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Molecular Oncology, Markers, Clinical Correlates |
Department of Oncology and Neurosciences, Section of Medical Oncology [I. I., N. T., C. N., D. A., S. I.], and Department of Surgery [E. C.], University G. D’Annunzio Medical School, 66100 Chieti, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BP, and other names, is isolated as
a monomer of Mr 
30,000 (1
, 2)
. Galectin-3 is found in the cytoplasm, but depending on cell
types and proliferative states, it can also be detected on the cell
surface (3)
, within the nucleus (4)
, and in
the extracellular compartment (5
, 6)
. Galectin-3 acts as a
receptor for ligands containing poly-N-acetyllactosamine
sequences. To date, several ligands for galectin-3, including
lysosomal-associated membrane proteins 1 and 2, IgE, laminin, and Mac-2
BP3
(also known as
90K), have been described (7
, 8)
. The biological functions of galectin-3 remain elusive. Studies from several groups suggest that galectin-3 may have a role in a variety of physiological and pathological processes. Of most relevance to the present study are experimental observations suggesting a relationship between galectin-3 and tumor progression and metastasis. For example, tumor cell variants demonstrating higher potential for lung colonization were found to express higher levels of galectin-3 on the cell surface (9) . Similarly, increased galectin-3 expression has been correlated with the metastatic potential of several tumorigenic cells, possibly by affecting cell motility and invasion of extracellular matrices (10 , 11) . However, the generality of these findings in relation to human tumors of epithelial origin is not fully clear. For example, in human colorectal carcinoma, galectin-3 has been reported to increase (12) or decrease (13) with progression toward a metastatic state. In addition, decreased expression of this lectin compared with the normal tissue has been associated with the metastatic propensity of cancer cells in breast (14) , endometrial (15) , and ovary (16) carcinomas. Thus, the ability of galectin-3 to promote or inhibit invasion and metastasis may depend upon tumor-specific factors. To further address the role of galectin-3 in tumor progression and metastasis, we developed an immunoligand assay for the determination of soluble galectin-3 in the circulation. Using this assay, we measured the levels of galectin-3 in the serum of patients with various types of cancer. Serum specimens from healthy blood donors were used as controls. In addition, we looked for a correlation between galectin-3 levels, tumor type, and disease state.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human Recombinant Galectin-3 Expression and Purification.
Recombinant galectin-3 was expressed in Escherichia coli and
purified by affinity chromatography as described in detail
(17)
.
Production and Purification of Recombinant Mac-2 BP.
Human embryonic kidney EBNA 293 cells transfected with the full-length
cDNA encoding human Mac-2 BP/90K (18)
were kindly supplied
by Dr. Rupert Timpl (Martinsried, Germany). Mac-2 BP was isolated from
cell culture supernatant as described previously (19)
. In
brief, cells were seeded into cell factories in DMEM containing
10% FCS. When the cells reached confluence, the serum
concentration was gradually reduced to 1%. Conditioned medium was
collected every day for 3 consecutive days, centrifuged at 10,000 x g for 15 min, and stored at -80°C after adding EDTA
and Pefabloc (Boehringer Mannheim) to 1 and 0.4
mM, respectively. Mac-2 BP was precipitated from
this medium by adding solid ammonium sulfate to 43% saturation
(19)
. The precipitate was dissolved in PBS containing
protease inhibitors and passed over a 3 x 12-cm column loaded
with Sepharose CL-4B conjugated to anti-Mac-2 BP monoclonal antibody as
described (20)
. After extensive washing with 0.5
M NaCl in PBS, Mac-2 BP was eluted with 20 ml of
Actisep elution medium (Sterogene Bioseparation, Inc., Arcadia, CA).
Purified material was aliquoted and stored at -80°C.
Western Blotting.
Serum (20 ml) from a healthy blood donor was adsorbed with
lactosyl-Sepharose 4B (17)
, and the bound proteins eluted
with 0.2 ml of 0.1 M lactose in PBS. Aliquots of the eluate
were resolved by 12% SDS-PAGE and transferred to nitrocellulose. The
membranes were then blocked with 1% BSA overnight at 4°C and probed
at room temperature for 2 h with rat anti-galectin-3 monoclonal
antibody M3/38 (1 µg/ml). Biotinylated goat antirat IgG was added as
secondary antibody, followed by peroxidase-conjugated streptavidin
(DAKO, Glostrup, Denmark). After extensive washing, the membranes were
processed for chemiluminescent detection using an enhanced
chemiluminescence kit (ECL; Amersham Life Sciences, Arlington
Heights, IL) according to the manufacturer’s instructions.
Membranes were also probed with purified recombinant Mac-2 BP (5
µg/ml; 2 h), followed by sequential incubation with mouse
anti-Mac-2 BP monoclonal antibody 1.A422 (Ref. 21
; 1
µg/ml; 45 min), biotinylated goat antimouse IgG,
peroxidase-conjugated streptavidin, and enhanced chemiluminescent
reagents as above.
Immunoligand Assay for Galectin-3.
Serum galectin-3 concentrations were assayed with a newly developed
immunoligand assay. The assay uses Mac-2 BP immobilized to plastics as
galectin-3 capture protein, followed by incubation with rat
anti-galectin-3 antibody and peroxidase-labeled goat antirat IgG as
detecting antibody. Ninety-six-well microtiter plates (Nunc-Maxisorp;
Life Technologies, Inc., Rockville, MD) were coated with 5 µg/ml
purified Mac-2 BP in PBS by incubation overnight at 4°C and then
blocked with 1% BSA (Sigma Chemical Co., St. Louis, MO) in PBS for
2 h at room temperature. For the assay, 100 µl of samples and
serially diluted human recombinant galectin-3 (standards) in dilution
buffer (PBS containing 0.1% BSA and 5% glycerol) were added to the
wells and incubated for 2 h at room temperature. After washing
with PBS containing 0.02% Tween 20 (three 5-min washes each,
with constant shaking), 100 µl of monoclonal antibody M3/38 (1
µg/ml) were added and incubated for 1 h at room temperature.
After washing the wells three times as above, 100 µl of
peroxidase-labeled antirat IgG were added to each well and incubated
for 45 min at room temperature. Wells were then washed three times, and
then the enzyme reaction was carried out at room temperature for 30 min
with diaminobenzidine as a substrate. The reaction was then stopped
with 1 N H2SO4,
and absorbance at 450 nm was read on a microplate reader. The amount of
galectin-3 from the samples was estimated by extrapolation from a
log:log linear regression curve determined from the serially diluted
human recombinant galectin-3 ranging from 625 to 0 ng/ml. Samples
containing galectin-3 >625 ng/ml were diluted with dilution buffer and
reassayed.
Statistical Analyses.
Beacause galectin-3 values were not normally distributed, the 5th and
95th percentile values were chosen for data description. Differences
between patient groups were tested with the nonparametric Mann-Whitney
(nonparametric) U test. All Ps given are used in
a descriptive manner, because no adjustment for multiple testing was
performed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, immunostaining of blotted serum
proteins with anti-galectin-3 monoclonal antibody M3/38 revealed one
major band with Mr 31,000
and a minor component of Mr
60,000 representing galectin-3 homodimers (17)
.
A superimposable staining pattern was obtained after reacting the
blotting membrane with Mac-2 BP. These results indicate that Mac-2 BP
does not react with serum components other than galectin-3.
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. When plates were coated with
human serum albumin instead of Mac-2 BP or when rat antigalectin-3
antibody M3/38 was substituted with a rat antitumor necrosis factor-
antibody, no signal above background level was observed. The
intra-assay and interassay coefficients of variation were less than 12
and 18% throughout the range, respectively. Long-term storage (>6
months) of frozen serum or 1 week of storage at 4°C failed to
significantly alter galectin-3 content. No difference in galectin-3
measurements was observed after two freeze-thaw cycles.
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). The 95th percentile of galectin-3
levels (184.3 ng/ml) was arbitrarily taken as the upper limit of
normal. There was no significant difference in galectin-3 serum
concentration between males (median, 61.5 ng/ml; range, 20–313 ng/ml)
and females (median, 64 ng/ml; range, 20–230 ng/ml), nor was there a
correlation between serum galectin-3 levels and age or blood
group. Serum galectin-3 concentrations of healthy individuals
and patients with cancer are summarized in Table 1
.
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Three (30%) of 10 patients with nonmetastatic gastrointestinal
carcinomas but 10 (66%) of 15 with metastatic disease had serum
galectin-3 concentrations above the upper limit of normal. Like the
breast carcinoma patients, patients with metastatic colorectal
carcinomas showed significantly higher galectin-3 serum levels than did
those with nonmetastatic disease (P < 0.026). Notably,
maximum serum concentrations of galectin-3 occurred in patients with
metastatic gastrointestinal carcinoma (median, 320 ng/ml; range,
20–950 ng/ml; Table 1
). Galectin-3 serum levels did not differ among
the various types of gastrointestinal neoplasia.
Thirteen (50%) of 26 patients with metastatic non-small cell lung cancer, 1 (25%) of 4 patients with metastatic melanoma, 1 (25%) of 4 patients with metastatic ovarian carcinoma, and 1 (20%) of 5 patients with stage IV non-Hodgkin’s lymphoma showed increased galectin-3 concentrations over the upper normal limit.
In five colorectal carcinoma patients, serum galectin-3 levels were
measured before and 2 days after tumor resection. In four of these
cases, there was a decrease in the galectin-3 serum concentrations
after surgery (Fig. 3)
. One patient with
regional lymph node involvement had a preoperative galectin-3 serum
level of 570 ng/ml that dropped to 185 ng/ml within 3 days after
surgery. In contrast, in one patient with localized tumor and normal
galectin-3 serum level before surgery, no significant change was
observed in galectin-3 serum concentration.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological role of galectin-3 remains elusive. Galectin-3 is found at elevated levels in a variety of neoplastic cells, and several experimental observations suggest that it is involved in tumor metastasis in vivo (22 , 23) . Different modalities have been proposed to explain how this lectin might be involved in the metastatic process: (a) the potential of galectin-3 to interact with extracellular matrix proteins, such as laminin, fibronectin, and vitronectin (24 , 25) , on one hand, and with cell surface proteins such as lysosomal-associated membrane proteins (1) , on the other hand, suggests that galectin-3 acts as a bridge, linking cells to the extracellular matrix or other cells; (b) the data demonstrating that galectin-3 is able to mediate homotypic cell-cell adhesion through interaction with complementary glycoproteins (26 , 27) lend credence to the theory that this lectin is involved in the formation of tumor emboli and dissemination of tumor cells in the circulation; and (c) the recent observations that galectin-3 is able to protect against apoptosis induced by the loss of cell anchorage (anoikis; Refs. 25 and 28 ) suggest that the expression of galectin-3 in tumor cells may provide a critical determinant for cell survival of disseminating cancer cells in the circulation during metastasis.
The results of the present study could imply that the metastatic spread of malignant tumors involves, among other factors, a higher level of expression of galectin-3 in the circulation. We are proposing that changes in the level of expression of galectin-3 may favor metastasis by either one or all of the above-mentioned modalities, i.e., by: (a) enhancing the adhesive interactions between tumor cells and the extracellular matrix; (b) promoting tumor cell embolization through increased cell-cell adhesion; and (c) conferring a selective survival advantage to metastatic cells. Alternatively, galectin-3 serum levels may reflect an immune reaction to the tumor load from inflammatory cells that are known to express galectin-3. However, this seems unlikely because we found no correlation between the extent of the inflammatory response in operable breast cancer and further disease progression.4
The source of increased serum galectin-3 in cancer patients remains unclear. According to our results, that removal of the tumor decreased serum galectin-3 concentrations, tumor tissues are likely to produce and secrete galectin-3 in sera. However, immunostaining of cancerous tissue with antigalectin-3 antibody showed that galectin-3 was expressed not only on malignant cells but also in macrophages and stromal cells (mainly fibroblasts) near cancer nests, and the stromal cells immediately adjacent to cancer nests have a higher galectin-3 expression in comparison to those cells farther away from the nests.5 These results suggest that circulating galectin-3 is generated not only by tumor cells but also from peritumoral inflammatory cells and stromal cells.
In summary, the data presented above indicate that the detection of increased galectin-3 levels in the serum of certain patients with cancer may reflect biological aspects of tumor behavior associated with a metastasizing phenotype. Additional studies are warranted to determine the clinical value of circulating galectin-3 in patients with early-stage cancer as a predictor of tumor invasion and metastasis.
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FOOTNOTES |
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1 This work was supported by a grant from
Associazione Italiana per la Ricerca sul Cancro and by a grant from
Ministero dell’Universit e Ricerca Scientifica e Tecnologica Cofin
1998. 
2 To whom requests for reprints should be
addressed. Fax: 39-0871-3556707; E-mail: iacobell{at}unich.it
3 The abbreviation used is: Mac-2 BP,
Mac-2-binding protein.
Received 9/30/99; revised 1/ 4/00; accepted 1/ 6/00.
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REFERENCES |
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