Oncogene (2007) 26, 1468–1476
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ORIGINAL ARTICLE
Interleukin-18 is a critical factor for vascular endothelial growth factorenhanced migration in human gastric cancer cell lines
K-E Kim1,8, H Song2,8, TS Kim3, D Yoon4, C-w Kim5, SI Bang6, DY Hur2, H Park7 and D-H Cho1
1
Department of Life Science, Sookmyung Women’s University, Yongsan-ku, Seoul, Korea; 2Department of Anatomy, Inje University
College of Medicine, Pusan, Korea; 3School of Life Science and Technology, Korea University, Seoul, Korea; 4Division of Bioscience
and Biotechnology, Konkuk University Graduate School, Seoul, Korea; 5Department of Pathology and Tumor Immunity Medical
Research Center, Seoul National University, College of Medicine, Seoul, Korea; 6Department of Plastic Surgery, Samsung Medical
Center, Sungkyunkwan University School of Medicine, Seoul, Korea and 7Department of Dermatology, St Mary’s Hospital, College
of Medicine, The Catholic University of Korea, Seoul, Korea
Cell migration and angiogenesis are key steps in tumor
metastasis. However, the mechanism of migration regulated by vascular endothelial growth factor (VEGF), a
potent regulator of angiogenesis, is not completely understood. This study examined the relationship between
VEGF and migration, along with the mechanism involved
in the VEGF-regulated migration of human gastric cancer
cells. The level of cell migration was increased by
recombinant human (rh)VEGF-165 in the VEGF receptor-2-expressing SNU-601 cells. Interleukin (IL)-18 is
associated with the malignant progression of tumors.
Accordingly, this study examined the effect of IL-18 on
the migration of cancer cells in order to identify the
factors involved in VEGF-enhanced migration. Inhibiting
IL-18 markedly reduced the level of VEGF-enhanced
migration, and IL-18 increased cell migration directly
through filamentous-actin polymerization and tensin
downregulation. It was confirmed that rhVEGF-165
increased IL-18 production significantly. An antioxidant
and an extracellular signal-regulated kinase (ERK)1/2specific inhibitor blocked rhVEGF-165-enhanced IL-18
production. Accordingly, rhVEGF-165 increased the
generation of region of interest (ROI) and activated the
ERK1/2 pathway. These results suggest that rhVEGF165 enhances IL-18 production via the generation of ROI
and ERK1/2 phosphorylation, which results in the
increased migration of gastric cancer cells.
Oncogene (2007) 26, 1468–1476. doi:10.1038/sj.onc.1209926;
published online 25 September 2006
Keywords: ERK1/2; F-actin; IL-18; migration; tensin;
VEGF
Correspondence: Dr H Park, Department of Dermatology, St Mary’s
Hospital, College of Medicine, The Catholic University of Korea,
Seoul, Korea. E-mail: [email protected] or Dr D-H Cho,
Department of Life Science, Sookmyung Women’s University,
Chungpa-Dong 2-Ka, Yongsan-ku, Seoul 140-742, Korea. E-mail:
[email protected]
8
These authors contributed equally to this task.
Received 29 May 2006; revised 12 July 2006; accepted 14 July 2006;
published online 25 September 2006
Introduction
Malignant tumors, such as melanoma, breast cancer and
gastric cancer, metastasize. This process promotes
tumor progression by allowing malignant tumor cells
to invade the blood or lymphatic vessels, and migrate
to other tissues or organs. A metastasis is generally
associated with a poor prognosis in tumor patients.
Angiogenesis is a key step in metastatic process.
Solid tumors cannot grow to more than 1–3 mm3
without requiring an independent supply of essential
nutrients and oxygen. During tumor growth, the tumor
cells are under hypoxic conditions, which activate the
hypoxia-inducible factor that increases the expression
of various angiogenic factors. These factors are involved
in increasing the angiogenic activity and disease
progression in many carcinomas. Tumor cells can
metastasize after new blood vessels form around a
tumor. This study investigated the angiogenic factors
in tumor cells focusing mainly on vascular endothelial
growth factor (VEGF), which is a major factor
mediating the survival, migration and proliferation
of endothelial cells during angiogenesis (Carmeliet
and Jain, 2000). An increase in VEGF expression is
associated with increased tumor growth and the metastatic spread of solid malignancies (Takahashi et al.,
1995). In humans, VEGF exists in six isoforms through
alternative splicing, such as VEGF-121, VEGF-145,
VEGF-165, VEGF-183, VEGF-189 and VEGF-206.
Among these VEGF isoforms, VEGF-121 and VEGF165 are the main isoforms. It has been reported that
VEGF exerts its cellular functions by interacting
with VEGF receptors, which are found in many gastric
cancer cell lines (Zhang et al., 2002). Interleukin-18
(IL-18) is a proinflammatory cytokine and is considered
to be a member of the IL-1 cytokine family on account
of it possessing a structure similar to IL-1. IL-18 is
converted from a 24 kDa inactive form, which does not
contain any signal peptides, to an 18 kDa bioactive form
as a result of cleavage by an IL-1b-converting enzyme.
The 18 kDa active form of IL-18 is then secreted. This
active IL-18 stimulates interferon (IFN)-g production
directly, which is known as the IFN-g-inducing factor.
Effect of IL-18 on VEGF-enhanced migration
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IL-18 is produced by macrophages, keratinocytes,
dendritic cells as well as by tumor cells (Okamura
et al., 1995; Stoll et al., 1997; Udagawa et al., 1997).
It has been reported that IL-18 is involved in the
immune escape of tumor cells, such as melanoma
cells (Cho et al., 2000). In addition, IL-18 increases
tumor cell proliferation in gastric cancer, and plays a
prometastatic role in human and murine melanoma
cell lines (Carrascal et al., 2003; Majima et al., 2006).
The ability of cells to migrate is essential during the
metastatic process. Cell migration plays an important
role in the inflammatory immune response and tumor
formation (Horwitz and Parsons, 1999). It was demonstrated that VEGF regulates the migration of metastatic
prostate cancer cells through VEGF receptor-2 (Chen
et al., 2004), and IL-18 induces the migration of
Langerhans cells and natural killer (NK) cells (Cumberbatch et al., 2001; Ishida et al., 2004). Although both
angiogenic factors, particularly VEGF and IL-18, are
related to the metastatic potential of some tumor cells,
the relationship between VEGF and IL-18 and with
the effect of IL-18 on enhancing tumor cell migration
in gastric cancer are not completely understood.
The results show that VEGF increases the migration
of human gastric cancer cells through VEGF-enhanced
IL-18 production.
Results
IL-18 is a critical mediator of VEGF-enhanced migration
The expression of VEGFR-2 and the effect of rhVEGF165 on the migration were examined to determine if
rhVEGF-165 increases the level of SNU-601 cell
migration. Figure 1a shows that VEGFR-2 was strongly
expressed on the SNU-601 cells. A dose titration of a
specific VEGFR-2 inhibitor, SU5416, was performed in
order to determine if rhVEGF-165 has specific functions
via VEGFR-2. As shown in Figure 1b and c, when
50 ng/ml of rhVEGF-165 was treated for 48 h, the
migration of SNU-601 cells was decreased, and the
spontaneous cell migration was decreased significantly
by SU5416 in a dose-dependent manner within 24 h. In
addition, SNU-601 cells produced VEGF-165 spontaneously (Figure 1d). It was hypothesized that IL-18 is an
important mediator in VEGF-enhanced migration
because it was reported that VEGF and IL-18 are
related to the metastatic potential of many malignant
cancers (Takahashi et al., 1995; Carrascal et al., 2003).
As a first step to study the involvement of IL-18 in
VEGF-enhanced migration, we investigated the effect
of a specific inhibitor of IL-18, IL-18-binding protein
(IL-18BP). Figure 2a and b show that VEGF-enhanced
migration was reduced significantly by IL-18BP. In
Figure 1 rhVEGF-165 increases the migration of VEGFR-2 expressing-SNU-601 cells. (a) SNU-601 cells were stained with mouse
anti-human VEGFR-2 antibody, and further stained with FITC-conjugated goat anti-mouse IgG antibody (bold line). The control was
not stained with any antibody (thin line). Mouse IgG was stained with only FITC-conjugated goat anti-mouse IgG antibody (dot line).
The cells were analysed by indirect FACS. A representative experiment of three experiments performed independently is shown.
(b) SNU-601 cells were treated with 50 ng/ml of rhVEGF-165 for 48 h. A migration assay was performed after incubation. (c) SNU-601
cells were treated with 0, 2.5, 5 and 10 mM of SU5416, a specific VEGFR-2 inhibitor, for 24 h. A representative experiment of three
experiments performed independently is shown; bars, mean7s.e. *Po0.05 versus control. (d) SNU-601 cultured supernatants were
collected, and the spontaneous VEGF-165 concentration was determined by ELISA.
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Effect of IL-18 on VEGF-enhanced migration
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Figure 2 VEGF-enhanced migration of SNU-601 cells is markedly reduced by IL-18 blockade. SNU-601 cells were pretreated with
500 ng/ml of IL-18BP. After incubation for 1 h, the cells were treated with 50 ng/ml of rhVEGF for 48 h. A migration assay was
performed. (a) Image of the migrated cells. (b) Relative migrated cells (%). (c) SNU-601 cells were transfected with siRNA targeting
IL-18 or negative sequence. After cell stabilization, the cultured supernatant and cells were collected. IL-18 siRNA transfection was
confirmed using IL-18 ELISA (white bars). The relative percentage of migrating cells is shown (black bars). (d) Control and transfected
cells were incubated with (black bars) or without (white bars) 50 ng/ml of rhVEGF-165 for 48 h. A migration assay was performed.
A representative experiment of three experiments performed independently is shown; bars, mean7s.e.*Po0.05 versus control.
addition, the migration rate of IL-18 small interfering
RNA (siRNA) transfectant was considerably lower than
the control cells (Figure 2c). Moreover, rhVEGF-165
did not enhance the migration ability of the IL-18
siRNA transfectant (Figure 2d), suggesting that IL-18
plays a key role in the VEGF-enhanced migration of
SNU-601 cells.
IL-18 increases directly the migration of gastric cancer
cells through F-actin polymerization and tensin
down-regulation
To examine the direct effect of IL-18 on the migration of
SNU-601 cells.rhIL-18 was used As shown in Figure 3a
and b, the addition of 150 ng/ml of rhIL-18 to IL-18
receptor-expressing SNU-601 cells increased the rate of
cell migration significantly. The difference in the level of
SNU-601 cell migration between the control and rhIL18-treated cells was most obvious when SNU-601 cells
were incubated in the Transwell chambers for 24 h.
rhIL-18 increased the migration of SNU-601 cells in a
manner both dose- and time-dependent (Figure 3c and
d). In addition, rhIL-18 increased the invasive ability of
SNU-601 cells approximately twofold (Figure 3e). The
mechanism of IL-18-increased migration in SNU-601
cells was investigated. It is known that f-actin is
polymerized in migrating cells, and the expression level
of tensin, which is a capping protein of f-actin, is
markedly lower in malignant tumor cells (Lo et al.,
Oncogene
1994; Meili and Firtel, 2003; Lo, 2004). Based on these
reports, this study examined whether or not f-actin
polymerization and tensin expression were associated
with IL-18-increased migration. The cells pretreated
with 1 mg/ml of cytochalasin D, an f-actin-depolymerizing agent, for 1 h showed a significant decrease in IL-18increased migration (Figure 4a). F-actin polymerization
was detected mainly in cortical region by IL-18,
resulting in change in the cell morphology to an annular
structure (Figure 4b). According to f-actin polymerization, it was hypothesized that IL-18 may decrease the
expression of tensin in migrating cells. As shown in
Figure 4c, tensin expression was much higher in the
IL-18 siRNA transfectant than in the control cells. This
effect was inhibited by rhIL-18. This suggests that IL-18
increases the migration of SNU-601 cells through tensin
downregulation, which enhances f-actin polymerization.
rhVEGF-165 enhances IL-18 production in gastric
cancer cells
The effect of rhVEGF-165 on IL-18 production in SNU601 cells was examined by reverse transcriptase–polymerase chain reaction (RT–PCR) and enzyme-linked
immunosorbent assay (ELISA) to confirm that
rhVEGF-165 enhances IL-18 production, which results
in increased migration. IL-18 mRNA expression was
increased significantly by a treatment with 50 ng/ml
rhVEGF-165 for 1 h (Figure 5a). IL-18 production was
Effect of IL-18 on VEGF-enhanced migration
K-E Kim et al
1471
Figure 3 rhIL-18 increases the migration of SNU-601 cells in a dose- and time-dependent manner. (a) SNU-601 cells were stained
with mouse anti-human IL-18R-a antibody, and further stained with FITC-conjugated goat anti-mouse IgG antibody (bold line). The
control was not stained with any antibody (thin line). Mouse IgG was stained with FITC-conjugated goat anti-mouse IgG antibody
only (dot line). The cells were analysed by indirect FACS. A representative experiment of three experiments performed independently is
shown. (b) SNU-601 cells were incubated with 150 ng/ml of rhIL-18 for 24 h. (c) SNU-601 cells were treated with 150 ng/ml of rhIL-18
for 24 h. The cells were collected and a migration assay was performed for 6, 12, 24 and 36 h. (d) rhIL-18 dose titration and time kinetic
were performed. SNU-601 cells were treated with 50, 100, 150 and 200 ng/ml of rhIL-18 for 24 h or 150 ng/ml of rhIL-18 for 12, 24 and
48 h. A migration assay was performed for 24 h. (e) SNU-601 cells were treated with 150 ng/ml of rhIL-18. After incubation, an
invasion assay was performed. A representative experiment of three experiments performed independently is shown; bars, mean7s.e.
*Po0.05 versus control.
also enhanced by rhVEGF-165 in both a time- and a
dose-dependent manner (Figure 5b and c). A dose
titration of SU5416 was performed to determine if
VEGF enhances IL-18 production via VEGFR-2. In the
absence of rhVEGF-165, SU5416 reduced the spontaneous production of IL-18 in a dose-dependent manner
(Figure 5d). The effect of rhVEGF-165 on IL-18
production in other gastric cancer cell lines, such as
MKN-28 and SNU-719 cells, was examined to determine if the effect of rhVEGF-165 on IL-18 production is
common to a variety of gastric cancer cells. Table 1
shows that MKN-28 and SNU-719 cells also express
VEGFR-2. Compared with the control for each cell line,
rhVEGF-165 increased IL-18 production. In addition,
the migration rate of these gastric cancer cell lines was
increased by rhVEGF-165 and rhIL-18 (Table 2). This
Oncogene
Effect of IL-18 on VEGF-enhanced migration
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Generation of ROI and activation of ERK1/2 MAPK
pathway are required for VEGF-enhanced IL-18
production
Region of interest (ROI) plays an important role in the
intracellular signal-transduction pathways, including
the regulation of inflammatory cytokine production,
such as tumor necrosis factor-a (Gossart et al., 1996).
An antioxidant, N-acetyl-L-cysteine (NAC), was used
to determine if ROI are involved in VEGF-enhanced
IL-18 production. NAC significantly decreased VEGFenhanced IL-18 production. rhVEGF-165 stimulation
increased the level of ROI, indicating that rhVEGF-165
enhances IL-18 production through the generation of
ROI (Figure 6a and b). The signaling pathway for
VEGF-enhanced IL-18 production was further examined by investigating whether or not extracellular signalregulated kinase (ERK)1/2 and c-Jun NH2-terminal
kinase (JNK) mitogen-activated protein kinase pathways are involved in VEGF-enhanced IL-18 production
using a specific inhibitor of ERK1/2 (PD98059) and
JNK (SP600125). PD98059 increased VEGF-enhanced
IL-18 production in a dose-dependent manner, but
SP600125 had no effect (Figure 6c and d). Accordingly,
SNU-601 cells were incubated with 50 ng/ml of
rhVEGF-165 for 2.5, 5, 10 and 15 min. ERK1/2
phosphorylation was determined using Western blot
analysis. Figure 6e shows that rhVEGF-165 increased
ERK1/2 phosphorylation, and reached a maximum at
5 min, which indicates that rhVEGF-165 stimulates
ERK1/2 phosphorylation directly. This shows that
rhVEGF-165 enhances IL-18 production via the activation of ERK1/2.
Discussion
Figure 4 IL-18 affects migration via f-actin polymerization and
tensin downregulation. (a) SNU-601 cells were pretreated with or
without 1 mg/ml of cytochalasin D. The cells were then treated with
150 ng/ml of rhIL-18. A migration assay was performed. A
representative experiment of three experiments performed independently is shown; bars, mean7s.e. *Po0.05 versus control.
(b) SNU-601 cells were incubated with or without 150 ng/ml of
rhIL-18 on slides. After incubation, immunostaining was performed. Image was collected using a confocal microscopy system;
bars, upper panels: 20 mm, lower panels: 5 mm. (c) IL-18 siRNAtransfected cells were treated with or without 150 ng/ml of rhIL-18
for 24 h. After cell lysis, immunoprecipitation was performed. The
expression of tensin was determined by Western blot analysis.
Actin was used to confirm the equal volume of the cell lysates.
indicates that rhVEGF-165 can enhance the migratory
properties in many gastric cancer cells by upregulating
IL-18 production.
Oncogene
IL-18 was identified as being an IFN-g-inducing factor.
Moreover, the active IL-18 protein increases IFN-g
production from immune cells, such as T or NK cells, to
develop Th1 cells and augment the activity of NK cells
(Okamura et al., 1995). Some reports have suggested
that IL-18 inhibits angiogenesis, suppresses tumor
growth and enhances the anti-tumor responses (Cao
et al., 1999; Ju et al., 2001). Although IL-18 can enhance
the immune response, it was recently reported that
tumors could counterattack the immune system by
secreting IL-18 (Cho et al., 2000). The serum IL-18
concentration was reported to be higher in metastatic
cancer patients than in early cancer patients, and the
metastatic ability of melanoma is regulated by IL-18 via
vascular cell adhesion molecule-1 (Lissoni et al., 2000;
Vidal-Vanaclocha et al., 2000). This suggests that the
IL-18 derived from cancer cells is involved in the cancer
metastatic process. In contrast to a study demonstrating
that IL-18 plays a role as an angiogenesis inhibitor, it
was recently reported that IL-18 induces angiogenesis in
rheumatoid arthritis, which suggests a novel proangiogenic function of IL-18 (Park et al., 2001). Angiogenesis
is essential for tumor progression and metastasis, and
proangiogenic factors are beneficial to tumor cells. In
particular, VEGF is the most potent mediator of
Effect of IL-18 on VEGF-enhanced migration
K-E Kim et al
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Figure 5 rhVEGF-165 enhances both IL-18 expression at the mRNA level and production at the protein level. (a) SNU-601 cells were
incubated with 50 ng/ml of rhVEGF-165, and the IL-18 mRNA level was then analysed by RT-PCT. (b) SNU-601 cells were treated
with 50 ng/ml of rhVEGF-165 for 6, 12 and 24 h. The control represents the cultured supernatants that were incubated without
rhVEGF-165 (open square), and rhVEGF-165 represents the cultured supernatants that were incubated with 50 ng/ml of rhVEGF-165
(closed square). (c) SNU-601 cells were treated with 25, 50, 100 and 200 ng/ml of rhVEGF-165 for 24 h. (d) SNU-601 cells were treated
with 0, 2.5, 5 and 10 mM of SU5416 for 24 h. The cultured supernatants were collected, and the IL-18 concentration was determined by
ELISA. A representative experiment of three experiments performed independently is shown; bars, mean7s.e. *Po0.05 versus
control.
Table 1 Effect of rhVEGF-165 on IL-18 production in the other
gastric cancer cell lines, SNU-719 and MKN-28
Table 2
Effect of rhVEGF-165 and rhIL-18 on cell migration in the
other gastric cancer cell lines, SNU-719 and MKN-28
Cell lines
VEGFR-2
expression
Control
(pg/ml)
rhVEGF-165
(pg/ml)
Cell lines
Control (%)
rhVEGF-165 (%)
rhIL-18 (%)
SNU-601
SNU-719
MKN-28
Yes
Yes
Yes
775.7722.8a
1768.5715.7
1054.8718.8
1451.279.7
2585.7742.8
1474.8712.3
SNU-601
SNU-719
MKN-28
101.275.5a
99.474.7
106.377.1
216.975.9
167.4710.2
187.579.4
243.673.4
152.475.9
198.1710.5
Abbreviations: VEGF, vascular endothelial growth factor; IL, Interleukin. aEach value represents the mean of IL-18 concentration7s.e.
Abbreviations: VEGF, vascular endothelial growth factor; IL, Interleukin. aEach value represents the relative percentage of cell
migration7s.e.
angiogenesis. The results in this study showed that
rhVEGF-165 enhanced IL-18 production in gastric
cancer cells, and the VEGF-enhanced IL-18 production
significantly increased the migration of SNU-601 cells.
This suggests that rhVEGF-165 affects the metastasis of
gastric cancer cells by not only regulating angiogenesis
but also enhancing the ability of these cells to migrate as
a result of VEGF-enhanced IL-18 production. This
study found that IL-18 induced the migration of SNU601 cells through f-actin polymerization and tensin
downregulation (Figure 4). F-actin polymerization by
IL-18 was mainly distributed in the cortical region
around the nuclei. IL-18 induced a change in cell
morphology to a round form, suggesting weaker
adherence. In addition, tensin expression, which inhibits
actin polymerization, was increased markedly in IL-18
siRNA-transfected cells, and was reduced by rhIL-18.
This means that IL-18 downregulates tensin expression
for promoting f-actin polymerization. It was reported
that tensin retards the initial f-actin polymerization, and
considerably decreases f-actin elongation (Lo et al.,
1994). Taken together, rhIL-18 can induce f-actin
polymerization directly, and reduce the expression of
tensin for f-actin polymerization. Besides migration,
IL-18 affects the invasion of SNU-601 cells (Figure 3e).
It was reported that IL-18 increased myeloid leukemia
and human NK cell invasion through the upregulation
of matrix metalloproteinases (MMP) expression (Ishida
et al., 2004; Zhang et al., 2004). Because MMP are
important proteins involved in the processes of tumor
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Effect of IL-18 on VEGF-enhanced migration
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Figure 6 rhVEGF-165 enhances IL-18 production through the generation of ROI and the activation of ERK1/2 pathway. (a) SNU601 cells were pretreated with or without 5 mM of NAC, and incubated with 50 ng/ml of rhVEGF-165. The cultured supernatants were
collected, and the IL-18 concentration was determined by ELISA. (b) SNU-601 cells (9 104 cells/well) were incubated with (VEGF) or
without (control) 50 ng/ml of rhVEGF-165 in wells of 96-well plate, and 50 mM of DCFH-DA was then added to indicate the level of
ROI. After incubation for 10 min, the level of ROI was measured using a fluorometer. (c) Before incubation with rhVEGF-165, SNU601 cells were pretreated with or without a specific inhibitor of ERK1/2 or JNK for 1 h. (d) SNU-601 cells were pretreated with or
without 12.5 or 25 mM PD98059. The cultured supernatants were collected, and the IL-18 concentration was determined by ELISA. A
representative experiment of three experiments performed independently is shown; bars, mean7s.e. *Po0.05 versus control. (e) The
cells were treated with 50 ng/ml of rhVEGF-165 for 2.5, 5, 10 and 15 min. After cell lysis, the level of ERK1/2 phosphorylation was
determined by Western blot analysis. The total ERK1/2 was used to confirm the equal volume of the cell lysates.
invasion and metastasis, it is believed that IL-18 can
regulate MMP expression, resulting in enhanced invasion. In this study, rhVEGF-165 was found to enhance
IL-18 production, suggesting that rhVEGF-165 stimulates the intracellular pathway of IL-18 production.
Previously, it was reported that ultraviolet (UV)
radiation increases IL-18 production in the human
keratinocyte cell line, HaCaT. UV-enhanced IL-18
production requires the generation of ROI (Cho et al.,
2002). It was hypothesized that VEGF might enhance
IL-18 production in gastric cancer cells via ROI
generation. VEGF-enhanced IL-18 production was
reduced significantly by NAC, and ROI level was
increased by rhVEGF-165 (Figure 5), indicating that
ROI are common factors for the intracellular mechanism of IL-18 production. ERK1/2 occurs downstream of
Oncogene
VEGF-induced VEGFR-2 phosphorylation (Pedram
et al., 1998). Therefore, this study examined whether
or not ERK1/2 pathway is involved in the intracellular
pathway of VEGF-enhanced IL-18 production mechanism. Figure 6 shows that PD98059 decreased the level of
VEGF-enhanced IL-18 production, and that rhVEGF165 can activate ERK1/2 directly. This suggests that
ERK1/2 is involved in the intracellular pathway of
VEGF-enhanced IL-18 production. Although NAC
decreased the spontaneous production of IL-18 from
SNU-601 cells, PD98059 had no affect in the absence of
rhVEGF-165 (data not shown). This indicates that
ERK1/2 is specifically associated with the VEGFenhanced IL-18 production. In conclusion, rhVEGF-165
increases IL-18 production in the gastric cancer cell
line, SNU-601, through the generation of ROI and the
Effect of IL-18 on VEGF-enhanced migration
K-E Kim et al
1475
activation of ERK1/2 pathway. As a result of VEGFenhanced IL-18 production, the migration of SNU-601
cells was increased through f-actin polymerization and
tensin downregulation. This suggests that rhVEGF-165
regulates the migratory process by enhancing IL-18
production. Taken together, it is proposed that VEGFenhanced IL-18 production plays an important role in
the pathogenesis of gastric cancer.
Materials and methods
Cell line
The human gastric cancer cell lines, SNU-601, SNU-719 and
MKN-28, were obtained from the Korean Cell Line Bank of
the Cancer Research Center, Seoul National University
College of Medicine. These cell lines were cultured in Roswell
Park Memorial Institute 1640 medium (Invitrogen Corporation, San Diego, CA, USA) containing 2 mM L-glutamine,
100 U/ml penicillin, 100 mg/ml streptomycin and 10% heatinactivated fetal bovine serum (FBS), and were maintained in a
5% CO2 incubator at 371C.
Flow cytometry
Indirect fluorescence-activated cell sorter (FACS) analysis was
performed to detect VEGFR-2 and IL-18R-a expression.
Briefly, the cells were washed twice with phosphate-buffered
saline (PBS), and stained with either mouse anti-human
VEGFR-2 antibody (Sigma, St Louis, MO, USA) or mouse antihuman IL-18R antibody (R&D systems Inc., Minneapolis, MN,
USA) for 30 min on ice. The cells were further stained with
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
immunoglobulin G (IgG) antibody (DiNonA) for 30 min on ice,
followed by two washes with PBS. An FACS Calibur (Becton
Dickinson, Mountain View, CA, USA) flow cytometer was used
for analysis.
Migration and invasion assay
Migration assay was performed using a Transwell chamber
(Corning) with 8 mm pore polycarbonate filters. Briefly, the
cells were suspended in serum-free media. One hundred
microliters of the cell suspension (5 105 cells/ml) was placed
into the upper chamber, and a medium containing serum was
placed into the lower chamber. After incubation for 24 h, the
cells that penetrated through the pores in the membrane were
stained with a staining solution (0.1% crystal violet in
ethanol). The stained cells were resolved in 10% acetic acid.
The OD values at 570 nm were measured using an ELISA
reader (Molecular Devices, Sunnyvale, CA, USA). Invasion
assay was determined using an extracellular matrix (ECM)based assay. The upper chamber was coated with an ECM gel
(Sigma), which forms a reconstituted basement membrane at
371C. After washing three times, migration assay was
performed.
RT–PCR
Total RNA was isolated, and the cDNA was then made. After
reverse transcription, the cDNA was incubated with either
human IL-18 primers (sense, 50 -AGGAATAAAGATGG
CTGCTGAAC-30 ; antisense, 50 -GCTCACCACAACCTCTA
CCTCC-30 ; target size, 837 bp) or human b-actin primers
(sense, 50 -TAGCGGGGTTCACCCACACTGTGCCCCATC
TA-30 ; antisense, 50 -CTAGAAGCATTTGCGGTGGACCG
ATGGAGGG-30 ; target size, 661 bp) for PCR amplification.
The cycling conditions for IL-18 were 1 min at 951C, 1 min at
58.51C, 45 s at 721C for 35 cycles, and for b-actin were 30 s at
941C, 30 s at 561C, 1 min at 721C for 30 cycles.
ELISA
In anti-human IL-18 antibody-coated wells, the cultured
supernatants were added along with biotinylated detection
antibody. After incubating the cultured supernatant for 3 h,
the wells were washed three times with a washing solution.
Streptavidin-horseradish peroxidase (HRP) was then added
for 30 min, and substrate 3,30 ,5,50 -tetramethyl benzidine
solution was added for 40 min at 371C in the dark. Color
development was stopped using H2SO4 stop solution, and the
OD values at 450 nm were measured by using an ELISA reader
(Molecular Devices).
Measurement of intracellular ROI level
Cells (9 104 cells/well) were incubated in 96-well plate for
30 min, and 50 mM 20 ,70 -dichlorofluorescein diacetate (DCFHDA; Sigma) was then added. After incubation, the intracellular
ROI levels were measured using a fluorometer (Molecular
Devices) at an excitation and emission wavelength of 485 and
538 nm, respectively.
Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in PBS for 20 min,
and then permeabilized for 30 min with 0.5% Triton X-100 in
PBS. After blocking with 3% FBS in PBS, the level of f-actin
polymerization was detected by incubating the cells with
FITC-conjugated phalloidin (Molecular Probes Inc., Eugene,
OR, USA) for 1 h. F-actin was detected using a confocal
microscope system (Olympus FV-300, 400 objective,
FLUOVIEW imaging program).
Immunoprecipitation and Western blot
Cells were washed with ice-cold PBS and extracted with an icecold lysis buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25%
deoxycholic acid sodium salt, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid and protein inhibitor cocktail). For the
detection of tensin expression, 1 mg of the protein was exposed
to 2 mg of mouse anti-human tensin antibody and protein-G
agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
The protein was separated by 8% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose transfer membrane (Schleicher &
Schuell Inc., Keene, NH, USA). The membrane was blocked
with 5% non-fat dried milk for 1 h, and incubated overnight
with anti-human tensin antibody. After washing, the membrane was incubated with rabbit anti-mouse IgG antibody
conjugated with biotin for 1 h. After incubation with HRP for
30 min, each of the proteins was detected using an enhanced
chemiluminescence system (Amersham Pharmacia Biotech,
Buckinghamshire, UK). For the detection of ERK1/2, the
protein was separated by 12% SDS–PAGE, and examined
using mouse anti-human phospho-ERK1/2 antibody or rabbit
anti-human total ERK1/2 antibody (Cell Signaling Technology, Danvers, MA, USA).
Acknowledgements
This work was supported by the Korea Science & Engineering
Foundation (KOSEF) through the Tumor Immunity Medical
Research Center (TIMRC) at Seoul National University
College of Medicine and through the Research Center for
Women’s Disease (RCWD; SRC program (R11-2005-01702003-0)) at Sookmyung Women’s University.
Oncogene
Effect of IL-18 on VEGF-enhanced migration
K-E Kim et al
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