Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells.
ABSTRACT Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt-2 is well known for its overexpression in colorectal cancer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with beta-catenin. In this study, we investigated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/beta-catenin signaling. We found that siRNA silencing of either Wnt-2 or Gal-3 expression inhibited TCF-reporter activity, decreased cytosolic beta-catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt-2 and Gal-3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/beta-catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt-2 and Gal-3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.
- [Show abstract] [Hide abstract]
ABSTRACT: Galectin-3, a member of β-galactoside-binding gene family is a multi-functional protein, which regulates pleiotropic biological functions such as cell growth, cell adhesion, cell- cell interactions, apoptosis, angiogenesis and mRNA processing. Its unique structure enables it to interact with a plethora of ligands in a carbohydrate dependent or independent manner. Galectin-3 is mainly a cytosolic protein, but can easily traverse the intracellular and plasma membranes to translocate into the nucleus, mitochondria or get externalized. Depending on the cell type, specific experimental conditions in vitro, cancer type and stage, galectin-3 has been reported to be exclusively cytoplasmic, predominantly nuclear or distributed between the two compartments. In this review we have summarized the dynamics of galectin-3 shuttling between the nucleus and the cytoplasm, the nuclear transport mechanisms of galectin-3, how its specific interactions with the members of β-catenin signaling pathways affect tumor progression, and its implications as a therapeutic target.Seminars in Cancer Biology 08/2014; · 9.14 Impact Factor
Article: Galectin-3 in cancer.[Show abstract] [Hide abstract]
ABSTRACT: Galectin-3(Gal-3) plays important roles in cell proliferation, adhesion, differentiation, angiogenesis and apoptosis in normal and pathologic tissues. Accumulated evidences indicate Gal-3 is closely involved in tumor cell transformation, migration, invasion and metastasis. In this review, the associations of the expression and localization of Gal-3 as well as its potential action mechanism in tumorigenesis in a variety of cancers were summarized and concluded. Gal-3 is gaining its attraction as a potential new biomarker for the diagnosis, treatment and prognosis of certain tumors.Clinica chimica acta; international journal of clinical chemistry 02/2014; · 2.54 Impact Factor
Article: Galectins in epithelial functions.[Show abstract] [Hide abstract]
ABSTRACT: Galectins are a family of animal lectins comprising 15 members in vertebrates. These proteins are involved in many biological processes including epithelial homeostasis and tumor progression by displaying intracellular and extracellular activities. Hence Galectins can be found either in the cytoplasm or the nucleus, associated with membranes or in the extracellular matrix. Current studies aim at understanding the roles of Galectins in cell-cell and cell-matrix adhesion, cellular polarity and motility. This review discusses recent progress in defining the specificities and mechanisms of action of Galectins as cell regulators in epithelial cells. Physiological, cellular and molecular aspects of Galectin specificities will be treated successively.Tissue barriers. 05/2014; 2:e29103.
Inhibition of Wnt-2 and galectin-3 synergistically destabilizes b-catenin and induces
apoptosis in human colorectal cancer cells
Yihui Shi, Biao He, Kristopher M. Kuchenbecker, Liang You, Zhidong Xu, Iwao Mikami, Adam Yagui-Beltran,
Genevieve Clement, Yu-Ching Lin, Junichi Okamoto, Dawn T. Bravo and David M. Jablons*
Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California,
San Francisco, CA
Constitutive activation of the Wnt pathway as a result of APC,
AXIN1 or CTNNB1 mutations has been found in most colorectal
cancers. For a long time, this aberrant Wnt activation has been
thought to be independent of upstream signals. However, recent
studies indicate that upstream signals retain their ability to regu-
late the Wnt pathway even in the presence of downstream muta-
tions. Wnt-2 is well known for its overexpression in colorectal can-
cer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding
protein implicated in a variety of biological functions, has recently
been reported to interact with b-catenin. In this study, we investi-
gated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/
b-catenin signaling. We found that siRNA silencing of either Wnt-
2 or Gal-3 expression inhibited TCF-reporter activity, decreased
cytosolic b-catenin level and induced apoptosis in human colorec-
tal cancer cells containing downstream mutations. More interest-
ingly, we showed that inhibition of both Wnt-2 and Gal-3 had
synergistic effects on suppressing canonical Wnt signaling and
inducing apoptosis, suggesting that aberrant canonical Wnt/b-cat-
enin signaling in colorectal cancer can be regulated at multiple
levels. The combined inhibition of Wnt-2 and Gal-3 may be of
superior therapeutic advantage to inhibition by either one of
them, giving rise to a potential development of novel drugs for the
targeted treatment of colorectal cancer.
' 2007 Wiley-Liss, Inc.
Key words: Wnt-2; galectin-3; Wnt signaling pathway; apoptosis;
human colorectal cancer
Colorectal cancer is the third most common cancer in the
United States. The overall 5-year survival rate from colorectal
cancer is ?60%, whereas for metastatic colorectal cancer, the 5-
year survival rate is only about 5% (www.cancer.org). Better sys-
temic treatment is therefore needed for this deadly disease.
Colorectal cancer develops via a multistage process involving
the accumulation of mutations in both oncogenes and tumor sup-
pressor genes.1Constitutive activation of the canonical Wnt sig-
naling pathway is an early progression event in tumorigenesis in
more than 90% of colorectal cancers.2In the canonical Wnt sig-
naling pathway, Wnt binds to frizzled receptor, activates dishev-
elled (Dvl) and disassembles the b-catenin ‘‘destruction com-
plex,’’ which prevents the phosphorylation and subsequent ubiqui-
tination of b-catenin, resulting in b-catenin stabilization and
accumulation in the cytoplasm. Stabilized b-catenin enters the nu-
cleus, where it complexes with TCF/LEF transcription factors to
regulate the transcription of downstream target genes.3The aber-
rant canonical Wnt signaling pathway occurs mainly through
mutations in the adenomatous polyposis coli (APC) gene, the
b-catenin/CTNNB1 gene or in the AXIN gene.2,4–6Originally,
these mutations were thought to lead to accumulation of free b-
catenin and activation of downstream target genes independent of
upstream signals. However recently, an increasing number of
upstream Wnt signaling components have been found to be misre-
gulated in colorectal cancer. For example, WNT-2 upregulation
was found in colorectal cancer tissue.7,8In addition, overexpres-
sion and inhibition of Wnt-1 activates or suppresses Wnt
signaling, respectively, even in the presence of downstream mu-
tations.9,10Furthermore, expression of Wnt antagonists, SFRPs,
WIF-1 and DKK-1, was reported to be silenced because of pro-
moter hypermethylation in colorectal cancer.9,11–13These findings
suggest that upstream Wnt signals at the cell surface level may
play important roles in colorectal tumorigenesis.
Galectin-3 (Gal-3), a member of the b-galactoside-binding pro-
teins family, has a variety of biological functions, involving RNA
processing, cell growth, differentiation, adhesion, apoptosis and
malignant transformation.14–16Aberrant expression of Gal-3 has
been found in a number of human cancers.17–19In human colorec-
tal carcinomas, the expression of Gal-3 has been extensively stud-
ied, leading to conflicting results. Various studies observed
increased Gal-3 expression in colorectal cancer and suggested a
correlation with disease progression and metastasis.20–22On the
contrary, decreased Gal-3 expression has also been reported in
colorectal cancer compared with corresponding normal tissue.23,24
Recently, Gal-3 was identified as a novel binding partner of
b-catenin,25which therefore linked Gal-3 to the Wnt signaling
To further investigate the upstream regulatory mechanisms
responsible for b-catenin dependent transcription in colorectal
cancer in this study, we examine how Wnt-2 and Gal-3 affect the
canonical Wnt signaling in the presence of downstream mutations.
Material and methods
Human colorectal cancer cell lines SW480, CaCO2, HCT116,
HT29 and LOVO were purchased from American Type Culture
Collection (Manassas, VA). These cell lines were cultured in
RPMI 1640 (SW480, HCT11 and HT29) or DMEM (CaCO2 and
LOVO) supplemented with 10% fetal bovine serum, penicillin
(100 IU/mL) and streptomycin (100 lg/mL). All cells were
cultured at 37?C in a humid incubator with 5% CO2.
Fresh human metastatic colorectal cancer tissue in lung was col-
lected from patients undergoing resection of their tumors as
described previously26and approved by the Committee on Human
Research at the University of California, San Francisco. These tis-
sue samples were snap-frozen in liquid nitrogen immediately after
resection and kept at 2170?C before use. The metastatic colo-
rectal tumor tissue specimens were exclusively tumor tissues.
Grant sponsor: National Institutes of Health; Grant number: RO1 CA
093708-01A3; Grant sponsors: The Larry Hall and Zygielbaum Memorial
Trust; The Kazan, McClain, Edises, Abrams, Fernandez, Lyons & Farrise
*Correspondence to: Department of Surgery, Comprehensive Cancer
Center, 1600 Divisadero Street, C322C, Box 1674, University of Califor-
nia, San Francisco, CA 94115, USA. Fax: 1415-502-3179.
Received 1 November 2006; Accepted after revision 17 April 2007
Published online 29 May 2007 in Wiley InterScience (www.interscience.
Int. J. Cancer: 121, 1175–1181 (2007)
' 2007 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Quantitative real-time reverse transcription-PCR
Total RNA from cell lines and fresh tumor samples was isolated
using an extraction kit (RNeasy Mini kit; Qiagen, Valencia, CA).
Normal colon tissue RNA was purchased from BD Biosciences,
Clontech (Palo Alto, CA). First-strand cDNA was synthesized
from total RNA using iScript cDNA synthesis kit (Bio-Rad,
Hercules, CA) according to the manufacturer’s instructions.
Transcript analysis was done by real-time reverse transcription-
PCR using the Taqman assay. Hybridization probes and primers
(Inventoried, chosen from the online catalog) were purchased
from Applied Biosystems (Foster City, CA). All samples were
amplified simultaneously in triplicate and amplifications were run
in a 7900 real-time PCR System (Applied Biosystems). Each
value was normalized to its GAPDH level.
An Atlas human cancer profiling array II was purchased from
Clontech Laboratories. The array contains pairs of cDNAs that are
generated from tumor and corresponding normal tissue samples
from individual patients, spotted side-by-side on a nylon
membrane. The array also contains human Ubiquitin cDNAs as
negative controls. The membrane was hybridized to a [32P]dCTP-
labeled human Wnt-2 cDNA probe as described previously.27The
hybridized membrane was exposed to X-ray film for 3 days. The
membrane was also hybridized to a [32P]dCTP-labeled human
Ubiquitin cDNA probe to normalize the amount of the spotted
cDNAs. The density of the signals was determined by Imagequant
Software (Molecular Dynamics, Sunnyvale, CA).
Cells were plated into a 6-well plate with fresh media without
antibiotics 24 hr before transfection. The HPLC-purified small
interfering RNAs [siRNAs (nonsilencing siRNA control, Wnt-2
and Gal-3 siRNA; >97% pure)] were purchased from Qiagen-
Xeragon (Germantown, MD). The control (nonsilencing) siRNA
does not target any known mammalian gene (sequence 50-
AATTCTCCGAACGTGTCACGT-30). The siRNA specific for
human Wnt-2 is derived from the mRNA sequence (50-GAA-
GATGGGAAGCGCCAAG-30) of human Wnt-2 as described pre-
viously.27The siRNA specific for human Gal-3 is derived from
the mRNA sequence (50-CCCACGCTTCAATGAGAAC-30) of
human Gal-3. As described previously,28the siRNA specific for
human Dishevelled (Dvl)-1 and -3 is derived from the mRNA
sequence (50-CAAGATCACCTTCTCCGAG-30), which is identi-
cal in both human Dvl-1 and Dvl-3. The siRNA specific for human
Dvl-2 is derived from the mRNA sequence (50-CTTTGAGAA-
CATGAGCAAC-30) of human Dvl-2. The lyophilized siRNAs
were dissolved in annealing buffer, reheated to 95?C for 1 min,
and then incubated for 1 hr at 37?C, following the protocol
described previously.29Transfection was performed by using oli-
gofectamin (Invitrogen, Carlsbad, CA) according to the manufac-
turer’s instructions. After siRNA transfection, the plates were
incubated at 37?C for 3–5 days before further analysis. Experi-
ments were performed in triplicate.
Cells were plated in 12-well plates with fresh media without
antibiotics, 24 hr before transfection. One microgram of the TOP-
FLASH or FOPFLASH reporter plasmid (kindly provided by
Dr. Hans Clevers) and 0.05 lg of internal control plasmid pRL-
TK (Promega, Madison, WI) were cotransfected transiently into
cells as described previously.4Transfection was performed by
using lipofectamin 2000 (Invitrogen) according to the manufac-
turer’s instructions. The cells were incubated at 37?C for 24 hr,
washed once with PBS, and then lysed to measure luciferase
reporter gene expression by using dual-luciferase reporter assay
system (Promega). TCF-dependent transcriptional activity was
determined by the ratio of pTOPFLASH/pFOPFLASH luciferase
activity, each normalized to luciferase activities of the pRL-TK
reporter. All experiments were performed in triplicate and a
minimum of 3 times.
Cytosolic protein extraction and Western blotting were
performed as described previously.30Anti-Gal-3 antibody was
purchased from Fitzgerald Industries International (Concord,
MA). Anti-b-catenin antibody was purchased form Transduction
Laboratories (Lexington, KY). Anti-cyclin D1 antibody was pur-
chased from Cell Signaling Technology (Beverly, MA). Anti-sur-
vivin antibody was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-cytochrome c antibody was purchased
from BD Biosciences (San Diego, CA). Anti-b-actin antibody was
purchased from Sigma Chemical (St Louis, MO).
Cells were harvested by trypsinization and stained using an
Annexin V FITC Apoptosis Detection Kit (Biosource, Camarillo,
CA) according to the manufacturer’s protocol. Stained cells were
immediately analyzed by flow cytometry (FACScan, Becton Dick-
inson, Franklin Lakes, NJ). Early apoptotic cells with exposed
phosphatidylserine but intact cell membranes bound to Annexin
V-FITC but excluded propidium iodide. Cells in necrotic or late
apoptotic stages were labeled with both Annexin V-FITC and
The data shown represent mean values 6 SD. Student’s t-test
was used for comparing activities of different treatments.
Results and discussion
We used quantitative real-time RT-PCR to examine Wnt-2 gene
expression in 5 colorectal cancer cell lines and confirmed that
Wnt-2 was overexpressed in all cell lines (Fig. 1a). Next, we meas-
ured Wnt-2 expression by cDNA expression array analysis in
primary colorectal cancer tissue and matched normal tissue
samples. No detectable Wnt-2 expression was found in normal col-
orectal tissue samples. In contrast, we observed dramatic overex-
pression of Wnt-2 in cancer tissue samples (Fig. 1b). We also
found that Wnt-2 was overexpressed in metastatic colorectal can-
cer tissue in lung compared to normal colorectal tissue by using
real-time RT-PCR (Fig. 1c). Furthermore, our real-time RT-PCR
and Western blot data showed that Gal-3 was overexpressed in all
the colorectal cell lines (Figs. 2a and 2b) and in the metastatic col-
orectal cancer tissue samples compared to normal colon tissue
(Fig. 2c). Taken together, we demonstrated that both Wnt-2 and
Gal-3 are upregulated in both colorectal cancer cell lines and can-
cer tissue samples.
Previous studies in our lab10demonstrated that inhibition of
Wnt-1 signaling induced significant apoptosis in colorectal cancer
cells that express Wnt-1 and contain downstream mutations, sup-
porting the hypothesis that constitutive Wnt signaling may be
required to complement downstream mutations in colorectal tu-
morigenesis. Because of the overexpression of Wnt-2 in colorectal
cancer cells containing downstream mutations, we asked whether
silencing of Wnt-2 could attenuate Wnt-2 signaling through the
canonical Wnt/b-catenin pathway. In addition, we questioned
whether perturbation of Gal-3 expression affects the canonical
Wnt signaling pathway based on the newly discovered function of
Gal-3 in Wnt signaling pathway. We hypothesized that both Wnt-
2 and Gal-3 overexpression play important roles in the constitu-
tive activation of Wnt signaling pathway in colorectal cancer and
that double-silencing of both genes may lead to synergistic sup-
pression of the Wnt signaling pathway.
To test these hypotheses, we first used Wnt-2 and/or Gal-3
siRNA to silence the gene expressions and examined whether inhi-
bition of Wnt-2 and/or Gal-3 expression affected transcriptional
activity of the TCF-reporter. siRNA knockdown of Wnt-2 and
SHI ET AL.
Gal-3 expression was confirmed by real-time RT-PCR (data not
shown). We found that TCF-reporter activities were slightly inhib-
ited after 50-nM Wnt-2 siRNA treatments in SW480 and CaCO2
cells (95.4 and 78.9%, respectively, of the control siRNA treat-
ment which was set to 100%; Fig. 3a). The TCF-reporter activities
were reduced more significantly when cells were treated with 100
nM Wnt-2 siRNA in SW480 and CaCO2 cells (70.6 and 67.1%,
respectively). These results support the hypothesis that upstream
Wnt signal at the ligand level retain its ability to regulate Wnt sig-
naling even in the presence of downstream mutations. Previous
studies have shown that Gal-3 forms a ternary complex with b-
catenin and TCF-4 independent of either APC or b-catenin muta-
tions. In addition, when introducing Gal-3 cDNA into HT29 cells,
the TCF-reporter activities were augmented in a dose-dependent
manner.25Therefore, we hypothesized that silencing of Gal-3
expression alone may lead to inhibition of TCF-reporter activity in
colorectal cancer cells. Indeed, we observed decreased TCF-re-
porter activities when 50 nM Gal-3 siRNA was transfected in
SW480 and CaCO2 cells (84.7 and 80.3%, respectively; Fig. 3a).
When 100 nM Gal-3 siRNA was used, we found that luciferase
activities were inhibited to 77.2% for SW480 and 62% for
CaCO2. More interestingly, when combination treatment of
50 nM Wnt-2 and Gal-3 siRNA was used, luciferase activities
were synergistically reduced to 56.8% for SW480 (p < 0.002) and
56.1% for CaCO2 (p < 0.001), suggesting that the TCF-dependent
transcriptional activity in colorectal cancer cells may be regulated
by multiple upstream events at different levels.
Dvl has been implicated as a key factor in Wnt signaling trans-
duction. It is believed that Dvl deciphers signals coming from the
plasma membrane and distributes them into divergent intracellular
FIGURE 1 – Wnt-2 is overexpressed in cell lines, primary colorectal
cancer tissue and metastatic colorectal cancer tissue in lung. (a) Real-
time reverse transcription-PCR (RT-PCR) analysis in 5 colorectal can-
cer cell lines: SW480, HCT116, HT29, LOVO and CaCO2. Percent
expression of Wnt-2 was normalized to GAPDH. (b) Expression of
Wnt-2 in matched normal colon and rectum tissue and primary colon
and rectum tumor tissue. Upper panel, NT represents 10 normal colon
tissue samples and 10 normal rectum tissue samples. Lower panel, TT
represents 10 primary colon tumor tissue samples and 10 primary rec-
tum tumor tissue samples. The dot blot was hybridized to [32P]dCTP-
labeled human Wnt-2 cDNA probe. No detectable Wnt-2 expression
was found in normal colorectal tissue samples, whereas dramatic over-
expression of Wnt-2 was found in cancer tissue samples. (c) Real-time
RT-PCR analysis of Wnt-2 expression in normal colon tissue and 5
samples of metastatic colorectal cancer tissue in lung. Percent expres-
sion of Wnt-2 was normalized to GAPDH.
FIGURE 2 – Gal-3 is overexpressed in cell lines and metastatic col-
orectal cancer tissue in lung. (a) Real-time RT-PCR analysis of Gal-3
expression in 5 colorectal cancer cell lines. Percent expression of Gal-
3 was normalized to GAPDH. (b) Western blot analysis of Gal-3
expression in 5 colorectal cancer cell lines (same serials as in a).
b-Actin served as loading control. (c) Real-time RT-PCR analysis of
Gal-3 expression in normal colon tissue and 5 samples of metastatic
colorectal cancer tissue in lung. Percent expression of Gal-3 was
normalized to GAPDH.
SYNERGISTIC ACTION OF Wnt-2 AND Gal-3
signaling pathways, including the canonical and noncanonical
Wnt signaling pathways.31To establish whether Wnt-2 transduces
its signal through Dvl to manipulate the canonical Wnt/b-catenin
pathway in colorectal cancer cells with downstream mutations, we
used siRNA to knockdown Dvl-1, -2 and 3 simultaneously.28
siRNA knockdown of the gene expressions was confirmed by real-
time RT-PCR (data not shown). To our surprise, Dvl siRNA treat-
ments at both 50 and 100 nM had no significant effects on TCF
transcriptional activity in either SW480 or CaCO2 cells (Fig. 3b).
Combination treatment of 50 nM Dvl siRNA with either 50 nM
Wnt-2 or 50 nM Gal-3 siRNA also had no additive or synergistic
effect on suppressing TCF-dependent transcription (Figs. 3b and
3c). These findings suggest that Dvl may play an insignificant role
in regulating downstream transcriptional activity in SW480 and
CaCO2 cells when mutations are present. In other words, the Wnt
pathway downstream mutations in SW480 and CaCO2 cells may
constitutively activate the Wnt signaling pathway despite
upstream perturbation at the Dvl level. This raises the possibility
that, in colorectal cancer cells, Wnt-2 may regulate the canonical
Wnt/b-catenin signaling pathway through several different mecha-
nisms. Our results indicate the possibility of unidentified proteins,
which function in a manner similar to Dvl, conducting signals
from the frizzled receptors. Alternatively, it is also possible that
Wnt-2 regulates other signaling pathways, which crosstalk with
the Wnt signaling pathway and affect b-catenin/TCF transcrip-
To verify that the decrease in TCF-reporter activity that we
observed (Fig. 3) is due to the depletion of b-catenin, we next
examined intracellular b-catenin level in SW480 and CaCO2 cells
after different siRNA treatments. After 50 nM Wnt-2 or Gal-3
siRNA treatment, a significant decrease in the level of cytosolic b-
catenin was observed (Figs. 4a and 4c). More interestingly, at a
low dose combination of Wnt-2 and Gal-3 siRNA (50 nM each)
and at a high dose of Wnt-2 or Gal-3 (100 nM alone) siRNA treat-
ments, cytosolic b-catenin levels were completely depleted. To
rule out the possibility that the siRNA treatments lead to repres-
sion of the b-catenin gene transcription, we used real-time RT-
PCR to measure the b-catenin/CTNNB1 gene expression after
Wnt2 and/or Gal-3 siRNA treatments. No significance change at
b-catenin/CTNNB1 transcriptional level was observed (Figs. 4b
and 4d). Taken together, our data showed for the first time that
silencing of Wnt-2 and Gal-3 synergistically decreased cytosolic
b-catenin level, suggesting that destabilization of b-catenin may
be responsible for the inhibition of TCF-dependent transcriptional
activity by siRNA treatment in colorectal cancer cells.
Cyclin D1 has been identified as a direct target gene of the
canonical Wnt signaling pathway in colorectal cancer cells.32We
therefore examined cyclin-D1 level in these cells after treatment
with different siRNAs. As expected, we observed decreased level
of cyclin D1, which correlated with the level of cytosolic b-cate-
nin in those treatments (Figs. 4a and 4c). It has been previously
reported that Gal-3 induces cyclin D1 promoter activity.33Consis-
tently, our results showed that cyclin D1 expression was com-
pletely depleted in the presence of low dose of Gal-3 siRNA
(50 nM). Interestingly, Gal-3 siRNA treatment appeared to be
more effective than Wnt-2 siRNA treatment in the inhibition of
cyclin D1 expression (Figs. 4a and 4c). This may be explained by
the finding that Gal-3 forms a protein/DNA complex at the cAMP-
responsive element site of cyclin D1 promoter region,33in addi-
tion to its interaction with b-catenin.25,34
Also, we analyzed the b-catenin and cylin D1 levels after the
Dvl siRNA treatment in SW480 and CaCO2 cells. We observed
slight decreases of b-catenin and cyclin D1 level after Dvl siRNA
treatments at 50 and 100 nM in SW480 (Fig. 5a) and CaCO2
(Fig. 5b) cells. When cells were treated with a combination of Dvl
and Wnt-2 or Dvl and Gal-3 siRNA, we saw no additive or syner-
gistic effects on cytosolic b-catenin and cyclin D1 levels. Consist-
ent with the luciferase assay data, this result again questions the
role of Dvl in stabilizing cytosolic b-catenin in colorectal cancer
cells with Wnt downstream mutations and also suggests that
FIGURE 3 – TOP/FOPFLASH assay of TCF-dependent transcrip-
tional activity in SW480 and CaCO2 cells after Wnt-2 and Gal-3
siRNA treatments. (a) The cells were transiently cotransfected with
pTOPFLASH or pFOPFLASH, internal control plasmid pRL-TK and
different siRNAs of control, Wnt-2 and/or Gal-3. The cells were har-
vested 24 hr after transfection, and TCF-reporter activity was deter-
mined by the ratio of pTOPFLASH/pFOPFLASH, each normalized to
luciferase activity of the pRL-TK internal control. Gray bars represent
the activities in SW480 cells; White bars represent those in CaCO2
cells. (b) Luciferase assays in cells treated with different combination
of Wnt-2 and Dvls siRNA as indicated. (c) Luciferase assays in cells
treated with different combination of Dvls and Gal-3 siRNA as indi-
cated. The methods to calculate the reporter activity in (b) and (c)
were the same as described in (a).
SHI ET AL.
unknown Wnt components or other signaling pathways may be
involved in modulating the canonical b-catenin/TCF transcription
in these cells.
Finally, to investigate whether the inhibitory effect of Wnt-2
and Gal-3 siRNA treatments on the Wnt canonical pathway could
induce apoptosis in colorectal cancer cells, we measured the levels
of apoptotic pathway effectors after different treatments. We
found that the protein level of survivin, an inhibitor of apoptosis
and also a Wnt downstream target gene,35was downregulated and
cytosolic cytochrome c level was increased by either 50 nM Wnt-
2 or 50 nM Gal-3 siRNA treatment (Fig. 6a). With combination
treatment of 50 nM Wnt-2 and 50 nM Gal-3 siRNA, we found
undetectable level of survivin and dramatically increased level of
cytochrome c, which is similar to the effect of 100 nM Wnt-2 or
100 nM Gal-3 siRNA treatment. Consistently, flow cytometry
analysis showed apoptosis induction in 13.35 or 5.63% of cells
after 50 nM Wnt-2 or 50 nM Gal-3 siRNA treatment alone. With
combination treatment of 50 nM Wnt-2 and 50 nM Gal-3 siRNA,
apoptosis was seen in 31.12% of cells (Fig. 6b). Taken together,
our results demonstrated that Wnt-2 and Gal-3 siRNA treatment
synergistically induced apoptosis in colorectal cancer cells.
Intracellular Gal-3 is well known to exhibit antiapoptotic activ-
ity. For example, Gal-3 protects T cells from apoptosis induced
by anti-Fas antibodies or the protein kinase inhibitor, staurospor-
ine.36Additionally, BT549, a breast cancer cell line, in which
Gal-3 is overexpressed, was found to render resistance to apopto-
sis induced by various stimuli.37,38It was proposed that this antia-
poptotic activity of Gal-3 is attributed to its structural similarities
with an antiapoptotic protein, Bcl-2.36Both Gal-3 and Bcl-2
contain the Asp-Trp-Gly-Arg (NWGR) motif at the C-terminus.
This NWGR motif is also called the ‘‘antideath’’ motif, and it is
believed to be critical for the antiapoptotic function of Bcl-2.39
However, the molecular mechanism by which Gal-3 exerts its
antiapoptotic activity cannot be explained solely on its NWGR
motif nor by its potential interaction with Bcl-2 through the
NWGR motif.40Recently, Shimura et al. demonstrated that Gal-3
is a novel binding partner of b-catenin.25Gal-3 complexes with
b-catenin/TCF, and thus induces TCF-transcriptional activity and
stimulates Wnt downstream gene expression. This new finding
links the function of Gal-3 to the Wnt signaling pathway, indicat-
ing another explanation of the antiapoptotic function of Gal-3. In
this study, our data suggests that the antiapoptotic activity of
Gal-3 is, at least, partially due to the Gal-3 contribution to the
stabilization of cytosolic b-catenin and the activation of the
canonical Wnt signaling pathway. However, the same experiment
conducted in CaCO2 did not demonstrate significant apoptosis
in any of the treatments (data not shown). CaCO2 has been
previously described as insensitive to Fas-mediated apoptosis
as compared to other colorectal cell lines.41In addition, CaCO2
is resistant to 5-flurorouracil treatment in combination with
20deoxyinosine, whereas the combined treatment increased the
sensitivity of HT29 and fluorouracil-resistant SW620 cells by 38-
to 700-fold.42On the basis of the previous findings, we argue that
CaCO2 may have developed multiple survival strategies to over-
come various apoptotic stimuli.
FIGURE 4 – Analysis of the Wnt downstream effectors after Wnt-2 and Gal-3 siRNA treatments. Western blot analysis of the Wnt downstream
effectors was performed after Wnt-2 and Gal-3 siRNA treatments in SW480 (a) and CaCO2 (c), respectively. b-Actin served as loading control.
Cytosolic proteins were prepared and used in the analysis after 3 days treatment. Real-time RT-PCR analysis of CTNNB1 expression was
performed 3 days after Wnt-2 and Gal-3 siRNA treatments in SW480 (b) and CaCO2 (d), respectively. Percent expression of CTNNB1 was
normalized to GAPDH.
FIGURE 5 – Western blot analysis of the Wnt downstream effectors
after Dvls, Wnt-2 and Gal-3 siRNA treatments in SW480 (a) and
CaCO2 (b). b-Actin served as loading control. Cytosolic proteins
were prepared and used in the analysis after 3 days treatment.
SYNERGISTIC ACTION OF Wnt-2 AND Gal-3
In summary, our findings show that inhibition of Wnt-2 signal-
ing at the cell surface level could effectively induce apoptosis in
the presence of downstream mutations in human colorectal cancer
cells. Thus, better understanding of upstream Wnt-2 signaling
at the cell surface level and further studies on the regulation of
Wnt-2 expression at the transcriptional level may offer new targets
for developing novel molecular treatments for colorectal cancer.
Moreover, the interaction between Gal-3 and the Wnt pathway
may also provide potential targets for drug development. The
binding sequences between b-catenin and Gal-3 have been
mapped to the N-terminus of b-catenin (amino acid residues 1-
131) and the C-terminus of Gal-3 (amino acid residues 63–250).25
It is particularly interesting that the C-terminus of Gal-3 contains
both the carbohydrate-binding motif and the NWGR motif. In this
study, we were unable to address the importance of the different
motifs on the C-terminus of Gal-3. However, we predict that fur-
ther study of the motifs of Gal-3 and narrowing down the binding
region between Gal-3 and b-catenin will help in designing com-
pounds that attenuate the canonical Wnt signaling pathway. Most
importantly, the synergistic effect of the inhibition of Wnt-2 and
Gal-3 observed in this study holds promise for new strategies to
target the canonical Wnt pathway at multiple levels in the treat-
ment of colorectal cancer.
We thank Pamela Derish, the scientific publications manager in
the Department of Surgery at UCSF, for her careful editing of this
paper. This work was partially supported by a National Institutes
of Health Grant (RO1 CA 093708-01A3), the Larry Hall and
Zygielbaum Memorial Trust, and the Kazan, McClain, Edises,
Abrams, Fernandez, Lyons & Farrise Foundation (all to D.M.J.).
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SYNERGISTIC ACTION OF Wnt-2 AND Gal-3