99. Accepted 3/31/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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-O-serine/threonine) density in primary breast carcinoma: a functional predictor of aggressiveness. Mol. Immunol., 26: 1-5, 1989.[Medline]
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