Alteration of galectin-1 during tumorigenesis of Opisthorchis viverrini infection-induced cholangiocarcinoma and its correlation with clinicopathology.
ABSTRACT Galectin-1 is a beta-galactoside-binding lectin to function in cell adhesion, proliferation, differentiation, and might be involved in tumor progression and metastasis. In the present study, the expression kinetics of galectin-1 during the tumorigenesis of a parasite Opisthorchis viverrini infection-induced cholangiocarcinoma (CCA) was investigated in model animal hamsters, and the expression was confirmed in human CCA cases. It was found that galectin-1 was overexpressed at mRNA and protein levels with the tumor progression. The mRNA expression was elevated in very early stage during tumorigenesis and the increase was time dependent. Galectin-1 protein expression profiles indicated that the increased expression was mainly located in the epithelium of extensively proliferated and hyperplasia small bile ducts at early stage of CCA development in model animal and mainly in the extensive tumor stroma tissues in both model animals and human CCA cases at later stage. The analysis of correlation of the overexpression with clinicopathology in human cases suggested that high expression of galectin-1 was associated with advanced stage and metastasis and with shorter cumulative overall survival of the patients. Multivariate Cox regression analysis revealed that galectin-1 expression was of independent prognostic significance for CCA. Our results suggest that galectin-1 is likely involved in the tumorigenesis and expected to serve as a tumor stroma marker in diagnosis and prediction of metastasis and poor prognosis of the opisthorchiasis-associated CCA.
- SourceAvailable from: Zhiliang Wu[Show abstract] [Hide abstract]
ABSTRACT: Milk fat globule epidermal growth factor 8 (MFG-E8) is a pleiotropic secreted glycoprotein to play roles in mediating immune tolerance and homeostasis maintenance and enhancing angiogenesis. To evaluate its value as a biomarker in opisthorchiasis-associated cholangiocarcinoma (CCA), the present study investigated MFG-E8 expression kinetics during the tumorigenesis in Opisthorchis viverrini infection-induced CCA, and demonstrated its expression in the tumor tissues of CCA patients and its serum level among them. During the tumorigenesis of CCA, MFG-E8 expression was increased in a time-dependent manner with the pathological processes. Absolutely higher expression of MFG-E8 messenger RNA was detected in the tumor tissues from CCA patients, compared with those in adjacent tissues. Immunobiochemical analysis showed that more than 90 % CCA cases were positive and the positive reaction located in the membrane and cytoplasm of the tumor cells. Moreover, the average serum level in the CCA patients was significantly higher than that in healthy individuals and those with O. viverrini infection or other parasitosis. Correlation analysis of MFG-E8 expression with CCA clinicopathology revealed that a high expression of MFG-E8 protein was significantly bound with a poor differentiation, pathological advanced stage, and metastasis of CCA. The multivariation analysis indicated that MFG-E8 was an independent prognostic factor. In addition, short hairpin RNA-mediated MFG-E8 knockdown in CCA cell line obviously suppressed the cell proliferation. Our results strongly suggested that MFG-E8 is a promising biomarker for the diagnosis, prognosis, and therapy target of opisthorchiasis-associated CCA.Tumor Biology 10/2013; · 2.52 Impact Factor
Alteration of galectin-1 during tumorigenesis of Opisthorchis
viverrini infection-induced cholangiocarcinoma
and its correlation with clinicopathology
Zhiliang Wu & Thidarut Boonmars & Isao Nagano &
Sirintip Boonjaraspinyo & Somchai Pinlaor &
Chawalit Pairojkul & Yaovalux Chamgramol &
Received: 14 November 2011 /Accepted: 10 February 2012 /Published online: 29 February 2012
#International Society of Oncology and BioMarkers (ISOBM) 2012
Abstract Galectin-1 is a beta-galactoside-binding lectin to
function in cell adhesion, proliferation, differentiation, and
might be involved in tumor progression and metastasis. In the
present study, the expression kinetics of galectin-1 during the
tumorigenesis of a parasite Opisthorchis viverrini infection-
induced cholangiocarcinoma(CCA) was investigated in model
animal hamsters, and the expression was confirmed in human
CCA cases. It was found that galectin-1 was overexpressed at
mRNA and protein levels with the tumor progression. The
mRNA expression was elevated in very early stage during
tumorigenesis and the increase was time dependent. Galectin-
1 protein expression profiles indicated that the increased ex-
pression was mainly located in the epithelium of extensively
proliferated and hyperplasia small bile ducts at early stage of
tumor stroma tissues in both model animals and human CCA
cases at later stage. The analysis of correlation of the over-
expression with clinicopathology in human cases suggested
that high expression of galectin-1 was associated with ad-
vanced stage and metastasis and with shorter cumulative over-
revealed that galectin-1 expression was of independent prog-
nostic significance for CCA.Our results suggestthatgalectin-1
is likely involved in the tumorigenesis and expected to serve as
a tumor stroma marker in diagnosis and prediction of metasta-
sis and poor prognosis of the opisthorchiasis-associated CCA.
Cholangiocarcinoma (CCA) is the most common liver cancer
and is becoming one of most important health problems in
Southern Asia, including Thailand, Laos, Vietnam, and Cam-
bodia [1, 2]. Also as the second most common primary liver
cancer in the other countries, the incidence and mortality of
CCA are markedly increasing in recent decades [3, 4]. There
are many risk factors for CCA, including primary sclerosing
cholangitis, inflammatory disease of the bile ducts, congenital
liver abnormality, viral hepatitis, alcoholic liver disease, cir-
rhosis, and Opisthorchis viverrini infection which is a major
risk factor for the CCA patients in the endemic area of opis-
thorchiasis. Epidemiological investigation and animal experi-
ments have confirmed relationship between O. viverrini
infection and CCA [5–9].
Thidarut Boonmars is the co-first author and co-corresponding author.
Z. Wu (*):I. Nagano:Y. Takahashi
Department of Parasitology,
Gifu University Graduate School of Medicine,
Gifu 501-1194, Japan
T. Boonmars:S. Boonjaraspinyo:S. Pinlaor
Department of Parasitology, Faculty of Medicine,
Khon Kaen University,
Khon Kaen 40002, Thailand
T. Boonmars:S. Boonjaraspinyo:S. Pinlaor
Liver Fluke and Cholangiocarcinoma Research Center,
Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand
C. Pairojkul:Y. Chamgramol
Department of Pathology, Faculty of Medicine,
Khon Kaen University,
Khon Kaen 40002, Thailand
Tumor Biol. (2012) 33:1169–1178
CCA arises from the neoplastic transformation of chol-
angiocytes and belongs to adenocarcinoma with a poor
prognosis and limitation in therapeutic options because the
early diagnosis is so difficult due to lack of specific physical
signs and symptoms, laboratory indexes, and tumor markers
in early or premalignant stages. Complete surgical excision
is the only chance for survival, but unfortunately, existence
of distant and extensive metastasis before diagnosed usually
excludes the option for surgical excision. The survival of
patients with unresectable CCA is generally quite short, less
than 12 months after diagnosis . Although improvement
has been made in diagnosis techniques such as serum tumor
markers, radiological and endoscopic imaging, and patho-
logical analysis of biopsies or endoscopic brushings, the 5-
year survival is still extremely low [11, 12], which is now
the problem confronted by doctors and patients in endemic
areas of opisthorchiasis. Therefore, it is necessary to deter-
mine the molecular mechanism of CCA tumorigenesis and
develop novel biomarkers for the diagnosis, prognosis, me-
tastasis, and therapy of this malignancy.
Galectin-1 is a carbohydrate-binding protein which binds
14]. Studies have indicated that this galectin functions in a lot
of biological and oncogenic processes, including in cell adhe-
sion, interaction between cell and extracellular matrix, cell
transformation, proliferation, apoptosis, migration, metastasis,
and angiogenesis [14–16]. The significance of galectin-1 ex-
pression in neoplasm has been reported. Studies on human
epithelial tumors such as colorectal, ovarian, breast, gastric,
andthyroid cancers have indicated thatthe galectinis involved
in tumor progression, invasion, and metastasis [16, 17], and it
is described as a potential tumor marker for the prediction of
prognosis and metastasis . However, little has been known
on galectin-1 in CCA, and so far no report has been made on
the galectin in the opisthorchiasis-associated CCA.
The use of animal models in cancer research plays an
important role in demonstrating tumorigenesis process ki-
netically, including the initiation, promotion, and progres-
sion of tumor. Observation using animal model can provide
useful information, such as expression kinetics of target
genes, which is usually difficult to be obtained by using
human specimen because of the limitation of sampling and
the samples in advanced stages. Opisthorchiasis-N-nitroso-
dimethylamine (NDMA)-associated CCA animal model has
also been established [19, 20] and widely used in the study
of tumorigenesis of O. viverrini infection-induced CCA. In
this present study, we studied kinetics of galectin-1 expres-
sion at mRNA and protein levels during the O. viverrini
infection-associated CCA tumorigenesis in an animal model
of O. viverrini infection-induced CCA and human CCA
cases from opisthorchiasis endemic area and further ana-
lyzed the relationship between the galectin-1 overexpression
and their clinicopathological features.
Materials and methods
Parasite and infection
An animal model of O. viverrini infection-induced CCAwas
established following previousreports [19, 20].Metacercariae
of O. viverrini were collected as a conventional method.
Eighty Syrian hamsters (6 weeks) were divided into four
groups (n020 per group): (1) normal control—without any
treatment; (2) OV infected—each hamster was infected with
50 metacercariae; (3) NDMA treated—hamsters were admin-
istrated with drinking water containing 12.5 ppm of NDMA
(Wako, Japan) for 2 months and then maintained; and (4) OV
infected plus NDMA treated—hamsters were infected and
treated as those in groups 2 and 3. Syrian hamster livers were
collected at 1, 2, 3, and 6 months postinfection (p.i., five per
group) for total RNA isolation and immunohistochemical
Collection of human CCA and adjacent tissues
Seventy-eight pairs of liver samples of CCA patients from
opisthorchiasis-heavy epidemical areas were provided by
the Liver Fluke and Cholangiocarcinoma Research Center,
Faculty of Medicine, Khon Kaen University, Thailand. Each
pair of sample included the CCA tumor tissue and the
adjacent normal tissues from the same patient. The tissues
were collected in CCA operation. One part of each sample
was frozen in liquid nitrogen immediately for RNA isolation.
One part was fixed with 10% formalin for histological exam-
ination and immunohistochemical analysis. The samples were
defined pathologically and histologically (Table 1). The utili-
zation of the human specimens in the present study was
approved by the Human Ethics Committee of the Khon Kaen
University (ethical clearance no. HEKKU501153) and Gifu
One part of hamster liver tissues from hilar region was fixed
with 10% formalin solution and paraffinized for hematoxy-
lin and eosin staining. Their histological changes were ob-
served during CCA development in the animal model.
Total RNAwas isolated from 200 mg liver tissue (from hilar
region in each group hamster; tumor area and adjacent
normal tissue in a human specimen) with TRIZOL reagent
(Invitrogen, Carlsbad, CA, USA) according to the manufac-
turer’s instructions. The isolated RNA was treated with
DNase (RQ1 RNase-Free DNase, Promega, Co., Madison,
WI, USA) and purified with a conventional method.
1170 Tumor Biol. (2012) 33:1169–1178
Quantitative real-time PCR
Quantitative real-time RT-PCR was applied to determine
galectin-1 expression with Thermal Cycler Dice Real Time
System (TAKARA, Japan). Because there was no genetic
information on Syrian hamster galectin-1, its gene was cloned
and sequenced based on the mouse sequence. The specific
primers of hamster galectin-1 for this PCR were designed as
following: forward: 3′-tacacttcaacccccgcttc and reverse 3′-
agacctccacaatgcttccag (GenBank accession no. JN997458).
Hamster glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
was selected as an endogenous marker, and its primers were
designed based on the published sequence in GenBank (ac-
cession no. U10983, forward: 3′-gacatcaagaaggtggtgaagca
and reverse 3′-catcaaaggtggaagagtggga).
The primers of human galectin-1 and endogenous marker
glucuronidase-beta (GUSB) were designed based on the
published sequences in GenBank (galectin-1: accession no.
NM_002305, forward: 3′-tgggcaaagacagcaacaac and re-
verse: 3′-ctggaagggaaagacagcctc; and GUSB: accession
no. NM_000181, forward 3′-atggaagaagtggtgcgtagg and re-
Reverse transcription was performed using a Prime Re-
according to the manufacturer’s instructions. Real-time PCR
was ran as previously described . Briefly, optimal condi-
tions for all investigated genes were established using SYBR
Premix Ex Taq Kit (TAKARA BIO) according to manufac-
consisted of 2 μl of the template (appropriate dilution was
determined by genes), 10 μl of SYBR Premix Ex Taq, and
0.8 μl of 5 μM of each primer. PCR was conducted as 1 cycle
denaturing at 95°C for 30 s and 40 cycles amplifying at 95°C
C to 95°C with 0.1°C/s temperature transition.
Specific external controls were constructed for target
genes. Tenfold serial dilutions (101to 107copies/2 μl) of
the standard DNAwere used to generate standard curves for
each gene. Differences of cDNA amount among different
samples were normalized by quantification of the house-
keeping gene, and their expression levels were represented
as the copy number of target gene/106G3PDH copies. The
values present as mean±SD.
Immunohistochemical staining for galectin-1 protein
Immunohistochemistry procedure was performed according
to streptavidin–biotin–peroxidase complex principle, using
Table 1 The correlation between galectin-1 expression and clinicopathology in 78 CCA patients
n Expression of galectin-1 mRNA revealed by real-time PCR
Expression of galectin-1 revealed by immunostaining
≧3 folds<3 foldsP value
Moderate and poor
0.604 33 (73.3%)
0.523 16 (36.4%)
0.366 19 (42.2%)
Bolded P value with asterisk indicates that there was significant difference (P<0.05)
Tumor Biol. (2012) 33:1169–11781171
rabbit monoclonal anti-galectin-1 antibody (Epitomics Inc.,
CA, USA). Briefly, deparaffinized sections were autoclaved
at 121°C for 10 min in 10 mM citrate buffer (pH 6.0) for
epitope antigen retrieval. The sections were incubated with
3% H2O2in methanol for the inactivation of endogenous
peroxidase and then blocked with 5% skim milk. After
incubation with primary antibody at 37°C for 60 min, the
sections were incubated with the secondary biotinylated
anti-rabbit antibody for 60 min, followed by incubation with
streptavidin–peroxidasefor 30min.The immunereactionwas
visualized by AEC (9-Ethylcarbazol-3-amine) as a chromo-
gen, and the sections were counterstained with hematoxylin.
The immunohistochemical results were evaluated by observ-
ers by semiquantifying the percentage of immunopositivity of
ten fields per slide and scored as 0%0negative, <25% 01+,
25–50% 02+, >50–75%03+, and >75%04+.
The statistical analysis was carried out with SPSS software
package version 18.0. The one-way ANOVA was used for
comparison of galectin-1 expression among animal groups.
The paired t test was used for comparison of mRNA expres-
sion between human CCA and adjacent tissue. The chi-square
test was used to analyze the correlations of mRNA and protein
expression with clinicopathological features. Survival curves
were plotted with the Kaplan–Meier method, and significance
were significant in univariate analysis (P<0.05) were sub-
jected to a multivariate Cox regression model to evaluate the
increment statistical power and independence of prognostic
impact. P value<0.05 was considered statistically significant.
Kinetics of galectin-1 mRNA expression
during tumorigenesis of CCA in Syrian hamster
The galectin-1 expression in Syrian hamsters was observed
in different groups (normal control, OV infected, NDMA
treated, and OV infected plus NDMA treated) at 1, 2, 3, and
6 months p.i. As shown in Fig. 1, the galectin-1 expression
level was greatly increased in the group of OV-infected plus
NDMA-treated hamsters compared with those in normal
Syrian hamsters (6.5-, 19.8-, 16.0-, and 25.6-folds at 1, 2,
3, and 6 months p.i., respectively; P<0.001), OV infected
(2.4-, 7.6-, 5.6-, and 3.1-folds; P<0.001), and NDMA treated
(4.3-, 4.1-, 2.9-, and 6.1-folds; P<0.001), and the increased
expression was in time-dependent manner. Galectin-1 expres-
sion in the OV-infected alone group was slightly increased at
p.i., but the expression levels were much lower than those in
the OV-infected plus NDMA-treated group. Similar expres-
sion pattern was observed in the NDMA-treated one.
Expression of galectin-1 protein in Syrian hamster revealed
by immunohistochemical staining
Galectin-1 protein expression was investigated in the liver
tissues from the different hamster groups (normal control,
OV infected, NDMA treated, and OV infected plus NDMA
staining using anti-galectin-1 antibody. As shown in Fig. 2, in
the small bile ducts (Fig. 2a–d). In the group of NDMA alone,
there were pathologically aggregation of inflammation cells, a
low degree of proliferation of bile ducts, glandular lesions,
cystic lesions, mucinous cyst, and cholangiofibrosis. The pos-
itive immunostaining was mainly observed in the small bile
ducts at 1, 2, and 3 months p.i. (Fig. 2e–g), and locally in the
proliferated epithelium of small bile ducts and glandular
lesions at 6 months p.i (Fig. 2h), but there was no positive
immunostaining in inflammatory cells and hepatocytes.
In the OV-infected group, parasites induced apparent
pathological changes, including inflammatory reaction
around bile ducts and in the portal connective tissues, hyper-
plasia of the bile duct epithelium, adenomatous formation,
proliferation of bile duct, and cirrhosis at 6 months p.i. The
positive immunostaining was shown mainly in the epithelium
of the proliferatedsmall bileducts (Fig. 2m–o),the fibroblasts
Copy numbers / 106 G3PDH copy
Months after infection
Fig. 1 Kinetics of galectin-1 mRNA expression in the animal groups
of normal control (normal), O. viverrini infected alone (OV infection),
NDMA treated alone, and OV infected plus NDMA treated (OV
+NDMA) at 1, 2, 3, and 6 months postinfection. The expression level
was determined with quantitative real-time PCR and was presented as
copy numbers within 106glyceraldehyde 3-phosphate dehydrogenase
(G3PDH) copies. The value was expressed as the mean±S.D from five
hamsters in each group. The expression in the group of OV+NDMA at
each time point was significantly higher than that in the group of
Normal, OV infection, or NDMA (P<0.001). ***P<0.001
1172 Tumor Biol. (2012) 33:1169–1178
around the hyperplastic bile ducts (Fig. 2i–l), and cholangio-
fibrosis in a small area (Fig. 2p).
In the OV-infected plus NDMA-treated group, tubular
and papillary CCA was developed from 2 months p.i. Ex-
tensive proliferated, hyperplastic, and dysplastic bile ducts,
various sizes of irregular glands with neoplastic cells, tubu-
lar formation in many regions of liver and papillary forma-
tion in main intrahepatic ducts, glandular and cystic lesions,
mucinous cysts, and cholangiofibrosis were prominent. Cor-
respondingly, the positive immunostaining was observed in
the stroma of periductal fibrosis and tumor at early stage of
CCA development (1 and 2 months p.i.) (Fig. 2p, r, u, and
v), which became stronger and more extensive at late stage
of CCA development (3 and 6 months p.i.) (Fig. 2t, x).
Positive immunostaining was also observed in the epitheli-
um of proliferated, hyperplastic, and dysplastic small bile
ducts at early stage of CCA development (Fig. 2u, v). There
was no staining in the inflammatory cells and epithelium of
large intrahepatic bile ducts (Fig. 2q, r, t and u).
Galectin-1 mRNA expression human CCA
The expression of galectin-1 mRNA in the pair of sample
(tumor and adjacent normal tissues) from 78 human CCA
cases was quantitatively determined with real-time PCR. As
shown in Fig. 3, in most of cases (68%), the galectin-1 gene
expression in the tumor tissues were threefolds higher than
those in the adjacent normal tissues (the average increase
was 17.7-folds). The paired t test analysis indicated that the
expression of galectin-1 in tumor tissues was significantly
higher than that in adjacent tissues (P<0.001).
Fig. 2 Immunohistochemical staining for galectin-1 protein in the
animal groups of normal control (Normal), O. viverrini infected alone
(OV infection), NDMA treated alone, and OV infected plus NDMA
treated (OV+NDMA) at 1, 2, 3, and 6 months postinfection. Panels A–
D: normal control group, showing weak staining in small bile ducts; E–
H: NDMA-treated alone group (NDMA), showing the positive staining
in proliferated small bile ducts; I–L: OV-infected along group (OV),
showing the positive staining in hyperplastic big bile ducts near to liver
fluke; M–P: OV-infected alone group (OV), showing the positive
staining in proliferated and hyperplastic small bile ducts; Q–T: OV-
infected plus NDMA-treated group (OV+NDMA), showing the posi-
tive staining in hyperplastic and dysplastic big bile ducts near to liver
fluke; and U–X: OV-infected plus NDMA-treated group (OV+NDMA),
showing the positive staining in hyperplastic and dysplastic small bile
ducts and tumor stroma tissues. P parasite; b normal bile duct; m
Tumor Biol. (2012) 33:1169–1178 1173
Expression of galectin-1 protein in human CCA
revealed by immunohistochemical staining
The galectin-1 protein was also detected in tumor specimens
of 78 CCA human cases. A weak expression was observed
in the small bile ducts in the adjacent normal tissue but no
positive immunostaining in the hepatocytes and blood ves-
sels (Fig. 4a). Forty-four cases (56.4%) showed extensive
and strong immunoreactivity in tumor tissues (≧3+), where
there were usually a lot of fibroblasts and some polymor-
phonuclear cell in the stroma of papillary (Fig. 4b–e) and
tubular types of CCA (Fig. 4f–i), but not in the inflamma-
tory cells. Cytochemically, those positive immunostaining
signals were located in both the nuclear and cytoplasm of
the fibroblast, while stronger staining was observed in the
nuclei, as shown in Fig. 4c, e, g, and i.
Correlation between galectin-1 expression and CCA
As mentioned above, galectin-1 expression at mRNA and
protein levels showed apparent differences among 78 CCA
parameters among the patients with high (≧3 folds at mRNA
and ≧3+ at protein levels) and low (<3 folds at mRNA level
and ≦2+ at protein level) expression, it was found that the
extent of galectin-1 expression, no matter at gene or protein
level, was correlated with CCA patient’s mortality, survival
13579 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77
Fig. 3 Fold change of galectin-1 mRNA expression in the tumor and
adjacent normal tissues of 78 human CCA. Total RNA was isolated
from the tumor and adjacent normal tissues. The expression level was
determined with quantitative real-time PCR, and was presented as copy
numbers within 106GUSB copies. The fold was calculated as copy
numbers in tumor tissue/copy numbers in adjacent normal tissue from
Fig. 4 Representative results of
for galectin-1 in the tumor and
adjacent tissues of 78 human
CCA. Panel A adjacent tissue,
weak staining in small bile duct;
B, C tubular type tumor tissue,
well differentiation without
metastasis; and C is high view
of the framed area in B. D, E
tubular type tumor tissues, poor
differentiation with metastasis,
and E is high view of the
framed area in D. F, G
papillary-type tumor tissue,
well differentiation without
metastasis, and G is high view
of the framed area in F. H, I
papillary-type tumor tissue,
poor differentiation with metas-
tasis, and I is high view of the
framed area in H
1174Tumor Biol. (2012) 33:1169–1178
rate, CCA pathological advancing stage, and its metastasis.
Statistical analyses showed that a mortality of the patients (by
the end of follow-up) with the high galectin-1 mRNA expres-
sion was significantly higher (42/53, 79.2%) than those with
Similarly, the mortality of the patients with high protein ex-
pression (34/45, 75.6%) was significantly higher than those
with low expression (17/33, 51.5%, P<0.027). Those patients
with high protein expression showed more advanced in stages
(III and IV) (32/45, 71.1%) than those with low expression
profile (15/33. 45.5%, P<0.022). The patients with high pro-
tein expression profile had higher metastasis rate (26/45,
57.8%) than those with low expression (11/33, 33.3%, P0
Univariate and multivariate analysis
Results regarding the clinicopathological variables are pre-
sented in Table 2. Univariate analysis indicated that metas-
tasis (with vs. without, P00.003) and histological grade
(well differentiated vs. moderate and poor differentiated,
P00.026) were significant prognostic factors. Overexpres-
sion of galectin-1 mRNA (≧3 fold vs. <3 fold, P00.031)
and protein (≧3+ vs. ≦2+, P00.015) were significantly
associated with survival. The Kaplan–Meier analysis also
showed poor survival rates in the patients with high galectin-
1 expression. The media survival time of the patients with
high mRNA expression was 0.73 year, which was significant-
ly shorter than those with low expression (1.77 years) (P0
0.031, Fig. 5). The survival time for the patients with high
protein expression profile (0.83 year) was shorter than those
with low expression (1.34 year, P00.015).
ard ratio 0.429, P00.004) and high expression of galectin-1
(hazard ratio, 2.202, P00.028) were found to be independent
prognostic factors for overall survival (Table 2).
galectin-1 during the tumorigenesis of O. viverrini infection-
induced CCA in animal model and human CCA cases and
confirmed a correlation between galectin-1 overexpression
and clinicopathology of CCA in human patients. The early
elevated expression of galectin-1 during tumorigenesis, and
the correlation of its overexpression with survival, tumor
stage, and metastasis indicate that this factor might be a
promising tumor stroma marker in diagnosis and prediction
ofmetastasis and poorprognosis ofopisthorchiasis-associated
CCA is a high lethal cancer because of the late appearance
of symptoms and lack of biomarkers for early diagnosis as
well as less option in treatment. Intensive studies are carrying
on because CCA is becoming a crucial public health problem
in the endemic area of O. viverrini due to its extremely high
prevalence. Therefore, searching novel tumor markers for the
early diagnosis is inevitably becoming a focus. Although it is
still a little conflicted, various efforts have been tried to test
galectin-1 as a potential target in some types of cancers
[21–24]. In the current study, we observed the kinetics of
galectin-1 expression during the tumorigenesis of O. viverrini
infection-associated CCA in model animal hamsters. The
early stage (1 month p.i. in OV-infected plus NDMA group)
during the period of our investigation. The immunohisto-
chemical staining with anti-galectin-1 antibody indicated that
the elevated expression came from the proliferation of epithe-
Table 2 Univariate and multivariate Cox regression analysis of prognostic factors associated with overall survival
VariableCategory Univariate analysisMutivariate analysis
P valueHazard ratio
(95% confidence interval)
galectin-1 protein expression
galectin-1 mRNA expression
Female vs. male
<60 vs. ≧60 years
I, II vs. III, IV
Tubular vs. papillary
With vs. without
Well vs. moderate and poor differentiation
≧3+ vs. ≦2+
≧3 fold vs. < 3 fold
Tumor Biol. (2012) 33:1169–1178 1175
lium, hyperplastic, and dysplastic small bile ducts, not from
the inflammatory filtration, which suggested that early
Although galectin-1 lacks recognizable secretion signal
sequence, it is well known that it is secreted and can be found
on the extracellular side of cell membranes as well as in the
extracellular matrices of neoplastic tissues [25, 26]. It is secret-
ed in a manner similar to fibroblast growth factor 2 via inside–
out transportation involving direct translocation across the
plasma membrane . Therefore, the elevated galectin-1 ex-
pression in the early stage of tumorigenesis of O. viverrini
infection-associated CCA and its extensive distribution in tu-
mor stroma tissues provide the possibility as a early diagnostic
marker for opisthorchiasis-associated CCA by serum detection
or tissue examination.
Galectin-1 overexpression was also confirmed in the
human CCA. Compared in the adjacent normal tissues, the
expression of galectin-1 in CCA tumor tissue was increased
more than 17-folds in average at mRNA level.
The expression pattern of galectin-1 in intrahepatic chol-
normal and CCA tissues as well as CCA cell line .
Reportedly, galectin-1 was not expressed in normal intrahe-
patic bile ducts, but stronglyexpressed intumor stroma and in
CCA cell line. The overexpression was related to neoplastic
progression and proliferative activities. Our present study
further confirmed the previous study. Based on the immuno-
histochemical analysis of the 78 CCA cases, strong positive
immunostaining mainly came from the cells in stroma and
was not or weakly detected in the normal bile ducts. Most of
staining was observed in the epithelium of well-differentiated
papillary and tubular CCA. Besides in the stroma, the positive
staining was observed in the proliferated and dysplastic small
bile ducts in the animal model. These observations also sup-
port that galectin-1 was expressed mainly in the epithelium of
proliferated, hyperplastic, and dysplastic small bile ducts in
the early stage of CCA and more extensively expressed in the
tumor stroma tissues with the CCA development. The results
indicate that galectin-1 staining patterns may be a signal of
tumor stages, which means galectin-1 may playdifferent roles
during the development of CCA.
Galectin-1 has multifunction in cell activities, including
cell growth, differentiation, migration, adhesion, motility,
and invasion . Reportedly, galectin-1 has different effects
on cell growth, depending on the dose involved [29, 30].
in fibroblasts controls the progression of fibrosis and cancer,
in which galectin-1 plays a critical role in stimulating the
transdifferentiation of fibroblasts to tumor-associated fibro-
blasts, and in promoting tumor progression and metastasis
bile ducts, proliferated and dysplastic bile ducts, and fibro-
blasts in tumor stroma tissues may play different roles in
normal cell and tumorigenesis process, and could be a signal
for tumor stage.
Fig. 5 Survival curves obtained by the analysis of the patients with
low and high mRNA expression revealed by real-time PCR (a) and low
and high expression of galectin-1 protein revealed by immunohisto-
chemical staining (b), with Kaplan–Meier method. a Survival curve
obtained from the analysis of mRNA expression showed that the
survival was significantly lower in the patients with high expression
(≧3 folds) than in the patients with low expression (<3 folds) (P0
0.031). b Survival curve obtained from the analysis of immunohisto-
chemical staining showed that the patients with high positive staining
(≧3+) showed significant low survival than those with low expression
1176Tumor Biol. (2012) 33:1169–1178
The significance of the expression of galectin-1 has been
evaluated in some other neoplasms, especially in human
epithelial tumors such as colorectal, thyroid, gastric, and
breast carcinoma. The studies have shown that the expression
pattern is related to the tumor progression, invasion, and
metastasis and some have shown that galectin-1 is a valuable
tumor marker for prediction of metastasis and poor prognosis
[16, 17, 23, 32–34]. In the present study, a significant corre-
lationwas found between mortality and galectin-1expression.
There were more death cases among the patients with high
mRNA expression (P<0.0001) and strong positive of immu-
nohistochemical staining(P00.027). TheKaplan–Meieranal-
mRNA expression (P00.031) and with strong immunohisto-
chemical staining (P00.015). High mRNA expression
showed no association with O. viverrini infection-associated
found between high protein expression and CCA tumor’s
stage and metastasis. The stronger the galectin-1 expression,
the more advanced the stage (stage III and VI) (P00.022) and
the more metastatic (P00.033). Multivariate Cox regression
analysis revealed that metastasis (P00.004) and high expres-
sion of galectin-1 (P00.028) were significant as independent
prognostic factors for overall survival. These results suggest
that galectin-1 is likely a promising biomarker for prognosis,
In many human tumors, the stromal microenvironment is
fundamentally different from that of normal tissues. The tu-
mor stroma, known as activated phenotype and characterized
by modified extracellular matrix, increased microvessel den-
sity, inflammatory cells, and tumor-associated fibroblasts, is
considered to play a central role in the complex process of
tumor–stroma interaction and consequently tumorigenesis
[35, 36]. Although there was no solid cancer found in O.
viverrini-infected alone animals during the period of our in-
vestigation, our immunohistochemical results indicated that
galectin-1 overexpressed in the proliferated small bile ducts
and the fibroblasts of hyperplastic intrahepatic bile ducts, but
not in the normal connective tissues, which suggested that the
a role in initialing bile ductal tumorigenesis. The chronic
inflammation by O. viverrini infection may contribute to the
tumorigenesis of CCA via promoting conversion of normal
fibroblasts to tumor-associated fibroblasts because the molec-
ular mechanismsin the stromal reaction of inflammation were
involve in the recruitment of cancer-associated fibroblasts in
which galectin-1 may involve .
Stroma is essential for the maintenance of epithelial tissues.
When the epithelium changes, the stroma inevitably changes
as well; this links with cancer progression and metastasis. It
important roles in the progression and metastasis in some
cancers. Galectin-1 secreted by activated stellate cells in
pancreatic ductal adenocarcinoma stroma promotes prolifera-
tion and invasion of pancreatic cancer cells . Suppression
of galectin-1 expression in carcinoma-associated fibroblasts
inhibits oral squamous cell carcinoma metastasis .
Galectin-1 expression in cancer-associated stromal cells corre-
lates invasion and progression in breast cancer . Our in-
vestigation revealed that galectin-1 wasincreasingly expressed
in the tumor stroma of the CCA animal models in a time-
dependent manner and the increase of galectin-1 expression
was related with tumor progression. Similar changes have also
the cell of tumor stroma was significantly correlated with the
tumor progression stages and metastasis. Clearly, these results
suggest that galectin-1 is likely involved in the tumorigenesis,
invasion, and metastasis of opisthorchiasis-associated CCA.
Galectin-1 overexpression inthe stroma canbe considered as a
sign of the malignant progression and consequently of a poor
prognosis of the CCA.
In conclusion, galectin-1 was overexpressed in the CCA of
significantly correlated with progression, stages, metastasis, and
survival, suggesting that galectin-1 likely involves in the tumor-
igenesis and is expected to serve as a reliable tumor stroma
and metastasis in the opisthorchiasis-associated CCA.
for Scientific Research (21590463) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan, and the Higher
Education Research Promotionand National Research University Project
of Thailand, Office of the Higher Education Commission, through the
Health Cluster (SHep-GMS), Khon Kaen University. The authors are
thankful to Dr Jianxin Sun, an epidemiologist at Connecticut Department
of Public Health, for his critical reading and editing of this manuscript.
This research was supported by the Grant-in-Aid
Conflicts of interest
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