SDF-1/CXCR7 axis regulates the proliferation, invasion, adhesion, and angiogenesis of gastric cancer cells
Ma et al. World Journal of Surgical Oncology
SDF-1/CXCR7 axis regulates the proliferation, invasion, adhesion, and angiogenesis of gastric cancer cells
De-Min Ma 2
Dian-Xi Luo 0 1
Jie Zhang 0 1
0 Department of Gastrointestinal Surgery, People's Hospital of Dezhou , 1751 Xin Hu Road, Dezhou, Shandong Province 253014 , People's Republic of China
1 Department of Gastrointestinal Surgery, People's Hospital of Dezhou , 1751 Xin Hu Road, Dezhou, Shandong Province 253014 , People's Republic of China
2 Department of Hepatobiliary and Vascular Surgery, People's Hospital of Dezhou , Dezhou, Shandong Province 253014 , People's Republic of China
Background: More recent studies have revealed that chemokine receptor CXCR7 plays an important role in cancer development. However, little is known about the effect of CXCR7 on the process of gastric cancer cell invasion and angiogenesis. The aim of this study is to investigate the expression of CXCR7 in gastric cancer cell lines and to evaluate the role of CXCR7 in the proliferation, invasion, adhesion, and angiogenesis of gastric cancer cells. Methods: Real-time PCR and Western blotting were used to examine the mRNA and protein levels of CXCR4 and CXCR7 in five gastric cancer cell lines (HGC-27, MGC-803, BGC-823, SGC-7901, and MKN-28). CXCR7-expressing shRNA was constructed and subsequently stably transfected into the human gastric cancer cells. In addition, the effect of CXCR7 inhibition on cell proliferation, invasion, adhesion, VEGF secretion, and tube formation was evaluated. Results: The mRNA and protein of CXCR7 were expressed in all five gastric cancer cell lines; in particular, the expression of CXCR7 was the highest in SGC-7901 cells. Stromal cell-derived factor-1 (SDF-1) was found to induce proliferation, invasion, adhesion, and tube formation. Moreover, the VEGF secretion in SGC-7901 cells was also enhanced by SDF-1 stimulation. These biological effects were inhibited by the silencing of CXCR7 in SGC-7901 cells. Conclusions: Increased CXCR7 expression was found in gastric cancer cells. Knockdown of CXCR7 expression by transfection with CXCR7shRNA significantly inhibits SGC-7901 cells' proliferation, invasion, adhesion, and angiogenesis. This study provides new insights into the significance of CXCR7 in the invasion and angiogenesis of gastric cancer.
Gastric cancer; Chemokines; Stromal cell-derived factor-1; CXC chemokine receptor-7; Metastasis
Gastric cancer is one of the most commonly
diagnosed malignancies and the main cause of
cancerrelated deaths in Asian populations . Most deaths
from gastric cancer are caused by metastasis, of
which lymph node metastasis is the most common
cause, which leads to the failure of surgery,
chemotherapy, or radiotherapy . Therefore, inhibition of
metastatic gastric cancer is an important therapeutic
strategy. However, the molecular mechanisms involved
in this process have not been fully elucidated.
Stromal cell-derived factor-1 (SDF-1, also called
CXCL12) is a member of the CXC subfamily of
chemokines and is expressed in a variety of tissues including
the lung, liver, bone marrow, and lymph nodes [3–5].
SDF-1 elicits biologic function through binding to its
receptor, CXCR4, which is present on the cell surface and
is a seven-transmembrane span G-protein-coupled
receptor . SDF-1 plays a role in a number of important
physiological processes including leukocyte trafficking
and vasculogenesis [7, 8]. More importantly, SDF-1 plays
a crucial role in the process of invasion and metastasis
of tumor cells . SDF-1 stimulates proliferation,
dissociation, migration, and invasion in a wide variety of
tumor cells, including breast cancer cells , pancreatic
cancer cells  and HCC cells , and gastric cancer
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Until recently, CXCR4 was considered to be the only
receptor for SDF-1. However, a recent study has shown
that chemokine receptor CXC chemokine receptor type
7 (CXCR7) can also bind to SDF-1, and it is identified as
a second receptor for SDF-1 . It has been
demonstrated that CXCR7 is expressed in a variety of tumor
cell lines and normal cells including activated
endothelial cells, fetal liver cells, T cells, B cells, and renal
multipotent progenitors [14, 15]. However, the function of
CXCR7 is still unclear and controversial. Some studies
suggested that CXCR7 is a non-signaling decoy
receptor and cannot activate intracellular signaling cascades,
while others considered that CXCR7 was a signaling
receptor and could activate (mitogen-activated protein
kinase (MAPK)) p42/44 and AKT phosphorylation
through binding with SDF-1 [16, 17].
There is increasing evidence that CXCR7 may
participate in tumor development. On the one hand,
overexpression of CXCR7 has been observed in various tumors,
including breast cancer, lung cancer, prostate cancer, and
pancreatic cancer [18, 19]. On the other hand, expression
of CXCR7 enhances the tumor cells’ proliferation,
adhesion, invasion, and blood vessel sprout formation in vitro
and promotes tumor growth in vivo [20, 21]. Although
the role of SDF-1 in the promotion of invasive
growth is well documented and the intracellular
signals triggered by CXCR4 activation have been
extensively investigated [22, 23], the role of SDF-1/CXCR7
axis in regulating tumor growth of gastric cancer is
not yet known.
Therefore, the present study was undertaken to test
the hypothesis that SDF-1/CXCR7 was involved in
malignant properties of gastric cancer cells. We have
studied the expression of CXCR7 in gastric cancer cell lines.
We have also evaluated the effect of specific inhibition
of CXCR7 on SDF-1-induced cell invasion, adhesion,
Human gastric cancer cell lines (HGC-27, MGC-803,
BGC-823, SGC-7901, and MKN-28) and human
umbilical vein endothelial cells (HUVECs) were purchased
from Cell Bank of Shanghai Institute of Biochemistry
and Cell Biology, Chinese Academy of Sciences
(Shanghai, China). Gastric cancer cell lines were grown in
Roswell Park Memorial Institute (RPMI) 1640 medium
(Sigma-Aldrich, USA) that contained 10 % fetal bovine
serum (FBS; HyClone, USA). HUVECs were maintained
in DMEM medium containing 10 % FBS. All the media
were supplemented with 100 U/ml penicillin and
100 μg/ml streptomycin (Invitrogen, USA) and
maintained in 5 % CO2 at 37 °C.
Construction of small hairpin RNA plasmid
Knockdown of CXCR7 was achieved by expression of
short hairpin RNA (shRNA) from the pGPU6/Neo
vector containing the human U6 promoter (GenePharma,
Shanghai, China). The sequence of the oligonucleotide
targeted to CXCR7 is 5′-GCATCTCTTCGACTACTC
AGA-3′, corresponding to positions 223 to 243 within the
CXCR7 mRNA sequence (accession no. NM_020311).
The following complementary oligonucleotide encoding
shRNA was designed to knock down CXCR7: (sense)
ATGC-3′. The pGPU6/Neo plasmid was linearized with
BamHI and BbsI to permit the insertion of the annealed
oligonucleotides. DNA oligonucleotides were annealed by
incubating the mixed oligonucleotides in the PCR
thermocycler using the following profile: 95 °C for 5 min, 80 °C
for 5 min, and 75 °C for 5 min, and gradually cooled to
room temperature. Annealed oligonucleotides were ligated
to the BbsI and BamHI sites of the pGPU6/Neo plasmid.
The scrambled shRNA was used as a negative control
(referred to as “NC” in the text), of which the
sequence was 5′-GACGAGCTTCTACACAATCAT-3′.
The recombinant constructs were verified by DNA
sequencing and by analyzing the fragments generated
from digestion with BamHI.
Generation of stable transfectants
SGC-7901 cells were seeded in six-well plates to 80–90 %
confluence. The cells were transfected with mixtures of
shRNA plasmids and Lipofectamine™ 2000 reagent
(Invitrogen, USA) according to the manufacturer’s instructions.
Forty-eight hours after transfection, the transfected cells
were grown in a growth medium containing 0.4 mg/ml
G418 (Gibco, USA) for selection. Stable transfectant
clones with low expression of CXCR7 were evaluated by
real-time PCR (RT-PCR) and Western blot analysis. Stable
transfectants were expanded for subsequent experiments.
SGC-7901 cells transfected by CXCR7shRNA were
referred to as CXCR7shRNA cells, while SGC-7901 cells
transfected by scrambled shRNA as NC cells.
Total RNA from gastric cancer cells was isolated using
TRIzol (Invitrogen, Carlsbad, CA, USA) and then reverse
transcribed with PrimeScript RT Master Mix (Takara,
Otsu, Japan). RT-PCR was conducted using an Eppendorf
Mastercycler ep realplex machine (Eppendorf, Germany)
and using SYBR Premix Ex Taq™ II Kit (Takara) according
to the manufacturer’s instructions. The primers were as
follows: CXCR7, forward (5′-TGGGTGGTCAGTCTC
GT-3′) and reverse (5′-CCGGCAGTAGGTCTCAT-3′);
CXCR4, forward (5′-CCTGAAGTACCCCATCGAGCA
C-3′) and reverse (5′-ATACCCCCTCGTAGATGGGC
ACA-3′); GAPDH, forward (5′-GAAGGTGAAGGTCGG
AGTC-3′) and reverse (5′-GAAGATGGTGATGGGA
TTTC-3′). Relative mRNA expression levels were
calculated by the 2-△△Ct method. GAPDH was used as a
For the preparation of lysates, the cells were washed
with ice-cold PBS solution and lysed in lysis buffer. Cells
were scraped into microcentrifuge tubes and centrifuged
at 10,000×g for 15 min at 4 °C. The supernatant was
collected, and protein concentrations were determined with
the BCA assay kit (Sigma-Aldrich, USA) according to
the manufacturer’s instruction. Samples were subjected
to 10 % PAGE analysis after they were boiled for 5 min
and electrophoretically transferred to polyvinylidene
difluoride (PVDF) membranes (Millipore, USA). Blocking
was performed in 5 % nonfat dried milk in Tris-buffered
saline containing 0.1 % Tween 20 at room temperature for
1 h. Membranes were then incubated with primary
antibody under constant agitation at antibody dilutions
suggested by the antibody supplier overnight at 4 °C.
After several washings, membranes were incubated
with horseradish peroxidase-conjugated secondary
antibody (anti-rabbit) for 1 h at room temperature
under constant agitation. Proteins were visualized by
using an enhanced chemiluminescence system (ECL;
Amersham Biosciences, USA).
Total protein extracts in a final volume of 250 ml were
incubated overnight at 4 °C with 5 μg rabbit
antiCXCR7 and 5 μg rabbit anti-SDF-1 antibodies,
previously bound to protein G magnetic beads (Millipore).
An irrelevant rabbit polyclonal antibody bound to
protein G magnetic beads was performed as a negative
control. The immune complexes were precipitated by
placing the tube into the magnetic stand (Millipore) and
washing three times with 500 μL of PBS containing
0.1 % Tween 20. Precipitated proteins were separated by
SDS-PAGE and analyzed by Western blotting with
mouse anti-CXCR7 or mouse anti-SDF-1 antibody.
Cell proliferation assay
SGC-7901 cells (including control, NC, and CXCR7shRNA
transfected groups) were seeded into 96-well plates at a
density of 5 × 103 cells per well without FBS. After 24 h,
the cultures were washed and re-fed with medium that
contained SDF-1 (100 ng/ml; Peprotech, UK). After
different time points (24, 48, 72, and 96 h), the number of viable
cells was counted using a CCK8 assay (KeyGen, China)
according to the manufacturer’s instructions. The quantity of
formazan product measured at 490 nm was proportional
to the number of live cells in the culture. The experiments
were repeated in triplicates.
Cell invasion assay
SGC-7901 cell invasion in response to SDF-1 was
assayed in the Biocoat Matrigel invasion chamber (Becton
Dickinson, USA) with 8-μm porosity polycarbonate filter
membrane that was coated with Matrigel. SGC-7901 cells
were suspended at 3 × 105 cells/ml in serum-free media,
respectively, and then 0.2 ml cell suspension was added to
the upper chamber. Next, 0.5 ml serum-free media with
SDF-1 (100 ng/ml) was added to the lower chamber.
The chambers were then incubated for 24 h at 37 °C
with 5 % CO2. After incubation, noninvasive cells were
gently removed from the top of the Matrigel with a
cotton-tipped swab. Invasive cells at the bottom of the
Matrigel were fixed in 4 % paraformaldehyde and
stained with hematoxylin. The number of invasive cells
was determined by counting the hematoxylin-stained
cells. For quantification, cells were counted under a
microscope in five fields.
Cell adhesion assay
Cell adhesion assay was carried out by using the
CytoSelect™ ECM Cell Adhesion Assay kit (Cell Biolabs
Inc., USA) following the instruction manual. Briefly, the
48-well plate precoated with laminin (LN) or fibronectin
(FN) were washed with PBS twice and blocked for 1 h at
37 °C with RPMI 1640 containing 0.1 % bovine serum
albumin (BSA) before plating cells. Plates were again
washed with PBS and air dried. SGC-7901 cells were
preincubated with SDF-1 (100 ng/ml) for 24 h at 37 °C. A cell
suspension containing 2 × 105 cells/ml was prepared in
serum-free media. The cell suspension (150 μl) was added
to the inside of each well (BSA-coated wells were provided
as a negative control). Cells were allowed to attach for 1 h
at 37 °C. Subsequently, unattached cells were removed by
gentle washing three times with PBS. Then, the attached
cells were stained with 1 % crystal violet. Each well was
gently washed three times with PBS. The total crystal
violet bound to the cells was eluted with 10 % acetic acid and
measured by the absorbance at 560 nm. All the
experiments were repeated three times in duplicate wells.
In vitro tube formation coculture assay
The ability of endothelial cells to align into tube-like
structures was measured using Matrigel™ tube formation
assay as described previously . Briefly, Transwell
chambers were precoated with growth factor-reduced
Matrigel (200 μL of 10 mg/mL). Control, NC, and
CXCR7shRNA transfected SGC-7901 cells were seeded
at a density of 2 × 104 cells/well in 24-well plates and
cultured for 24 h, respectively. HUVECs (2 × 104 cells/
well) were then seeded in Transwell chambers precoated
with the Matrigel. Subsequently, Transwell chambers
containing HUVECs were inserted into the 24-well
plates and cocultured for 24 h. After 24 h of cocultured
at 37 °C and 5 % CO2, the number of capillary-like tubes
from three randomly chosen fields was counted and
photographed under an Nikon inverted microscope
(Japan). Tubes were defined as endothelial cells that had
aligned to form >90 % closed structures .
ELISA for VEGF
SGC-7901 cells were plated in 24-well tissue culture
plates at a density of 1 × 105 cells per well and followed
with serum starvation for 24 h with RPMI 1640. Then,
cells were treated with recombinant human SDF-1
(100 ng/ml), and the supernatants were collected 24 h
after treatment. Vascular endothelial growth factor
(VEGF) concentration was determined using Quantikine
ELISA Kits according to the manufacturer’s instructions
(R&D Systems, Minneapolis, MN).
Data are reported as means ± SD. The one-way
ANOVA was used for data analysis. All statistics were
calculated using SPSS 16.0 software (SPSS, Chicago,
IL, USA). P < 0.05 was considered as statistically significant.
Expression of CXCR7 on gastric cancer cell lines and
To determine whether CXCR7 is expressed in gastric
cancer cell lines, we first evaluated the expression of
CXCR7 by Western blot in a panel of gastric cancer cell
lines (HGC-27, MGC-803, BGC-823, SGC-7901, and
MKN-28) and HUVEC. As shown in Fig. 1, CXCR7
protein expression was clearly detected in five gastric cancer
cell lines and HUVEC, with different amounts of CXCR7
transcripts; in particular, the expression of CXCR7 was
the highest in SGC-7901 cells.
Interaction between CXCR7 and SDF-1 in SGC-7901 cells
In order to prove the interaction between CXCR7 and
SDF-1 in SGC-7901 cells, the total protein extracts
from SGC-7901 cells were immunoprecipitated with
an anti-CXCR7 or anti-SDF-1 antibody, precipitated
proteins were analyzed by immunoblotting with
antibodies directed specifically to either CXCR7 or SDF-1.
Figure 2a showed that SDF-1 was pulled down together
with CXCR7 by the anti-CXCR7 antibody in SGC-7901
cells, whereas none of these two proteins was recovered
when an irrelevant antibody (IgG0) was used for
immunoprecipitation, thus establishing the specificity of the assays.
In Fig. 5b, CXCR7 was pulled down together with rabbit
anti-SDF-1 antibody. Co-immunoprecipitation with each
Fig. 1 Expression of CXCR4 and CXCR7 in gastric cancer cell lines
and HUVECs. a Western blot analysis was performed to detect CXCR7
and CXCR4 protein expression. β-Actin was used as a control to ensure
equal loading. b RT-PCR was performed on various cell lines to
determine CXCR7 and CXCR4 mRNA expression. GAPDH was used as a
control. Data shown is representative of means ± SD of three
specific antibody proved association between CXCR7 and
SDF-1 in SGC-7901 cells, which proved the formation of
SDF-1/CXCR7 protein complex in SGC-7901 cells.
The CXCR7shRNA causes effective and specific
downregulation of CXCR7 expression
In order to study the potential role of CXCR7 in gastric
cancer cell lines, CXCR7shRNA and scrambled shRNA
were used to transfect SGC-7901 cells. After G418
selection, the knockdown efficiencies were subsequently
tested using RT-PCR and Western blot. As shown in
Fig. 3b, CXCR7 mRNA levels were reduced by 80 % in
CXCR7shRNA-transfected cells, compared with the
control cells. Similar to the RT-PCR results, the expression
level of CXCR7 protein was significantly reduced in
CXCR7shRNA-transfected cells (Fig. 3a). The scrambled
sequence shRNA had no effect on CXCR7 expression.
These results demonstrated that the expression of
CXCR7 was specifically silenced in SGC-7901 cells.
Fig. 2 SDF-1 interacts with CXCR7 in SGC-7901 cells. Whole cell
extracts from SGC-7901 cells were immunoprecipitated with a a rabbit
anti-CXCR7 antibody and b rabbit anti-SDF-1 antibody. An irrelevant
antibody (IgG) was used as control. Immunoprecipitated proteins were
analyzed by Western blotting with mouse anti-CXCR7 and
CXCR7 silencing inhibits SDF-1-induced SGC-7901 cells
proliferation in vitro
To evaluate a role of CXCR7 in regulating the
proliferation of tumor cells, we selected the SGC-7901 cell line
as a model. After incubation for 24 to 96 h, cell
proliferation was significantly enhanced by SDF-1 (Fig. 4). We
next evaluated the effect of silencing of CXCR7 on
SGC7901 cells proliferation. The CXCR7shRNA cells
displayed decreased proliferation ability compared with the
control cells and NC cells (Fig. 4). Taken together, these
findings indicate that SDF-1 potently enhances the
proliferation ability of SGC-7901 cells and that silencing of
CXCR7 inhibits the proliferation ability of the cells
induced by SDF-1.
CXCR7 silencing inhibits SDF-1-induced SGC-7901 cell
invasion in vitro
Cell invasion experiments were performed with a Matrigel
invasion chamber, which is considered an in vitro model
system for metastasis. As shown in Fig. 5, SGC-7901 cells
spontaneously invaded through artificial basement
membrane in the absence of SDF-1. In addition, we found that
SDF-1-induced a significant increase of cancer cell
invasion through Matrigel. We next evaluated the effect of
silencing of CXCR7 on SGC-7901 cells invasion. The
Fig. 3 Downregulation of CXCR7 expression in SGC-7901 cells by
transfection with CXCR7shRNA. a After G418 selection, the protein
expression levels of CXCR7 were measured by Western blot using
anti-CXCR7 antibody and β-actin as a loading control. The experiment
was repeated three times with similar results. b Cellular RNA was
harvested after G418 selection, and CXCR7 mRNA was measured
using RT-PCR. GAPDH was used as a loading control
CXCR7shRNA cells displayed decreased invasive ability
compared with the control cells and NC cells (Fig. 5).
Taken together, these findings indicate that SDF-1
potently enhances the invasive ability of SGC-7901 cells and
that silencing of CXCR7 inhibits the invasive behavior of
the cells induced by SDF-1.
CXCR7 silencing inhibits SDF-1-induced SGC-7901 cell
adhesion in vitro
To analyze the effect of CXCR7 expression on the
adhesion of tumor cells to LN or FN, SGC-7901 cells were
examined by a cell adhesion assay. As shown in Fig. 6,
SGC-7901 cells displayed an enhanced cell adhesion to
LN or FN in the presence of SDF-1. Adhesion of
SGC-7901 cells to LN was greater than adhesion of
SGC-7901 to FN or BSA. However, cells transfected
by CXCR7shRNA showed significantly reduced ability
of adhesion to LN or FN compared with the control and
NC cells. Control, NC, and CXCR7shRNA transfected
cells adhered equally to BSA-coated dishes. Together,
these results indicate that treatment with SDF-1 increases
Fig. 4 Silencing of CXCR7 inhibits SDF-1-induced enhancement on SGC-7901 cell proliferation in vitro. Cell proliferation was measured by CCK-8
at different time points (0, 24, 48, 72, and 96 h). Each bar represents mean ± SD from three independent experiments. **P < 0.01 (as compared
with untransfected cells)
the adhesive ability of SGC-7901 cells and CXCR7
silencing results in decreased adhesive ability.
CXCR7 silencing inhibits SGC-7901 cell-induced tube
formation in vitro
To address whether SDF-1/CXCR7 interaction could
mediate in vitro tumor cell-induced tube formation, a
coculture system was used in which HUVECs were
induced by SGC-7901 cells to form capillary-like
structures. The tube formation of HUVECs on the
Matrigel was quantified by measuring the tube
number. As shown in Fig. 7, control and NC cells induced
HUVECs to differentiate into capillary-like structures
within 24 h. In contrast, SGC-7901 cells transfected
with CXCR7shRNA markedly inhibited tumor
cellinduced tube formation. HUVECs showed a significant
decrease in the number of tubes after transfecting
SGC7901 with CXCR7shRNA.
SDF-1 induces VEGF secretion through CXCR7 in
To evaluate whether SDF-1 contributes to proangiogenic
factor secretion in tumor cells, we treated SGC-7901
cells with SDF-1 and measured the secretion of
proangiogenic factor VEGF by ELISA analysis. As shown in
Fig. 8, VEGF secretion increased significantly when
SGC-7901 cells were treated with SDF-1 for 24 h. To
further investigate whether VEGF secretion was
mediated by CXCR7, CXCR7 expression was inhibited by
RNA interference before treatment with SDF-1.
Significant reduction in VEGF secretion was observed in
CXCR7shRNA cells compared with control and NC
cells. Thus, these findings indicate that SDF-1 can
induce VEGF secretion in SGC-7901 cells and that
CXCR7 can serve as a factor involved in regulation of
secretion of VEGF.
Tumor metastasis is a multistep process that involves
the coordinated events of invasion, adhesion, proteolysis,
and migration. Considerable efforts have been made in
recent years to elucidate the biological function of
chemokine receptors in cancer invasion and metastasis. To
date, the role of CXCR7 in regulating gastric cancer cell
invasion is unclear. In this study, we found that
expression of CXCR7 is elevated in all five gastric cancer cell
lines. In addition, we observed that treatment with
SDF1 enhanced proliferation and invasion, and silencing of
CXCR7 significantly inhibited the proliferation and
invasive ability of SGC-7901 cells. Our study indicated the
significance of CXCR7 on gastric cancer cell
proliferation and invasion. These results are consistent with
recent studies showing that CXCR7 mediates chemotaxis
of cancer cells toward SDF-1 [21, 26].
Tumor cells interact with ECM components and
basement membranes, an essential initial event during the
process of invasion. It also has been reported that
expression of CXCR7 can regulate adhesion of tumor cells
to endothelial cells [14, 27]. Our results demonstrated
that SDF-1 could induce adhesion of SGC-7901 cells to
FN and LN. The enhanced cell-matrix adhesion may
contribute to the metastasis of tumor cells. In addition,
we also found that RNA-mediated downregulation of
CXCR7 significantly inhibited SDF-1-induced adhesion
of SGC-7901 cells to LN or FN. Therefore, these
Fig. 5 Silencing of CXCR7 inhibits SDF-1-induced enhancement on SGC-7901 cell invasion in vitro. a CXCR7shRNA transfected, NC, and control
cells were treated with and without SDF-1 (0 or 100 ng/ml). b Mean number of invasive cells from five independent fields/well is indicated. Data
are expressed as means ± SD from three independent experiments. ***P < 0.001 (as compared with control cells)
findings clearly indicate that CXCR7 participate in
SDF1-induced cell-matrix adhesion.
Some studies have shown that CXCR7 cannot
trigger chemotaxis and activate calcium mobilization and
intracellular signaling cascades, such as PI3K and
ERK pathways [14, 28]. However, a recent study has
demonstrated that CXCR7 is not a decoy but a
functional seven-transmembrane span chemokine receptor
and can induce phosphorylation of MAPK p42/44 and
AKT in human rhabdomyosarcoma cell lines . In
this study, we did not elucidate the molecular
mechanisms by which CXCR7 regulated the proliferation,
adhesion, and invasion of gastric cancer cells. Another
recent study suggests that signaling pathways
mediated by CXCR7 are independent of those triggered
through CXCR4 . Therefore, it is reasonable to
speculate that CXCR7 may exert effects on other signaling.
Also, the different biological effects elicited by CXCR7
may depend on cell type. Thus, further studies elucidating
roles of CXCR7 in invasion and signaling cascades
activated by SDF-1/CXCR7 axis are required.
Cancer cells depend on angiogenesis to survive and
proliferate. We observed that gastric cancer cells could induce
in vitro tube formation, which could promote tumor
growth . Although SDF-1-induced VEGF secretion has
been reported in various cells, such as lung and prostate
cancer cells [21, 31], SDF-1-induced VEGF production in
gastric cancer cells has not been previously studied. In the
current study, we found that SDF-1/CXCR7 interaction
promoted the secretion of VEGF, a potent survival factor
for endothelial cells, and one of the most prominent
angiogenic factors produced by various tumor cells.
Furthermore, our data demonstrate that the knockdown of
CXCR7 inhibits secretion of VEGF and tube
formation, suggesting that CXCR7 may be involved in the
regulation of angiogenesis in gastric cancer.
Fig. 6 Effect of CXCR7 silencing on SGC-7901 cell adhesion in vitro. SGC-7901 cells were treated as described in the “Methods” section. Each bar
represents mean ± SD from three independent experiments. ***P < 0.05 (as compared with untransfected cells)
Fig. 7 Effect of CXCR7 silencing on tube formation induced by SGC-7901 cells. HUVECs were cocultured with SGC-7901 cells, as described in the
“Methods” section. a Representative images of tube-like structures are given for control, NC, and CXCR7shRNA transfected SGC-7901 cells with or
without SDF-1. b Quantitative analysis of the number of tubes. Each bar represents mean ± SD from three independent experiments. ***P < 0.001
(as compared with control cells)
Fig. 8 SDF-1 induces VEGF secretion through CXCR7 in SGC-7901
cells. SGC-7901 cells were plated in the 24-well plates. SGC-7901 cells
were serum starved for 24 h, and the cells were treated with SDF-1
(0 or 100 ng/ml). The cultured supernatants were harvested 24 h
after treatment, and VEGF was measured by ELISA assay. Each
bar represents mean ± SD from three independent experiments.
***P < 0.001 (as compared with control cells)
The above findings imply that SDF-1/CXCR7
interaction may regulate multiple processes in gastric cancer
gastric cancer invasion and tumor growth. First, CXCR7
was expressed in all gastric cancer cells. Second, CXCR7
could affect SDF-1-induced tumor cell proliferation,
adhesion, and invasion. Third, CXCR7 could regulate
gastric cancer invasive ability through angiogenesis and
VEGF secretion. Thus, we provide mechanistic evidence
that SDF-1/CXCR7 interaction may affect gastric cancer
progression by multiple mechanisms including
proliferation, adhesion, invasion, angiogenesis, VEGF production,
and tumor growth. Because CXCR4 is also a receptor for
SDF-1, we cannot exclude the possibility that CXCR4
may be involved in regulating these biological behaviors
triggered by CXCR7. Although our study shows the
importance of CXCR7 in gastric cancer proliferation,
adhesion, invasion, and angiogenesis, the role of SDF-1/
CXCR7 interaction in tumor progression are not fully
established. Moreover, a recent study has shown that
AMD3100, a small synthetic inhibitor of CXCR4, not
binds only to CXCR4 but also to CXCR7 . We
propose that more attention should be paid to SDF-1/
CXCR4 axis and SDF-1/CXCR7 axis. Thus, further
studies elucidating the role of SDF-1/CXCR7 axis in cancer
development are needed.
In summary, we presented the first evidence that CXCR7
was expressed in gastric cancer cells. We also observed
that suppression of CXCR7 expression by RNA
interference impairs in vitro cellular invasion, adhesion, VEGF
secretion, and angiogenesis. Taken together, this study
provides novel evidence that inhibition of CXCR7
expression may be an effective approach to suppressing
tumor growth of gastric cancer.
Availability of data and materials
The data will not be shared because not all authors agreed with this.
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