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From the Department of Tumor Progression,*
Karmanos
Cancer Institute, Detroit, Michigan; the Department of
Otolaryngology,
Osaka Teishin Hospital, Osaka,
Japan; the Division of Internal Medicine,
University of Michigan, Ann Arbor, Michigan; and the Department of
Pathology and Radiation Oncology,§
Wayne State
University, Detroit, Michigan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years numerous studies have focused on identifying
angiogenesis stimulators, leading to the identification of several
angiogenic factors. These can be divided into three groups of
extracellular signals. The first comprises soluble growth molecules
such as acid and basic fibroblast growth factors (aFGF
and bFGF) and vascular endothelial growth factor (VEGF), which affect
endothelial cell growth and differentiation.5,6
The second
group of factors inhibits proliferation and enhance differentiation of
endothelial cells and includes transforming growth factor ß
(TGFß),7-9
angiogenin, as well as several
low-molecular-weight substances.10,11
The third group
comprises extracellular matrix-bound cytokines released by proteolysis,
which may contribute to angiogenic regulation.11
Tumors
can also generate inhibitors of angiogenesis, including
angiostatin,12,13
thrombospondin,14,15
and
endostatin.16
In addition, a number of tumor-associated
macrophages that secrete bFGF, tumor necrosis factor 
(TNF-),
endothelial growth factor (EGF), and VEGF, among other cytokines, were
shown to play a role in tumor angiogenesis.17,18
All of
the above implies that angiogenesis is governed by a balance between
positive and negative regulators within the
microenvironment.19,20
In addition to the above, it was
shown that soluble forms of the carbohydrate-binding proteins (ie,
E-selectin,21,22
vascular cell adhesion
molecule-1,22
and P-selectin23
) can promote
endothelial cell migration and morphogenesis after binding to their
respective glycoconjugate ligands. Antibodies directed
against the carbohydrate epitopes of the ligand or E-selectin inhibited
this process.21,24
The clinical manifestation of elevated
concentration of circulating E-selectin in sera of cancer
patients25-28
lends credence to these experimental
observations. However, this premise was recently challenged by the
finding that experimental angiogenesis can be induced normally in P-
and E-selectin-deficient mice29
and that endothelial cells
from E-selectin-deficient mice undergo in vitro vascular
tube formation.30
Because endothelial cells’
glycoproteins containing N-linked oligosaccharide structures
are important for the tube formation process,31
we
questioned whether a different glycoprotein-binding protein, ie,
galectin-3, can replace E-selectin and thus introduce an alternative
signaling pathway into carbohydrate-mediated endothelial cell
morphogenesis while bridging the above discrepancies.
Galectin-3 is a member of a growing family of carbohydrate-binding
proteins with molecular masses ranging from 29 to 34 kd. They share an
affinity for ß-galactoside-containing glycoconjugates and a conserved
sequence of the sugar-binding motif (for reviews see refs.32–34).
Galectin-3 (also known as Mac-2, CBP-35, ml-34, L-29, hL-31, and 
BP)
is a Mr ~30,000 protein composed of
two distinct structural motifs, an amino-terminal half containing
Gly-X-Y tandem repeats characteristic of collagens and a
carboxyl-terminal half containing the carbohydrate-binding site.
Galectin-3 is expressed in a wide range of neoplasms and is involved in
multiple biological processes through interaction with specific
ligands, including cell growth, adhesion, differentiation,
inflammation, apoptosis, and metastasis.33,34
In human
tumors, a direct relationship between galectin-3 levels and the stage
of tumor progression in colon, gastric, thyroid, breast, and head and
neck carcinomas has been demonstrated.35-42
It is obvious that the formation of new capillary vessels in a tumor is critical for its continuous growth and provides a gateway for the dissemination of malignant cells. We have previously transfected galectin-3 null human breast carcinoma BT-549 cells with human galectin-3 cDNA and established expressing cell clones that, unlike the parental cells, progressively grew and metastasized in nude mice.38 This raised the question of a possible role for galectin-3 in angiogenesis.
We show here that soluble galectin-3 induces endothelial capillary tube formation in vitro and angiogenesis in vivo, and the specificity of the effect is established by antibodies and competitive sugars. In summary, these results suggest that angiogenesis could be mediated by carbohydrate recognition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human umbilical vein endothelial cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The human breast cancer cell line BT-549 was a gift from Dr. Eric W. Thompson, and its galectin-3-transfected clone (11-9-1-4) was established as previously reported.38 HUV-EC-C were cultured in Ham’s F12K medium (Irvine Scientific, Irvine, CA) supplemented with 100 µg/ml heparin (Sigma Chemical Co., St. Louis, MO), 50 µg/ml endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA), and 10% fetal calf serum (FCS) (Summit Biotechnology, Fort Collins, CO). BT-549 and 11-9-1-4 cells were maintained in Dulbecco’s minimum essential medium (Gibco, Rockville, MD) containing 10% heat-inactivated FCS, essential and nonessential amino acids (Gibco), vitamins, and antibiotics (Mediatech Inc., Herndon, VA). The cells were maintained in a humidified chamber with 95% air and 5% CO2 at 37°C. The HUV-EC-C were grown to confluence, detached from the monolayer with 0.25% trypsin, 2 mmol/L EDTA, and split 1:2; the breast cancer cell lines were split 1:4 for further cultivation.
For collection of the conditioned media, 4 x 106 human breast carcinoma cells were plated on a 60-mm tissue culture dish. The next day, the medium was replaced with serum-free medium after thorough washing with phosphate-buffered saline (PBS). The medium was collected 3 days later, concentrated 10-fold by centrifugation through ultrafree MC filter units (Millipore, Bedford, MA) with a 10,000-kd cut point, and analyzed for the presence of galectin-3 by Western blotting, using anti galectin-3 mAb.
Recombinant Galectin-3, Antibodies, and Modified Citrus Pectin
Human recombinant galectin-3 was isolated and affinity purified as described.43 Briefly, galectin-3 cDNA was expressed in Escherichia coli HMS174, using pET5a expression vector (Promega, Madison, WI). The protein was purified from the bacterial cell lysates, using an asialofetuin affinity column followed by extensive dialysis against PBS (pH 7.4). The pAb was prepared in rabbits against purified human recombinant galectin-3 (Genemed Biotechnologies, S. San Francisco, CA). The anti-galectin-3 mAb-producing hybridoma TIB-166 was purchased from ATCC. Mouse anti-ß3-integrin monoclonal antibody was purchased from Transduction Laboratories (San Diego, CA). Anti-von Willebrand factor polyclonal antibody raised in rabbit was obtained from Sigma Chemical Co. Citrus pectin was purchased from Sigma Chemical Co. and modified into modified citrus pectin (MCP) as already described.44
Capillary Tube Formation
To prepare Matrigel,45 200 µl of Matrigel (Collaborative Biomedical Products, MA) thawed on ice was added to each chamber of an eight-chamber slide. The air bubbles were carefully removed, and the slide was transferred to a 37°C incubator for 15 minutes. After gelation, 5 x 104 endothelial cells, which were separated from monolayers with trypsin treatment, were plated onto the gel in 200 µl medium. In some chambers, specific antibodies or PIS was added to the cells at the time of plating on Matrigel. The slides were incubated for 16 hours at 37°C. Alternatively, the Matrigel was diluted to 4.5 mg/ml with serum-free F12K medium and allowed to gel for 2 hours at 37°C. A total of 2 x 104 cells/200 µl medium were plated per chamber. Purified recombinant or secreted galectin-3 was added to some chambers. In some experiments galectin-3 was added with or without a competitive disaccharide (lactose), a competitive polysaccharide (MCP), and a noncompetitive disaccharide (sucrose), and endothelial cell migration and rearrangement was visualized after 4–6 hours.
Electrophoresis and Immunoblotting
Endothelial cells were incubated with 10 µg/ml galectin-3 and lysed at different time intervals. The cell lysates or, in some experiments, conditioned media were suspended in reducing Tris-sodium dodecyl sulfate sample buffer, boiled for 5 minutes, and separated on a 8% or 12.5% polyacrylamide separating gel and 3.5% stacking gel. The separated proteins were then electroblotted to polyvinyl pyrrolidine fluoride (PVDF) membranes (MSI, Westborough, MA) and quenched in 5% nonfat dried milk in PBS for 4 hours, followed by incubation with the first antibody for 1 hour at room temperature. Subsequently, the membrane was washed five times with quench solution containing 0.1% Tween-20 and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG or rabbit anti-rat IgG + IgM; Zymed, S. San Francisco, CA) for 1 hour and washed as above and processed for chemiluminescence.
Binding of Galectin-3 to HUV-EC-C and Scatchard Plot Analysis
To determine the binding affinity and number of receptors on the endothelial cell surface, galectin-3 was iodinated in the presence of chloramine T. Briefly, 1 µg galectin-3 was incubated with 250 µCi Na125I in the presence of 40 µg chloramine T and 100 µl H2O on ice for 1 minute. The reaction was stopped by the addition of 20 µl of 1 mol/L KI. To remove the unbound labeled iodine, the reaction mixture was spun through Ultrafree-CL centrifugal filters (Millipore) precoated with 0.1% bovine serum albumin (BSA) in PBS. The labeled galectin-3 was resuspended in 50 µl of 0.1% BSA in 1x PBS and stored at 4°C for a maximum period of 1 month. One day before the assay HUV-EC-C were plated at a density of 5 x 104 cells per well in a 24-chamber plate. After washing, the wells were blocked with 0.1% BSA in 1x PBS for 30 minutes and incubated with 1–6 ng iodinated galectin-3/well in the presence or absence of 50 mmol/L lactose. After 2 hours of incubation at 4°C with constant shaking, the solution was removed and the cells were washed three times with 0.1% BSA-PBS. To measure the bound galectin-3, the cells were lysed with 1 mol/L NaOH for 30 minutes at room temperature, and the radioactivity was measured with a gamma counter.
For Scatchard plot analysis, the cells were incubated with 0.5 ng of iodinated galectin-3 in 0.1% BSA and 100 µl of 0.1% BSA containing a serial dilution of cold galectin-3 ranging from 0 to 50 ng for 2 hours at 4°C with constant shaking at 60 cycles/minute. The washes and counting were done as above. Values of ligand-receptor and [ligand receptor]/free ligand were calculated as follows:
Ligand receptor: [cold galectin-3(ng) + iodinated galectin-3 (ng)] x [bound galectin-3 (cpm)]/[Total counts added (cpm)]
Ligand receptor/free ligand: bound galectin-3 (cpm)/total cpm - bound cpm
The affinity of the receptor (KD value) and receptor concentration (R value) were determined using the method of Scatchard.46
Chemotaxis
The assay was performed with a Boyden chamber. Briefly, 30 µl of conditioned medium from the galectin-3-secreting cells (11-9-1-4) and null galectin-3 cells (BT-549) and recombinant galectin-3 (0–20 µg/ml) in serum-free F12K medium were added to the lower chamber as a chemoattractant. HUV-EC-C (5 x 104) were added to the upper chamber. The two chambers were separated with polycarbonate filters (8-µm pore size) and incubated at 37°C for 5 hours. At the end of the incubation period, the cells attached to the lower surface of the filter were fixed and stained using the Diff-Quik Stain set (Baxter, IL). A total of 10 fields were counted from each chamber under a microscope, and their sum was plotted. Each assay was carried out in quadruplicate.
In Vivo Angiogenesis
Galectin-3-expressing (11-9-1-4) or nonexpressing (BT-549) cells38 were injected subcutaneously into the dorsolateral region of nude mice (five per group) in the presence of Matrigel (1 x 106 cells in 300 µl per mouse). After 10 days the tumors were removed along with the overlying skin, fixed with 10% buffered formalin, and processed for embedding, sectioning, and immunohistochemistry as described below.
NCR nude mice were also injected subcutaneously dorsolaterally with 0.4 ml Matrigel alone or in combination with 5 µg/ml bFGF or 10 µg/ml galectin-3 according to the method of Passaniti et al.47 The injected Matrigel rapidly formed a solid gel that persisted for at least 10 days in mice. The mice were euthanized after 6 days, and the mass of Matrigel was removed along with overlying skin and fixed with 10% formaldehyde for at least 24 hours before it was embedded in paraffin. The paraffin blocks were then cut into 4-µm-thick sections and processed for immunohistochemistry.
Immunohistochemistry
Immunohistochemistry was performed using a modification of the avidin-biotin peroxidase complex technique. Briefly, 4-µm tissue sections were deparaffinized, rehydrated, and placed in 3% hydrogen peroxide to inhibit endogenous peroxidase. The tissue sections were trypsinized with 0.1% trypsin and 0.1% CaCl2 for 30 minutes at 37°C to expose the antigenic sites masked by formalin fixation, blocked for 1 hour with 3% normal goat serum (Sigma Chemical Co.), and subsequently incubated with antibody against von Willebrand factor for 60 minutes at a dilution of 1:1000. The sections were then treated with biotinylated secondary antibody (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, followed by avidin biotin complex reagent for 30 minutes and diaminobenzidine (Sigma Chemical Co) for 1 minute. Counterstaining was performed with hematoxylin.
For galectin-3 staining, basically a similar protocol was followed with a few modifications. The sections were not trypsinized, as formaldehyde fixation did not mask the antigenic sites of galectin-3. Blocking was performed with 3% normal rabbit serum. The primary antibody used was monoclonal antibody produced by TIB-166 hybridoma at a dilution of 1:2 for 60 minutes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the tube-forming property of endothelial cells on
Matrigel, HUV-EC-C were plated on a gel formed by 200 µl of 14 mg/ml
or 4.5 mg/ml Matrigel. On a gel prepared with undiluted Matrigel there
was a rapid organization of endothelial cells into capillary-like
structures (Figure 1A)
, whereas on
diluted Matrigel, the cells underwent migration and organization into
elongated structures without an interconnecting network (Figure 2A)
.
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To study the effect of galectin-3 on endothelial cell tube
formation, we have analyzed the dose response of HUV-EC-C to soluble
human galectin-3. Cells (5 x 104) were
plated on a gel formed by diluted Matrigel, in the presence of varying
concentrations of galectin-3. Figure 1, A–D
, show a
concentration-dependent anastomosing network of endothelial cells.
Similar stimulation of organization into a network was observed when
HUV-EC-C were incubated with conditioned medium collected from the
transfected human breast carcinoma 11-9-1-4 cells (Figure 1E)
, which
secrete galectin-3 (inset: Lane 1). The conditioned medium collected
from the galectin-3 null parental BT-549 cell culture (inset: Lane 2)
failed to enhance the organization (Figure 1F)
, suggesting that this
stimulation is not only restricted to the recombinant form but could
also be mediated by cellular processed galectin-3. To further verify
the importance of galectin-3, HUV-EC-C were plated on undiluted
Matrigel in the presence and absence of a specific antibody or the
preimmune serum. Figure 2, A–C
, demonstrates that the
basement-membrane-induced HUV-EC-C differentiation could be inhibited
by anti-galectin-3 polyclonal antibody and not by preimmune serum
control. There was a 50–90% reduction in the number of capillaries
formed in the presence of pAb in various experiments. However, the mAb
against the amino-terminal domain failed to inhibit tube formation (not
shown).
Next, we questioned whether the effect of galectin-3 is mediated by its
carbohydrate-binding domain; thus the effect of competitive sugars on
tube formation was tested. We used MCP, a complex carbohydrate, and
lactose, a disaccharide, which have been shown previously to compete
with natural ligand (s) recognition by galectin-3.44,48,49
HUV-EC-C were plated on a gel formed by Matrigel, in the presence of
0.1% MCP, a competitive polysaccharide; 50 mmol/L lactose, a
competitive disaccharide; and 50 mmol/L sucrose, a noncompetitive
disaccharide (Figure 3, B, C, and D
,
respectively). In the presence of MCP, cell motility and organization
were completely inhibited (Figure 3B)
. The inhibitory effect was not
due to cell damage and was reversible. In the presence of lactose, a
40–60% inhibition in tube-forming capability was observed (Figure 3C)
, whereas sucrose did not affect galectin-3-induced cell migration
and organization (Figure 3D)
.
|
. This binding was specific, as it was
inhibited by 50 mmol/L lactose, indicating the involvement of
carbohydrate recognition in the process and confirming the results
shown in Figure 2B
. Scatchard plot analysis (Figure 5)
revealed the presence of two
galectin-3 receptors on the HUV-EC-C cell surface: a high-affinity one
with a KD of 0.537 x
10-9 and a low-affinity one with a
KD of 7.161 x
10-9. The R values for the two
receptors were 5.81 x 103
and 3.2 x
104, respectively.
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Chemotaxis is an integral part of angiogenesis.50
To
establish whether galectin-3 stimulates the cell motility of HUV-EC-C
by chemotaxis, like other carbohydrate-binding proteins,22
we performed a Boyden chamber chemotaxis assay. Conditioned media
containing galectin-3 (11-9-1-4 cells) or the conditioned medium devoid
of it (BT-549 parental cells) or galectin-3 in different concentrations
were added to the lower chamber as chemoattractants. The upper chamber
contained the HUV-EC-C, and the two chambers were separated by a
polycarbonate filter with 8-µm pores. After 5 hours of incubation the
experiments were terminated and the cells that had migrated from the
upper chamber through the filter toward the chemoattractants were
counted. The results (Figure 6)
show that
only galectin-3 and conditioned medium of cells secreting galectin-3
induced a dose-dependent chemotactic response by the endothelial cells.
P values were <0.001.
|
To test whether galectin-3 induces not only morphogenesis
(in vitro) but also angiogenesis (in
vivo), we injected the nude mice with galectin-3-expressing and
nonexpressing cells, as well as recombinant galectin-3. A total of
1 x 106
BT-549 or 11-9-1-4 cells were
injected subcutaneously into nude mice along with Matrigel. After 10
days the tumors were removed, embedded, sectioned, and stained with
anti-von Willebrand factor for the presence of blood vessels (Figure 7, A and C)
. A serial section was also
immunostained for the presence of galectin-3 (Figure 7, B and D)
. Our
results show the presence of tumor cells between the skin and the
underlying muscle layer. In Figure 7, A and D
, a few fat cells were
visible at the basal layer of the skin. In the galectin-3-positive
tumor (Figure 7, C and D)
, the fat cell layer was interrupted by
tumor mass and blood vessels. At a similar position in the
galectin-3-negative tumor (Figure 7, A and B)
, no blood vessels could
be detected. Capillary density was calculated in the area immediately
below the skin as the mean of the total number of vessels in five
independent fields counted in three sections. In the tumor formed by
BT-549 cells the capillary density was 3.3 ± 1.64 compared to
16.6 ± 7.86 in tumors formed by 11-9-1-4 cells (Figure 8)
. The P value was 0.0038, as
calculated by the Mann-Whitney test.
|
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.
Matrigel supplemented with either galectin-3 or bFGF revealed an
angiogenic reaction associated with the chemotactic response, as seen
by the presence of cells infiltrating the gel (Figure 9, B and C
,
respectively). Microvessel density was 5.2 ± 2.3 and 6.5 ±
1.8 for galectin-3 and bFGF, respectively, thus supporting the notion
that angiogenesis might be associated with an inflammatory
response51
and the conclusion that galectin-3 can act as
an angiogenic factor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Galectins are a family of differentiation regulatory molecules that participate in a variety of biological functions, depending on the organ microenvironment and their specific cellular complementary glycoconjugates. Here we report a novel function for this carbohydrate-binding protein, showing that soluble recombinant galectin-3 or secreted galectin-3 stimulates in vitro and in vivo angiogenesis. Furthermore, this process is inhibited in vitro by specific antibodies and competitive sugar inhibitors.
Tumor-associated macrophages have been implicated at least in part in the mediation of neovascularization.52,53 There is also a significant positive correlation between the number of macrophages in vascular tissues and angiogenesis.53 Furthermore, many of the secreted products of activated macrophages have been characterized as inducers or inhibitors of angiogenesis.18,54 It was shown previously that when monocytes differentiated into macrophages there was an induction of galectin-3 production and that galectin-3 could also be secreted by macrophages.55-57 We presume that tumor-associated macrophages may also contribute to the pool of galectin-3 secreted by tumor cells into the extracellular matrix (ECM). In ECM galectin-3 may bind to one of its ligands. There are two known ligands of galectin-3 in the ECM, namely fibronectin and laminin.58,59
Galectin-3 secreted by tumor cells as well as macrophages may also act
on endothelial cells to induce chemotaxis, facilitating their motility
during the initial phase of tube formation, as we have shown in our
results. Various angiogenic inducers selectively alter integrin
profiles and influence cellular interactions with the ECM during
neovascularization. It was shown that VEGF stimulates a type of
neoangiogenesis in which endothelial cell migration and growth are
dependent on attachment via vß5 integrins, whereas neoangiogenesis
induced by bFGF is dependent on vß3 integrins (for a review see
ref. 60
). Similarly, TGFß stimulates angiogenesis through
up-regulation of 2, 5, and ß1 integrins.61
Our
results suggest a significant up-regulation of vß3 integrins by
treating the endothelial cells with exogenous galectin-3 (not shown).
This could lead to cell motility in the initial phase of tube
formation. The diffusible angiogenic factors can be placed in two
categories. The factors in one category induce proliferation of
endothelial cells in vitro and their differentiation (eg,
bFGF, aFGF, VEGF, platelet-derived endothelial cell growth factor
(PD-ECGF), and TGFß). The second category of factors induces
differentiation but does not induce in vitro proliferation
of endothelial cells (eg, angiogenin, TGFß, platelet-activating
factor, soluble E-selectin, and TNF) (for a review see ref. 50
).
Galectin-3 appears to belong to the second category.
The formation of capillary tubes induced by galectin-3 was inhibited by the neutralization of the action of galectin-3 by its specific carbohydrate inhibitors or the specific antibodies. The effect of galectin-3 is comparable to that of other cell adhesion molecules (ie, VE-cadherin and E-selectin; for a review see ref. 62 ). Antibodies against VE-cadherin inhibited the capillary tube formation in fibrin or collagen gel over gel systems.63 It was previously demonstrated that sialylated fucosylated N-linked oligosaccharides, potential ligands for soluble E selectin, were preferentially synthesized in endothelial cells undergoing tube formation. In addition, antibodies against E-selectin, sialyl Lewis X, and sialyl Lewis A inhibited tube formation.22
Similarly, bFGF, aFGF and many other cytokines bind to heparan sulfate
proteoglycans via their sulfated oligosaccharide
domains.64-66
Inhibition of this binding interferes with
the angiogenic phenomenon and the in vivo mitogenic response
of the endothelial cells to this factor (for a review see ref. 66
).
These and the current observations, showing an inhibition of tube
formation by neutralization of the carbohydrate recognition domain of
galectin-3, indicate the importance of carbohydrate-protein
interactions for angiogenesis. It is possible that after secretion,
galectin-3 could either be stored in bound form to its ECM ligands or
interact directly with endothelial cells. Scatchard plot analysis for
the binding of galectin-3 to endothelial cells (Figure 5)
demonstrates
the presence of at least two cell surface receptors on EC. Inhibition
of this binding by lactose indicates the involvement of the
carbohydrate recognition domain in one or both of the receptor
bindings. Once galectin-3 binds to its cell surface receptors, it may
induce overexpression of integrin vß3, leading to endothelial cell
migration and attachment. Induction of in vivo angiogenesis
by galectin-3-expressing cells or soluble galectin-3 further emphasizes
the importance of carbohydrate-protein interactions during
angiogenesis.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants CA-46120 (to A. R.) and CA69568-04 (to K. J. P.).
Accepted for publication October 26, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. Szoke, K. Kayser, I. Trojan, G. Kayser, J. Furak, L. Tiszlavicz, J.-D. Baumhakel, and H.-J. Gabius The role of microvascularization and growth/adhesion-regulatory lectins in the prognosis of non-small cell lung cancer in stage II Eur. J. Cardiothorac. Surg., May 1, 2007; 31(5): 783 - 787. [Abstract] [Full Text] [PDF]
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J. Nieminen, A. Kuno, J. Hirabayashi, and S. Sato Visualization of Galectin-3 Oligomerization on the Surface of Neutrophils and Endothelial Cells Using Fluorescence Resonance Energy Transfer J. Biol. Chem., January 12, 2007; 282(2): 1374 - 1383. [Abstract] [Full Text] [PDF]
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 V. L. J. L. Thijssen, R. Postel, R. J. M. G. E. Brandwijk, R. P. M. Dings, I. Nesmelova, S. Satijn, N. Verhofstad, Y. Nakabeppu, L. G. Baum, J. Bakkers, et al. Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy PNAS, October 24, 2006; 103(43): 15975 - 15980. [Abstract] [Full Text] [PDF]
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 M. F. McCarty and K. I. Block Toward a Core Nutraceutical Program for Cancer Management Integr Cancer Ther, June 1, 2006; 5(2): 150 - 171. [Abstract] [PDF]
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M. R. Zubieta, D. Furman, M. Barrio, A. I. Bravo, E. Domenichini, and J. Mordoh Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies Am. J. Pathol., May 1, 2006; 168(5): 1666 - 1675. [Abstract] [Full Text] [PDF]
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T. Fukumori, N. Oka, Y. Takenaka, P. Nangia-Makker, E. Elsamman, T. Kasai, M. Shono, H.-o. Kanayama, J. Ellerhorst, R. Lotan, et al. Galectin-3 regulates mitochondrial stability and antiapoptotic function in response to anticancer drug in prostate cancer. Cancer Res., March 15, 2006; 66(6): 3114 - 3119. [Abstract] [Full Text] [PDF]
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G. A. Rabinovich, A. Cumashi, G. A. Bianco, D. Ciavardelli, I. Iurisci, M. D'Egidio, E. Piccolo, N. Tinari, N. Nifantiev, and S. Iacobelli Synthetic lactulose amines: novel class of anticancer agents that induce tumor-cell apoptosis and inhibit galectin-mediated homotypic cell aggregation and endothelial cell morphogenesis Glycobiology, March 1, 2006; 16(3): 210 - 220. [Abstract] [Full Text] [PDF]
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 Y. Higami, J. L. Barger, G. P. Page, D. B. Allison, S. R. Smith, T. A. Prolla, and R. Weindruch Energy Restriction Lowers the Expression of Genes Linked to Inflammation, the Cytoskeleton, the Extracellular Matrix, and Angiogenesis in Mouse Adipose Tissue J. Nutr., February 1, 2006; 136(2): 343 - 352. [Abstract] [Full Text] [PDF]
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G. Rennebeck, M. Martelli, and N. Kyprianou Anoikis and Survival Connections in the Tumor Microenvironment: Is There a Role in Prostate Cancer Metastasis? Cancer Res., December 15, 2005; 65(24): 11230 - 11235. [Abstract] [Full Text] [PDF]
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 D. Chauhan, Y. Chang, D. He, T. Hideshima, K. Podar, F. Tao, P. Neri, R. Schlossman, J. Schindler, B. Carver, et al. Expression and Modulation of Carbohydrate-Binding Protein Galectin-3 in Multiple Myeloma Cells by Combined Treatment with GCS-100 and Dexamethasone. Blood (ASH Annual Meeting Abstracts), November 16, 2005; 106(11): 4447 - 4447. [Abstract]
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 J. Nieminen, C. St-Pierre, and S. Sato Galectin-3 interacts with naive and primed neutrophils, inducing innate immune responses J. Leukoc. Biol., November 1, 2005; 78(5): 1127 - 1135. [Abstract] [Full Text] [PDF]
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D. Chauhan, G. Li, K. Podar, T. Hideshima, P. Neri, D. He, N. Mitsiades, P. Richardson, Y. Chang, J. Schindler, et al. A Novel Carbohydrate-Based Therapeutic GCS-100 Overcomes Bortezomib Resistance and Enhances Dexamethasone-Induced Apoptosis in Multiple Myeloma Cells Cancer Res., September 15, 2005; 65(18): 8350 - 8358. [Abstract] [Full Text] [PDF]
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 N. Ortega, D. J. Behonick, C. Colnot, D. N.W. Cooper, and Z. Werb Galectin-3 Is a Downstream Regulator of Matrix Metalloproteinase-9 Function during Endochondral Bone Formation Mol. Biol. Cell, June 1, 2005; 16(6): 3028 - 3039. [Abstract] [Full Text] [PDF]
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 A. W. Stitt, C. McGoldrick, A. Rice-McCaldin, D. R. McCance, J. V. Glenn, D. K. Hsu, F.-T. Liu, S. R. Thorpe, and T. A. Gardiner Impaired Retinal Angiogenesis in Diabetes: Role of Advanced Glycation End Products and Galectin-3 Diabetes, March 1, 2005; 54(3): 785 - 794. [Abstract] [Full Text] [PDF]
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 M. von Wolff, X. Wang, H.-J. Gabius, and T. Strowitzki Galectin fingerprinting in human endometrium and decidua during the menstrual cycle and in early gestation Mol. Hum. Reprod., March 1, 2005; 11(3): 189 - 194. [Abstract] [Full Text] [PDF]
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 J. Zou, V. V. Glinsky, L. A. Landon, L. Matthews, and S. L. Deutscher Peptides specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated cancer cell adhesion Carcinogenesis, February 1, 2005; 26(2): 309 - 318. [Abstract] [Full Text] [PDF]
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M. P.V. Shekhar, P. Nangia-Makker, L. Tait, F. Miller, and A. Raz Alterations in Galectin-3 Expression and Distribution Correlate with Breast Cancer Progression: Functional Analysis of Galectin-3 in Breast Epithelial-Endothelial Interactions Am. J. Pathol., December 1, 2004; 165(6): 1931 - 1941. [Abstract] [Full Text] [PDF]
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R. I. Zuberi, D. K. Hsu, O. Kalayci, H.-Y. Chen, H. K. Sheldon, L. Yu, J. R. Apgar, T. Kawakami, C. M. Lilly, and F.-T. Liu Critical Role for Galectin-3 in Airway Inflammation and Bronchial Hyperresponsiveness in a Murine Model of Asthma Am. J. Pathol., December 1, 2004; 165(6): 2045 - 2053. [Abstract] [Full Text] [PDF]
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 E. A. Partridge, C. Le Roy, G. M. Di Guglielmo, J. Pawling, P. Cheung, M. Granovsky, I. R. Nabi, J. L. Wrana, and J. W. Dennis Regulation of Cytokine Receptors by Golgi N-Glycan Processing and Endocytosis Science, October 1, 2004; 306(5693): 120 - 124. [Abstract] [Full Text] [PDF]
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 D. Houzelstein, I. R. Goncalves, A. J. Fadden, S. S. Sidhu, D. N. W. Cooper, K. Drickamer, H. Leffler, and F. Poirier Phylogenetic Analysis of the Vertebrate Galectin Family Mol. Biol. Evol., July 1, 2004; 21(7): 1177 - 1187. [Abstract] [Full Text] [PDF]
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N. Ahmad, H.-J. Gabius, S. Andre, H. Kaltner, S. Sabesan, R. Roy, B. Liu, F. Macaluso, and C. F. Brewer Galectin-3 Precipitates as a Pentamer with Synthetic Multivalent Carbohydrates and Forms Heterogeneous Cross-linked Complexes J. Biol. Chem., March 19, 2004; 279(12): 10841 - 10847. [Abstract] [Full Text] [PDF]
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 M. H. Melner, N. A. Ducharme, A. R. Brash, V. P. Winfrey, and G. E. Olson Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes Biol Reprod, February 1, 2004; 70(2): 406 - 414. [Abstract] [Full Text] [PDF]
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M. J. Abedin, Y. Kashio, M. Seki, K. Nakamura, and M. Hirashima Potential roles of galectins in myeloid differentiation into three different lineages J. Leukoc. Biol., May 1, 2003; 73(5): 650 - 656. [Abstract] [Full Text] [PDF]
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D. Riss, L. Jin, X. Qian, J. Bayliss, B. W. Scheithauer, W. F. Young Jr., S. Vidal, K. Kovacs, A. Raz, and R. V. Lloyd Differential Expression of Galectin-3 in Pituitary Tumors Cancer Res., May 1, 2003; 63(9): 2251 - 2255. [Abstract] [Full Text] [PDF]
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 P. Nangia-Makker, V. Hogan, Y. Honjo, S. Baccarini, L. Tait, R. Bresalier, and A. Raz Inhibition of Human Cancer Cell Growth and Metastasis in Nude Mice by Oral Intake of Modified Citrus Pectin J Natl Cancer Inst, December 18, 2002; 94(24): 1854 - 1862. [Abstract] [Full Text] [PDF]
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 T. K. Mandal and C. Mukhopadhyay Binding free energy calculations of galectin-3-ligand interactions Protein Eng. Des. Sel., December 1, 2002; 15(12): 979 - 986. [Abstract] [Full Text] [PDF]
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Y. K. Song, T. R. Billiar, and Y. J. Lee Role of Galectin-3 in Breast Cancer Metastasis : Involvement of Nitric Oxide Am. J. Pathol., March 1, 2002; 160(3): 1069 - 1075. [Abstract] [Full Text] [PDF]
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 Y. J. Lee and Y. K. Song Cooperative Interaction between Interleukin 10 and Galectin-3 against Liver Ischemia-Reperfusion Injury Clin. Cancer Res., January 1, 2002; 8(1): 217 - 220. [Abstract] [Full Text] [PDF]
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R.-Y. Yang, D. K. Hsu, L. Yu, J. Ni, and F.-T. Liu Cell Cycle Regulation by Galectin-12, a New Member of the Galectin Superfamily J. Biol. Chem., June 1, 2001; 276(23): 20252 - 20260. [Abstract] [Full Text] [PDF]
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) or absence
(•) of 50 mmol/L
lactose.
