Original Paper
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file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 349-355
doi:10.1111/j.1745-7270.2008.00392.x
High expression of osteoglycin
decreases the metastatic capability of mouse hepatocarcinoma Hca-F cells to
lymph nodes
Xiaonan Cui1, Bo
Song2, Li Hou2, Zhiyi Wei3, and Jianwu Tang2*
1 Department of Oncology, the First Affiliate
Hospital of Dalian Medical University, Dalian 116011, China
2 Department of narcotherapy, Dalian Medical
University, Dalian 116044, China
3 Department of Anesthesiology, The Second
Affiliate Hospital of Dalian Medical University, Dalian 116027, China
Received?February
14, 2008
Accepted?March
10, 2008
This
work was supported by a grant from the National Natural Science Foundation of
China (No. 30500586)
*Corresponding
author: Tel, 86-411-86110002; Fax, 86-411-86118866; E-mail, [email protected]
Osteoglycin,
one of the matrix molecules, belongs to the small leucine-rich proteoglycan
gene family and might play important roles in cell growth and differentiation
and in pathological processes such as fibrosis and cancer growth. In this
study, a eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was
constructed and transfected into mouse hepatocarcinoma Hca-F
cells to evaluate the contribution of osteoglycin to the malignant behavior of
Hca-F. It was found that Hca-F cells transfected with pIRESpuro3 osteoglycin(+)
showed significantly decreased potential for both migration and invasion.
Furthermore, Hca-F cells transfected with osteoglycin showed decreased
metastatic potential to peripheral lymph nodes. However, proliferation
potential and adhesive capacity of Hca-F cells to different protein substrates
were not influenced by osteoglycin transfection. In summary, high expression of
osteoglycin decreases the metastatic capability of Hca-F to lymph nodes.
Keywords osteoglycin;
transfection; hepatocellular carcinoma; neoplasm metastasis
Osteoglycin, one of the matrix molecules,
belongs to the small leucine-rich proteoglycan (SLRP) gene family characterized
by repeats of leucine-rich motifs (LRR) bounded by two cysteine clusters and a
conserved pattern of glycosylation [1]. Previous studies showed that many SLRPs
functioned as important components of the extracellular matrix. They served as
scaffolds for the cells of a given tissue and involved in collagen fibrillogenesis and cell adhesion [2,3]; as the cellular
macromolecular environment, they also interacted with collagens [4–6], growth
factors/growth factor receptors [7–11], cytokines, and signaling molecules [12–15] and played
roles in cell differentiation and proliferation and contributed to pathological
processes such as fibrosis and cancer growth [16,17]. Osteoglycin, one of the
SLRP gene family members, played a role in collagen fibrillogenesis, a process
essential in development, tissue repair, and metastasis, as illustrated by in
vivo studies using osteoglycin-deficient mice [18,19]. Studies in vitro
also suggested that osteoglycin might regulate cellular growth because: (1) the
level of osteoglycin mRNA was high in corneal keratocytes maintained in low
serum or serum-free media, but rapidly decreased if these cells were grown in
media containing serum [20]; (2) osteoglycin mRNA was absent or at low levels
in the majority of cancer cell lines and tumors [21]; (3) the tumor suppressor
protein p53 activated transcription of bovine and human osteoglycin genes,
however, osteoglycin expression was also absent in different cancer cell lines
and tumors where p53 was inactivated/mutated [21,22]; (4) growth factors, such
as basic fibroblast growth factor modulated osteoglycin mRNA expression in
corneal keratocytes and vascular smooth muscle cells [23,24]; and (5)
interferon-g regulated osteoglycin transcription in different cell types [25].However, there are few reports about the effects of osteoglycin on
tumor metastasis. Hca-P and Hca-F cells are gynogenetic mouse hepatocarcinoma
cell lines. When inoculated subcutaneously in 615 mice, they metastasized only
to the lymph nodes and not to other organs. Hca-P cells showed a low metastatic
potential (lymphatic metastasis rate<30%), whereas Hca-F cells showed a high metastatic potential (lymphatic metastasis rate>80%) [26,27]. In our
previous study, we used the suppressive subtracted hybridization technique to
identify differentially expressed genes between the two cell lines to obtain
candidate genes of metastasis, especially for lymphatic metastasis, and
osteoglycin was one of the genes that we obtained that low expression in Hca-F
cells and high expression in Hca-P cells. In this study, we transfected osteoglycin into Hca-F cells to evaluate
the contribution of osteoglycin to the malignant behavior of Hca-F with high
metastatic potential.
Materials and Methods
Cell lines, cell cultures, and
animals
Mouse hepatocarcinoma cell line Hca-F (established by Department of
Pathology, Dalian Medical University, Dalian, China) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, USA)
supplemented with antibiotics (100 U/ml penicillin/streptomycin; Gibco BRL) and
10% fetal bovine serum (FBS; Gibco BRL), and cultured in a humidified incubator
at 37 ?C with 5% CO2. Inbred 615 mice (8-week-old males) were
provided by the Animal Facility of Dalian Medical University.
Construction of targeting
vector
The osteoglycin coding sequence was amplified by polymerase chain reaction
(PCR). Briefly, total RNA from 1?107
Hca-F cells was isolated with Trizol (Invitrogen, Carlsbad, USA). A High
Fidelity PrimeScript RT-PCR kit (TaKaRa, Dalian, China) was used to synthesize
the cDNA according to the manufacturer’s protocol. PCR was carried out with
primer sets P1, 5‘– GAATTCATGGAGACTGTGCACTCTA-3‘ (forward),
and P2, 5‘– GCGGCCGCTTAGAAGTATGACCCTA-3‘ (reverse),
containing EcoRI and NotI sites, respectively (underlined), using
obtained cDNA as a template. PCR was carried out under the following
conditions: 30 cycles of denaturation for 10 s at 98 ?C, annealing for 15 s at
55 ?C, and extension for 60 s at 72 ?C. After digestion by EcoRI and NotI
enzymes, the PCR product was cloned into pIRESpuro3 vector digested by the same
enzymes and designated pIRESpuro osteoglycin(+). Sequence and orientation were
confirmed by DNA sequencing using a BigDye Terminator V3.1 cycle sequencing kit
(Applied Biosystems, Foster City, USA).
Cell transfection and
screening
Hca-F cells were incubated in antibiotic-free medium with 10% FBS,
then transferred to a 6-well tissue culture and incubated at 37 ?C in a CO2 incubator to obtain 60%80% confluence. Then cells were stably
transfected with pIRESpuro3 and pIRESpuro3 osteoglycin(+) (2 mg cDNA) by
TransIT-LT1 transfection reagent (TaKaRa) according to the
manufacturer’s protocol. The transfected Hca-F cells were selected by puromycin
(Clontech, San Jose, USA) for 2 weeks and maintained in medium containing 0.5 mg/ml puromycin.
Reverse transcription (RT)-PCR
analysis of osteoglycin mRNA
For RT-PCR analysis of osteoglycin mRNA levels, total RNA was
isolated from cells using Trizol and cDNA was synthesized with a High Fidelity
PrimeScript RT-PCR kit according to the manufacturer’s instructions. The
sequences of the primers were 5‘-TTCTCCTGCTACTCTTCGTG-3‘
(forward) and 5‘-AAGCAGACACACAACAGGCA-3‘ (reverse) for
osteoglycin, and 5‘-CGGGACCTGACAGACTACCT-3‘ (forward) and 5‘-AGCACTGTGTTGGCATAGAG-3‘
(reverse) for b-actin. PCR was carried out under the following conditions: 30
cycles of denaturation for 10 s at 98 ?C, annealing for 15 s at 55 ?C, and
extension for 30 s at 72 ?C. The amplified products were analyzed by agarose
gel electrophoresis using 1.6% gel with ethidium bromide staining. The bands
were analyzed with LabWorks (version 4.6; UVP BioImaging Systems, Cambridge,
UK).For RT-PCR analysis of osteoglycin mRNA levels, total RNA was
isolated from cells using Trizol and cDNA was synthesized with a High Fidelity
PrimeScript RT-PCR kit according to the manufacturer’s instructions. The
sequences of the primers were 5‘-TTCTCCTGCTACTCTTCGTG-3‘
(forward) and 5‘-AAGCAGACACACAACAGGCA-3‘ (reverse) for
osteoglycin, and 5‘-CGGGACCTGACAGACTACCT-3‘ (forward) and 5‘-AGCACTGTGTTGGCATAGAG-3‘
(reverse) for b-actin. PCR was carried out under the following conditions: 30
cycles of denaturation for 10 s at 98 ?C, annealing for 15 s at 55 ?C, and
extension for 30 s at 72 ?C. The amplified products were analyzed by agarose
gel electrophoresis using 1.6% gel with ethidium bromide staining. The bands
were analyzed with LabWorks (version 4.6; UVP BioImaging Systems, Cambridge,
UK).
Western blot analysis
Western blot analysis was carried out to evaluate osteoglycin
protein levels. Cellular protein was extracted from near-confluent 35 mm
culture dishes with lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 2 mM EGTA, 10% glycerol, 0.15% sodium dodecylsulfate, 1%
deoxycholate, 1% Triton X-100, and 1% anti-protease cocktail (Sigma, St. Louis,
USA)]. The extracted proteins (50 mg total protein) were subjected to 10% sodium
dodecylsulfate-polyacrylamide gel electrophoresis, blotted onto polyvinylidene
difluoride membranes (Invitrogen), then detected by an ECL Western blotting kit
(Amersham Biosciences, Little Chalfont, UK). Goat anti-mouse osteoglycin and b-actin
polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz,
USA). The bands were analyzed with LabWorks [28].
The
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
MTT assay was carried out as reported previously [29]. Approximately
1?106 cells in 200 ml DMEM were
seeded in duplicate into each well of the 96-well culture plates, and 100 ml MTT (5 mg/ml;
Sigma) was added at 24, 48, 72, 96, 120, and 144 h. After 4 h incubation at 37
?C with 5% CO2, 100 ml dimethylsulfoxide (Gibco)
was added to dissolve the formazan product for 30 min at room temperature. The
absorbency A570 was measured using a microplate reader
(Bio-Rad, Hercules, USA). Growth rate (%)=A570 of
transfectants/A570 of Hca-F?100%.
Migration assay
A Boyden chamber assay was carried out according to the
manufacturer’s protocol with minor modifications. Sterile 8 mm polyethylene
terephthalate filters (BD Pharmingen, San Diego, USA) were coated with gelatin (Sigma).
The filters were hydrated with 150 ml serum-free medium at room temperature for
120 min before use. The lower chambers of a 24-well plate were filled with 0.75
ml DMEM with 10% FBS and 0.5 ml serum-free DMEM containing 4?105 tumor cells was added to
the gelatin-coated trans-well chambers and incubated at 37 ?C in a 5% CO2 humidified atmosphere for 21 h. At the end of incubation, the cells
on the upper surface of the filter were completely removed gently with a cotton
swab. Then the filters were fixed in methanol and stained with Wright-Giemsa.
Cells on the lower surface of the filter were counted under a light microscope
with a magnification of 400 and photographed. Triplicate samples were acquired
and the data were expressed as the average cell number of five fields.
Migratory cells were calculated and analyzed with Image-Pro Plus 4.5 software
(Media Cybernetics, Silver Spring, USA).
Invasion assay
Cell invasion was measured with a Boyden chamber assay with minor modifications.
Matrigel-coated and sterile polyethylene terephthalate filters (8 mm) were
rehydrated with serum free medium at room temperature for 120 min. The lower
chambers of the 24-well plate were filled with 0.75 ml DMEM containing 10 mg/ml fibronectin
(BD Pharmingen) as a chemoattractant, and 0.5 ml serum-free DMEM containing 4?105 tumor cells was added to
the trans-well chambers and incubated at 37 ?C in a 5% CO2 humidified atmosphere for 46 h. Further staining and result
recording were carried out as in the migration assay.
Cell adhesion assay
The 96-well plates were coated by Fibronectin (Sigma), laminin
(Sigma) or collagen I (Upstate Biotechnology, Lake Placid, USA) according to
the manufacturer’s protocol with minor modifications. Suspended cells (3?104) were added to each well
for 60 min incubation at 37 ?C (120 min for collagen I), and unattached cells
were removed by washing twice with PBS. Cells were then fixed with 2%
paraformaldehyde in PBS for 10 min at room temperature. After washing the wells
with PBS, the cells were stained with crystal violet (0.1%) for 20 min at room
temperature. Wells were then washed three times with PBS and the cells lysed in
PBS containing 1% Triton X-100 and then well absorbance was read at A595.
In vivo tumor metastasis assay
A total of 90 inbred 615 mice were randomly divided into three
groups. Each mouse was inoculated subcutaneously with 0.1 ml cell suspension
(approximately 2?106 cells) of Hca-F cells, Hca-F cells transfected with pIRESpuro3, or
Hca-F cells transfected with pIRESpuro3 osteoglycin(+). At 28 d
post-inoculation, the implanted tumor and axillary and inguinal lymph nodes
were stained with hematoxylin-eosin and examined under a microscope by paraffin
sections. Mice with at least one metastatic axillary lymph node or one
metastatic inguinal lymph node were considered metastatic. The lymph node
metastatic rates in each group were calculated by the following equation: Lymph
node metastatic rate=metastatic mice/total mice.
Statistical analysis
Data are presented as the mean±SD and were analyzed by Student’s t-test,
ANOVA, and c2-test using spss
version 11.0 (SPSS, Chicago, USA). P<0.05 was considered statistically significant.
Results
Osteoglycin highly expressed
at mRNA and protein levels in Hca-F cells transfected with osteoglycin
The relative mRNA and protein levels of osteoglycin were determined
by RT-PCR and Western blot analysis, respectively (Fig. 1). Hca-F cells
transfected with pIRESpuro3 osteoglycin(+) showed significant high expression
of both mRNA and protein levels of osteoglycin compared to Hca-F cells and
Hca-F cells transfected with pIRESpuro3.
Effect of osteoglycin on
proliferative ability of Hca-F cells in vitro
No significant difference was seen in the growth rate of Hca-F
cells, Hca-F cells transfected with pIRESpuro3, and Hca-F cells transfected
with pIRESpuro3 osteoglycin(+), indicating osteoglycin could not influence the
proliferative ability of Hca-F cells in vitro (Fig. 2).
High expression of osteoglycin
decreased migration potential of Hca-F cells
A Boyden chamber assay was used to investigate whether osteoglycin
expression affected tumor cell migration. Results showed that the number of
Hca-F cells transfected with pIRESpuro3 osteoglycin(+) (456) migrated through
the filter was much lower than that of Hca-F cells (905; P<0.01), or Hca-F cells transfected with pIRESpuro3 (927; P<0.01) (Fig. 3),
indicating that high expression of osteoglycin decreased the migration
potential of Hca-F cells.
High expression of osteoglycin
decreased invasive potential of Hca-F cells
Relative invasiveness of tumor cells was observed with a Boyden
chamber assay with Matrigel-coated filters. As a result, the number of Hca-F cells
transfected with pIRESpuro3 osteoglycin(+) (257) that invaded through the
filter was much lower than that of Hca-F cells (6510; P<0.01) and Hca-F cells transfected with pIRESpuro3 (705; P<0.01) (Fig. 4),
indicating that high expression of osteoglycin decreased the invasive potential
of Hca-F cells.
Effect of osteoglycin on
adhesive capacity of Hca-F cells
There was no significant difference in Hca-F cells, Hca-F cells
transfected with pIRESpuro3, or Hca-F cells transfected with pIRESpuro3
osteoglycin(+) in their adhesive capability to fibronectin, laminin, or
collagen over a 1–2 h period (Fig. 5), indicating cell adhesion was not a
factor accounting for the major differences associated with high expression of
osteoglycin expression in cell migration and invasion.
Effect of osteoglycin on in
vivo tumor metastasis potential
Implanted
tumor of Hca-F cells, Hca-F cells transfected with pIRESpuro3, and Hca-F cells
transfected with pIRESpuro3 osteoglycin(+) in tumor-burdened mice were palpable
at 7 d post-inoculation. At 28 d post-inoculation, 53.3% (16/30) of
tumor-burdened mice inoculated with Hca-F cells transfected with pIRESpuro3
osteoglycin(+) developed metastatic axillary lymph nodes or/and inguinal lymph
nodes. In contrast, 80% (24/30; P<0.05) of tumor-burdened mice inoculated with Hca-F cells, and 83.3% (25/30; P<0.05) of tumor-burdened mice inoculated with Hca-F cells transfected with pIRESpuro3 developed metastatic axillary lymph nodes and/or inguinal lymph nodes. Hca-F cells with osteoglycin transfection showed a significant decrease in metastastic potential to lymph node (Fig. 6).
Discussion
Tumor metastasis is the major cause of tumor-related death [30], and
identification of metastasis-related genes might help to reveal its molecular
mechanisms [31]. Hca-P and Hca-F cells are gynogenetic mouse
hepatocarcinoma cell lines presenting a specific potential for lymphatic
metastasis, with a significant differential in their potential of metastasis
[26,27]. In our previous study, we used the suppressive subtracted
hybridization technique to identify differentially expressed genes between
Hca-P and Hca-F cells. Osteoglycin was one of the differentially expressed genes
we obtained that low expression in Hca-F cells and high expression in Hca-P
cells. Together with reports that: (1) osteoglycin is likely to play roles in
cellular growth, for example, growth factor and cytokine modulation of mRNA
expression of osteoglycin in corneal keratocytes and vascular smooth muscle
cells [23,24]; (2) the tumor suppressor protein p53 activated transcription of
osteoglycin gene in bovine and human; and (3) osteoglycin was absent in the majority of cancer cell lines and tumors [21,22], we
hypothesized that osteoglycin would likely contribute to suppression of tumor
metastasis. To test this hypothesis, we transfected the osteoglycin gene into
Hca-F cells to evaluate the contribution of osteoglycin to the malignant
behavior of Hca-F cells with high metastatic potential. As a result, in
vitro, Hca-F cells transfected with pIRESpuro3 osteoglycin(+) produced
effective, stable, high expression of osteoglycin at mRNA and protein levels
and, consequently, attenuation in both migration and invasion associated with
high expression of osteoglycin was found in Hca-F cells transfected with
pIRESpuro3 osteoglycin(+). Cellular adhesion to extracellular matrices was an
important determination for cell migration and invasion [32]. In our study, the
adhesive capability of Hca-F cells to fibronectin, laminin, or collagen was not
influenced by osteoglycin transfection, indicating that cell adhesion was not a
factor accounting for the major difference associated with high expression of
osteoglycin in cell migration and invasion. No proliferative difference was
shown among Hca-F cells, Hca-F cells transfected with pIRESpuro3, or Hca-F
cells transfected with pIRESpuro3 osteoglycin(+), suggesting that osteoglycin
was not a factor in cell proliferation. Furthermore, in vivo, Hca-F
cells transfected with pIRESpuro3 osteoglycin(+) showed decreased metastatic
potential to peripheral lymph nodes when inoculated subcutaneously in 615 mice.
The data provided compelling evidence as to the function of osteoglycin and
suggested that osteoglycin acted as a tumor lymphatic metastasis-associated
gene.
In vitro
observations showed that osteoglycin is likely to play roles in cellular
growth, however, little research has been carried out into the role of
osteoglycin in metastasis. Our study showed that osteoglycin inhibited tumor
metastasis in multiple processes such as migration and invasion. The mechanism
of osteoglycin in regulating metastasis was elusive. Our study showed that cell
adhesion was not likely a major factor of the contribution of osteoglycin to
cell migration and invasion. Reports that p53, basic fibroblast growth factor
and interferon-g interact with osteoglycin indicated that osteoglycin might inhibit
tumor metastasis in multiple processes [21–25]. Further studies are
needed to identify the mechanisms by which osteoglycin contributes to lymphatic
metastasis.
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