ABBS 2005,38(03): Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-b1

Abstract        Transforming growth factor-b (TGF-b), a
multifunctional cytokine, exerts contradictory roles in different kinds of
cells. A number of studies have revealed its involvement in the progression of
many types of tumors. To investigate the effect of TGF-b on gastric carcinoma, SGC7901, BGC823 and MKN28 (a TGF-b-resistant cell line) adenocarcinoma clones were
used. After pretreatment in serum-free medium with or without 10 ng/ml TGF-b1, their experimental metastatic potential,
chemotaxis, and invasive and adhesive ability were measured. Furthermore,
zymography for gelatinase was processed. Liver colonies were also measured 4
weeks after inoculation of SGC7901, BGC823 and MKN 

Key words        gastric cancer; transforming growth
factor-b; invasion;
metastasis

Transforming growth
factor-b (TGF-b), a family of 25 kDa homodimeric multifunctional
regulatory peptides, is involved in various processes, including development,
wound healing, and carcinogenesis [1]. Of the three isoforms of human TGF-b, TGF-b1 is the
predominant form [2,3]. In vitro, TGF-b1 potently inhibits proliferations of nearly all
epithelia and promotes adhesion by enhancing matrix production and decreasing
proteolysis [1]. In addition, TGF-b1 is also
involved in the events of apoptosis, replicative senescence, genomic stability,
a negative angiogenic regulator profile and cellular immortalization [4,5].
Resistances to the negative growth-regulating properties of TGF-b1 have been observed in epithelial and mesenchymal
tumors [6]. Tumor cell lines that lack TGF-b receptors lost responsiveness to TGF-b, and the escape of cells from TGF-b-mediated negative regulation was linked to tumor
progression [7,8]. Furthermore, TGF-b1 has also
been found to suppress the invasive potential of many types of cancer cell
lines in vitro, such as renal cell carcinomas, fibrosarcoma and thyroid
cancer cell lines [9–12]. All
these indicated that TGF-b1 might serve
as a tumor suppressor.

But interestingly, the
more aggressive forms of tumors are growth-stimulated by TGF-b [13]. In breast cancer, expression of TGF-b1 was positively associated with invasion and
metastasis [14,15]. Huang et al. reported that transfection of a TGF-b1 antisense expression plasmid decreased the
tumorigenicity of colon cancer cells [3]. These observations suggested that
TGF-b1 synthesized by tumor
cells might facilitate tumorigenesis and hence regulate tumor cell behavior.

The mortality of gastric
cancer patients is quite high in  

Materials and Methods

Cell lines and tissue
culture

Gastric cancer cell
lines SGC7901 and BGC823 were purchased from Type Culture Collection of the  

Expressions of TGF-b1 and TGF-b type II
serine/threonine (Ser/Thr) receptor in untreated SGC7901, BGC823 and MKN28
cells

Expression of TGF-b1 and
TGF-b type II Ser/Thr
receptor in untreated SGC7901, BGC823 and MKN28 cells was measured by Western
blotting. Briefly, total proteins were measured using a BCA protein assay kit
(Pierce,  

Treatment with TGF-b1

Subconfluent cultures
were seeded and, at the desired confluence (80%), the medium was removed. The
cells were washed with pre-warmed D-Hanks solution and the medium was replaced
with serum-free RPMI 1640 with or without 10 ng/ml of TGF-b1. The cells were cultured for 24 h before being
used in other assays [19].

Experimental metastasis

Forty-eight Balb/c nude mice
aged 4–6 weeks were
purchased from Shanghai Laboratory Animal Company ( 

In vitro invasion
assay

The Transwell chamber ( 

Chemotaxis assay

Chemotaxis assay was
performed with Transwell chambers containing 8 mM pores [24]. Eight-hundred microliters of RPMI 1640
containing 10 mg/ml
fibronectin was placed in the lower compartment of the Transwell chamber. Tumor
cells (1´10⁵ per well)
pretreated with or without TGF-b1 were added into the
upper compartment and incubated for 6 h, and then the migrated cells were
counted as described above.

Assay for adhesion of
SGC7901, BGC823 and MKN28 cells to HUVEC monolayer

Adhesion of SGC7901,
BGC823 and MKN28 cells to the HUVEC monolayer were assayed according to the
method described in previous reports [23,25] with some modification. Cells
treated with or without TGF-b1 for 24 h were
detached from culture dishes and suspended in RPMI 1640 containing 1% calf
serum. When HUVEC seeded on 96-well plates reached confluency, the medium was
replaced with previous cancer cell suspensions. The mixture was incubated at 37
ºC for 30 min, then fixed with 4% paraformaldehyde and stained with low keratin
in an immunocytochemical way. Adhesion was evaluated by the number of labeled
cells per field (magnification, 400´).

Adhesion to tissue
culture plates coated with Matrigel or fibronectin

Each well of the 96-well
tissue culture plate was coated with 50 ml of Matrigel (1:20 dilution), 10 mg/ml bovine serum albumin or 10 mg/ml fibronectin and left to air-dry in a
hood overnight. Before use, the plate was incubated with 50 ml of serum-free RPMI 1640 containing 10 mg/ml
bovine serum albumin per well for 1 h. One-hundred microliters of suspended
tumor cells pretreated with or without TGF-b1 were
seeded into each well and incubated in a CO₂ incubator.
After incubation for 1 h, suspended cells that had not adhered to the coated
plate were washed away, and the amounts of adhered cells were evaluated with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay
[23].

Zymography for
gelatinase

Zymographic analysis of
gelatinase activity in secreted medium was performed in 10% sodium dodecyl
sulphate-polyacrylamide gels containing 1 mg/ml gelatin, as described in
previous reports with some modification [20,26]. Briefly, cells were
subcultured in 50 ml tissue culture dishes. The next day, the cells were washed
extensively and cultured in serum-free RPMI 1640 with or without 10 ng/ml TGF-b1. After incubating for 24 h, supernatant was
collected and used for electrophoresis. The gels were incubated for 60 min in  

Statistical analysis

Results were expressed
as mean±SD. Student’s t-test was used for statistical analysis. In all
statistical comparisons, P<0.05 was used to indicate a statistically significant difference.

Results

Expression of TGF-b1 and TGF-b type II Ser/Thr
receptor in untreated SGC7901, BGC823 and MKN28 cells

Western blotting
analysis showed no definite basal expression of active TGF-b 

Effect of TGF-b1 on liver
metastasis

TGF-b1 (10 ng/ml) pretreatment increased liver
metastases in Balb/c nude mice in SGC7901 and BGC823 groups compared with the
untreated control (liver metastases numbers: 11.0±3.0 vs. 53.3± 

Effect of TGF-b1 on gastric
cancer cell invasion across reconstituted basement membrane

Matrigel is a kind of basement
membrane matrix extract, which is rich in laminin, type IV collagen, and
heparan sulfate proteoglycans. Its structural and functional characteristics
are similar to those of basement membranes in vivo, therefore it
constitutes a valuable in vitro basis for assessing the invasive
potential of tumor cells. Only cells that can digest the Matrigel matrix are
capable of moving across this artificial basement membrane. To investigate the
potential of TGF-b 

Effects of TGF-b1 on adhesion
to Matrigel and fibronectin

Pretreatment with TGF-b1 promoted the adhesiveness to Matrigel and
fibronectin significantly in SGC7901 and BGC823 groups, but it had no effect on
adhesion of MKN28 cells to the matrix (Table 1).

Effect of TGF-b1 on adhesion
of cancer cells to HUVEC

SGC7901 and BGC823 cells
pretreated with TGF-b1 attached to
the HUVEC monolayer at significantly higher rates than the untreated control
(15.97±1.38 vs. 6.57± 

Effect of TGF-b1 on
chemotaxis of gastric cancer cells

The motility of tumor
cells is an essential property to invade the endothelium or extracellular
matrix. Compared with untreated SGC7901 and BGC823 cells, TGF-b1-treated SGC7901 and BGC823 cells showed higher
chemotactic activity to fibronectin (78±8 vs. 35± 

Zymography for
gelatinase

To further elucidate the
phenotypic difference between treated and untreated cells, zymographic analysis
was used to assess whether their invasive nature correlated with their
gelatinase activity. Activity of 72 kDa and 92 kDa gelatinase was observed in
all cells. Compared with the untreated control, the activity of 72 kDa and 92
kDa gelatinase increased in treated SGC7901 and BGC823 groups (P<0.05). However, there were no differences between MKN28 groups (Table 2 and Fig.
9).

Discussion

Invasion and metastasis
are the major obstacles in the treatment of malignancy, and are the major
causes of death. Investigations showed that TGF-b participated in invasion and metastasis by modulating the
ability of cells in invading the extracellular matrix and basement membrane
[27]. For example, TGF-b could promote
the invasion and metastasis of mammary tumors [28]. But TGF-b was also found to suppress the invasive potential
of many kinds of cancer cell lines in vitro [9–12]. Recent studies indicated that TGF-b upregulates the expression of the
cyclin-dependent kinase inhibitors p15 and p21 and inhibits cell proliferation
by causing cell cycle arrest at the G1 phase [29]. So the exact role of TGF-b in tumor cells is still unclear. It seemed that
TGF-b might exert different,
even contradictory biological effects on different tumors. In this study we
focused on what kind of role TGF-b may exert
during the progression of gastric cancer, suppressor or accelerator?

SGC7901 and BGC823 are
sensitive to TGF-b [30,31];
however, MKN28 is a TGF-b-resistant
gastric carcinoma cell line. It is further confirmed by our data that liver metastatic
and invasive capacities of SGC7901 and BGC823 were enhanced by pretreatment with
TGF-b1, and the
metastatic and invasive potential of MKN28 did not alter after pretreatment
with TGF-b1. It is well
known that TGF-b initiates
its signal through binding the TGF-b type II
Ser/Thr receptor kinase, which in turn phosphorylates the type I Ser/Thr
receptor, forming an activated receptor complex. Then the activated receptor
complex transmits the TGF-b signal to a
set of intracellular mediators, known as the Smad proteins. Based on these
facts, we speculated that the TGF-b signal
pathway might be intact in SGC7901 and BGC823, which was further confirmed by
our finding that there was high-level expression of the TGF-b type II Ser/Thr receptor in SGC7901 and BGC823.
However, expression of the TGF-b type II
Ser/Thr receptor was also detected in MKN28. So the resistance of MKN28 to TGF-b might be due to other mechanisms such as low
binding activity of the type I receptor to TGF-b [18], mutation of RUN-X3 [32] and so on, which are
crucial in the transmission of the TGF-b signal.

Blood-borne tumor cells
must attach to the endothelium and matrix to establish metastatic colonies in a distant organ, therefore
the interaction of the tumor cells with the endothelium and matrix is very
important. The adhesion assay in vitro using the endothelial cell
monolayer revealed that TGF-b1 increased
the adherent capacity of SGC7901 and BGC823. We speculated that the augmented
adhesion to the endothelium monolayer by TGF-b might be due in part to increased expression of CD44H
and b1 integrin [21].
Furthermore, our data showed that pretreatment with TGF-b1 significantly promoted the adhesiveness of
SGC7901 and BGC823 cells to Matrigel and fibronectin.

It is well known that a
defect of the basement membrane of the epithelium is the signal of invasion and
that tumor cells invading the extracellular matrix rely on three sequential
biological events [33,34]: attachment to components of the matrix; production
of matrix degradative enzymes; and tumor cell locomotion into the region of
proteolyzed matrix. In this study, we focused on the interaction between these
three cell lines and the matrix in vitro and analyzed mechanisms of TGF-b1 on metastasis of SGC7901, BGC823 and MKN28
cells.

Degradation of the
matrix is the key point of invasion. During this process, matrix
metalloproteinases exert critical roles. Overexpression of gelatinase A
significantly correlated with the invasion of esophageal carcinoma, metastasis
of esophageal carcinoma and gastric cancer. Zymography indicated that activity
of gelatinase A positively correlated with the grading of breast carcinoma and
local invasion of gastric cancer. Compared with non-metastasis cases, pulmonary
carcinoma with nodal metastasis displayed higher gelatinase A activity [35].
The expression and activity of gelatinase B also positively correlated with the
grading of breast carcinoma [36]. In our study, higher levels of MMP-2 and
MMP-9 activity were observed in SGC7901 and BGC823 cells after pretreatment
with TGF-b1, indicating
TGF-b1 facilitates MMP
production. Extracellular matrix degradation of similar results have been
reported by Welch et al. [19].

Migration of tumor cells
is the rate-limiting step of metastasis. Generally, migration ability
positively correlated with metastasis potential. Yoshinaga et al. found
that the migratory ability of metastatic melanoma cells in lamin and collagen
was much greater than that of primary melanoma cells [37]. With their
chemotaxis and haptotaxis to stimulus, tumor cells metastasize to remote sites.
Studies showed that many kinds of substances, such as growth factors, could
stimulate the migration of tumor cells. Our research revealed that TGF-b1 could enhance the migration of both kinds of
TGF-b-sensitive gastric
cancer cells.

In this study, we found
that TGF-b1 was involved
in gastric cancer progression and metastases, in spite of the fact that TGF-b signaling is impaired in most gastric cancers
[38]. Recently, Santibanez et al. and Kim et al. found that TGF-b could mediate the expression of MMP-2 and MMP-9
through the mitogen-activated protein kinase signaling pathway [39,40].
Therefore the cross-talk between TGF-b-signaling
and other signaling pathways might be the mechanism responsible for TGF-b1-involved gastric cancer progression and
metastases. However, to clarify the issue, further research is needed.

In conclusion, our study
postulated that TGF-b might be
regarded as one of the factors that enhance malignant progression of gastric
cancer cell lines. In clinical gastric cancer cases, TGF-b-positive tumor cells seemed to express more
malignant phenotypes, for example, serosal invasion, infiltrative growth and
lymph node metastasis were more prominent in TGF-b1-positive cases [16]. So it is possible for TGF-b to serve as a potential target for gene therapy in
TGF-b positive gastric cancer
and blockage of TGF-b or TGF-b signaling might prevent gastric cancer cells from
invading and metastasizing. More evidence is needed to confirm this
postulation.

Acknowledgements

We thank Prof. Wei-Jun
CAI for generous assistance in preparing the manuscript.

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