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DOI 10.1007/s10147-005-0508-7
ORIGINAL ARTICLE
Yutaka Yonemura Yoshio Endo Kayoko Tabata
Taiichi Kawamura Hyo-Yung Yun Etsurou Bandou
Takuma Sasaki Masahiro Miura
Abstract
Background. The molecular mechanisms of lymphangiogenesis induced by vascular endothelial growth factor
(VEGF)-C and VEGF-D in gastric cancer were studied.
Methods. VEGF-C and VEGF-D gene expression vectors
were transfected into the gastric cancer cell line KKLS,
which did not originally express VEGF-C and VEGF-D,
and stable transfectants (KKLS/VEGF-C and KKLS/
VEGF-D) were established. The cell lines were inoculated
into the subserosal layer of the stomach and subcutaneous
tissue of nude mice.
Results. VEGF-C and VEGF-D expression in KKLS/
VEGF-C and KKLS/VEGF-D cells was found by reverse
transcription polymerase chain reaction (RT-PCR) and
Western blot analysis. Expression of mouse VEGF receptor
(VEGFR)-2 and mouse VEGFR-3 mRNA was detected in
the KKLS/VEGF-C and KKLS/VEGF-D gastric tumors.
Newly formed lymphatic vessels were detected not only in
the periphery but also in the center of the tumors. The
intratumor lymphatic vessels connected with the preexisting
lymphatic vessels in the muscularis mucosa. The average
numbers of lymphatic vessels in KKLS/VEGF-C (52.0
9.5) and KKLS/VEGF-D (16.4 0.6) gastric tumors were
significantly higher than that in the KKLS/control vector
tumors (4.0 1.4).
Conclusion. VEGF-C and VEGF-D may induce neoformation of lymphatic vessels in experimental gastric tumors by
the induction of VEGFR-3 expression.
Introduction
Gastric cancer is the fourth most common malignancy in the
world, and is the second leading cause of death after lung
cancer.1 About 60% of resectable gastric cancers have
lymph node metastasis at the time of diagnosis, and lymph
node metastasis is the strongest prognostic factor. However,
little is known about the molecular mechanisms of lymph
node metastasis in gastric cancer. The multistep process of
lymph node metastasis is initiated by cancer cell invasion
into lymphatic vessels. Proliferation of lymphatic vessels
is a common finding in gastric cancer tissue, and this
favors the permeation of cancer cells into the lymphatic
channels. Accordingly, lymphangiogenesis around cancer
cells may have a crucial role in the initial step of lymph node
metastasis.
Vascular endothelial growth factor (VEGF)-C is wellknown to induce lymphangiogenesis by activating VEGF
receptor (VEGFR)-3, which is expressed on lymphatic
endothelial cells.2
Activation of VEGFR-1 and VEGFR-2 induces mitogenic capacity in endothelial cells of blood vessels.3 VEGF-C
can also activate VEGFR-2 and VEGFR-3, and this activation stimulates both angiogenesis and lymphangiogenesis.3
Recently, we have found that a VEGF-C and VEGFR-3
paracrine loop has a big role in the lymphatic proliferation
of clinical gastric cancer, and that the overexpression of
VEGF-C induces lymph node metastasis.4,5 Induction of
lymphangiogenesis by VEGF-C has been demonstrated in
breast cancer and lung cancer.6,7
Yamada et al.8 identified a new member of the VEGF
family, designated VEGF-D; its amino-acid sequence
shows 23.3% identity with that of VEGF-C. Achen et al.9
reported that VEGF-D binds both VEGFR-2 and VEGFR3, and can activate the receptors similarly to VEGF-C.
319
with the vector alone (control vector) using Fugene 6 transfection reagent. Transfected cells were selected and maintained in growth medium containing 40 mg/ml Zeocin
(Invitrogen).
Stable transfectants of KKLS were designated KKLS/
VEGF-C and KKLS/VEGF-D.
Enzyme-linked immunosorbent assay (ELISA)
Culture medium and anti-VEGF-C antibody (diluted 1 : 200
with bovine serum albumin-phosphate buffered saline
[BSA-PBS], C-20, Santa Cruz Biotech, Santa Cruz, CA,
USA) or anti-VEGF-D antibody (1 : 500; C-18, Santa Cruz
Biotech) were added to a 96-well plate and incubated at
room temperature for 60 min. Wells were washed with a
TBS-Tween buffer. Alkaline phosphatase-conjugated antigoat secondary antibody (diluted 1 : 1000 with BSA-PBS)
was added to each well. After 60-min incubation, the plate
was washed, and p-nitrophenyl phosphate (Chemicon,
Temecula, CA, USA), was dispensed to each well, followed
by incubation for 60 min. The yellow color of nitrophenol
was measured at 405 nm. Serial dilutions (four fold) of
human recombinant VEGF-C (Santa Cruz Biothech) and
VEGF-D (Santa Cruz Biothech), from 20 mg/ml to 5 pg/ml,
were used to make a calibration curve.
Reverse transcription (RT)-polymerase chain
reaction (PCR)
RT-PCR analysis was performed using a modification of
the method of Conboy et al.10 After the isolation of RNA
by Isogen (Nippon Gene, Tokyo, Japan),11 the RNA was
mixed with oligo-dT primer and reverse-transcribed with
AMV reverse transcriptase (Life Sciences, St. Petersburg,
FL, USA), followed by PCR amplification (Perkin-Elmer
Cetus, Norwalk, CT, USA) with primers specific for human
genes and mouse genes specific for lymphangiogenesis.4
The primers specific for human and mouse genes are shown
in Table 1.
Western blot analysis
Protein samples were mixed with sample buffer and
dithiothreitol (DTT) prior to separation with the NuPAGE
Electrophoresis System (Novex, San Diego, CA, USA).4
After the completion of electrophoresis, samples were
transferred to polyvinglidene (PVDF) membranes
(Immobilon; Millipore, Bedford, MA, USA). The membranes were incubated with antibodies (anti-human VEGFC [H190], anti-human VEGF-D [C-18], anti-mouse CD-31
[M-20; Dako, Copenhagen, Denmark], anti-mouse LYVE1 [Santa Cruz Biotech], and anti-mouse VEGFR-3 (Ab
3127; Chemicon) for 2 h. Then the membranes were incubated with the secondary antibodies specific for each
primary antibody for 40 min, rinsed, and treated with
an electrochemi luminescence (ECL) detection reagent
(Amersham Life Science, London, UK).
320
Table 1. Primers specific for anglogenesis- and lymphanglogenesis-related human and mouse genes
Target gene
Sequence
Human VEGF-A
Sense: 5-TCGGGCCTCCGAAACCATGAACTTTCT-3
Antisense: 5-GGTTTCTGGATTAAGGACTGTTCTGTC-3
Human VEGF-C
536
Sense: 5-ATTTGCTGCAGCACATTATAATACAGAGAT-3
Antisense: 5-TCACTATATGAAAATCCTGGCTCACAAGCC-3
Human VEGF-D
514
Sense: 5-TGAGAGTGGTCTTCTGTTCCAGCAAGTGGA-3
Antisense: 5-GCTCAGCATCCCATCGGTCCACTAGGTTTG-3
Human TFR
415
Sense: 5-ACAGACTCTACATGTAGGAT-3
Antisense: 5-AAACCTTGAAGTTGCTGGTA-3
Human VEGFR-1
545
Sense: 5-GCTGCAAATATCTAGCTGTACCT-3
Antisense: 5-GGAATTGCTTTGGTCAATTCGTC-3
Human VEGFR-2
311
Sense: 5-AGACTTTGAGCATGGAAG-3
Antisense: 5-CCATTCCACCAAAAGATG-3
Human VEGFR-3
298
Sense: 5-AGCCATTCATCAACAAGCCT-3
Antisense: 5-GGCAACAGCTGGATGTCATA-3
Human b-actin
591
Sense: 5-GAAAATCTGGCACCACACCTT-3
Antisense: 5-TTGAAGGTAGTTTCGTGGAT-3
Mouse VEGF-A
269
Sense: 5-GGGTTTCGGGAACCAGACCTCTCAC-3
Antisense: 5-CAACCCTAATCTTCCGGGCTTGGCG-3
Mouse VEGFR-1
397
Sense: 5-ATGGCTCGCCTGCAACATTGAAGTC-3
Antisense: 5-CTCTCAATTCTGTTTCCTAAGTTGCTGC-3
Mouse VEGFR-2
700
Sense: 5-TCTGTCAGTGACCAGCATGGCATCGT-3
Antisense: 5-GGAGCTGGGTAACTGAGATACTTCACA-3
Mouse VEGFR-3
699
Sense: 5-CTCCATGACCCCTCCAACCCTGAA-3
Antisense: 5-CTCAGCCCACACTGTACAGTTCAAA-3
Mouse CD31
322
Sense: 5-TCAGAATTTCAGCAAGATCGCCGAGG-3
Antisense: 5-GCACTGGTATTCCATGTCTCTGGTGG-3
Mouse b-actin
540
Sense: 5-GTGGGCCGCTCTAGGCACCAA-3
Antisense: 5-CTCTTTGATGTCACGCACGATTTC-3
Animal experiments
The lymphatic vessel count was assessed by light microscopy in the area of the tumor containing the highest numbers of lymphatic vessels. The areas containing the highest
numbers of lymphatic vessels were identified by scanning
tumor sections. After the area with highest number of lymphatic vessels was identified, a vessel count was performed
in a 100 field.
Evaluation of metastatic ability of cancer cells in chick
embryos by PCR
Cancer cells (106 cells) were injected into the vein of the
chorioallantoic membrane (CAM) of 11-day chick embryos
in fertilized eggs.32,33 After incubation for 7 days, the embryonic livers and lungs were dissected and DNA was extracted
using a rapid DNA preparation.32 The 576-bp product of the
human b-globin gene segment was amplified by PCR. The
sense and anti-sense primers for the human b-globin gene
were: Hu b1 (5-AGAGCCATCTATTGCTTACA-3) and
Hu b8 (5-TATGACATGAACTTAACCAT-3). DNA
from the embryonic organs was amplified by 25 cycles of
PCR with the primers Hu b1 and Hu b8 and analyzed by
agarose gel electrophoresis. The relative density of the
DNA fragments of the b-globin and b-actin genes on a
scanned gel image were quantified using NIH image.
321
Fig. 1. Vascular endothelial growth factorA (VEGF-A), VEGF-C, and VEGF-D
mRNA expression in gastric cancer cell
lines, a fibroblast cell line (KMST-6), an
osteosarcoma cell line (HT1080), and a prostatic cancer cell line (PC-3), TFR, transferrin receptor
Fig. 2. Protein expression in KKLS control vector cells and stable clones of KKLS cells transfected with VEGF-C or VEGF-D expression
vector
Statistical analysis
All statistical calculations were performed using SPSS
statistical software. Data values are expressed as means
plus or minus SDs. The c2 test and Students t-test
were used to determine the significance of intergroup
differences.
Results
VEGF-A, VEGF-C, and VEGF-D mRNA expression in
human cell lines
As shown in Fig. 1, VEGF-A was expressed in all cell lines.
In contrast, VEGF-C mRNA was expressed in two gastric
322
Fig. 3. Western blot analysis of VEGFC and VEGF-D in conditioning
medium of stable clones. After 1-h
treatment with plasmin, 29-kDa Cterminal peptides of VEGF-C and
VEGF-D were detected, but the 58kDa and 53-kDa C-terminal peptides
of VEGF-C and VEGF-D precursor
disappeared
323
Fig. 4. mRNA expression of angiogenesis- and
lymphangiogenesis-related factors in gastric
tumors bearing KKLS/control vector, KKLS/
VEGF-C, and KKLS/VEGF-D cell stable
clones. mVEGFR-1, mouse VEGF receptor-1
324
Fig. 6. Enzyme histochemical evaluation of blood and lymphatic vessels with enzyme-histochemical staining for alkaline phosphatase
(ALPase) and 5-Nase activity in gastric tumor-bearing KKLS/VEGFC cells on day 14. Arterial blood vessels are stained blue. Lymphatic
vessels are scarce. 10
Discussion
VEGF-C is known to have a major role in lymphangiogenesis in cancer tissue, but the relationship between
VEGF-D and lymphangiogenesis is controversial.1315 In the
embryo, VEGF-D is expressed by melanocytes, lung connective tissue, and fibroblasts. In normal adult tissue, it is
detected in lymph nodes, heart, and small bowel.24,25
The present study aimed to clarify the mechanisms of
lymphangiogenesis and the metastatic ability of VEGF-C
and VEGF-D in gastric cancer, using KKLS cells and the
stable transfectants of VEGF-C and VEGF-D genes.
Among the ten gastric cancer cell lines tested, VEGF-C
mRNA was expressed in two cell lines, and it was also
expressed in a fibroblast cell line, KMST-6. Accordingly,
not only cancer cells but also fibroblasts may have a role in
lymphangiogenesis in gastric cancer tissue. We have already
reported that VEGF-C mRNA was expressed in 47% of
primary gastric cancers.4
VEGF-D mRNA, in contrast was not expressed in any
gastric cancer cell lines or in fibroblasts. Ishikawa et al.33
reported that VEGF-D mRNA expression was detected
in only 17% of clinical gastric cancers.23 In colon tissue,
VEGF-D is expressed in the normal gland rather than
in colon cancer or adenoma.31 In contrast, VEGF-D expression is found in 60%80% of breast cancers and oral
cancers.21,22
From these results, it seems that VEGF-D may not have
a main role in lymphangiogenesis in gastric cancer, because
of the low expression rate of VEGF-D in gastric cancer,
although, as shown in the present study, once gastric cancer
cells express VEGF-D, lymphangiogenesis is induced.
Table 2. Metastatic ability of KKLS cells overexpressing VEGF-C and VEGF-D in chick embryo
Clone
Liver metastasis
Lung metastasis
Control vector
35.4 26.3
5.7 8.6
KKLS/VEGF-C
75.1 16.2
P = 0.006 (vs control vector)
93.2 37.8
P = 0.001 (vs control vecctor)
Control vector
14.8 8.8
17.7 14.4
KKLS/VEGF-D
37.0 14.9
P = 0.007 (vs control vecctor)
58.8 11.4
P = 0.0002 (vs control vecctor)
325
Fig. 7AG. Enzyme histochemical
evaluation of lymphatic vessels with
enzyme-histochemical staining for
5-Nase activity. Lymphatic vessels
are stained brown. A Gastric tumorbearing KKLS/control vector cells
on day 28. Lymphatic vessels are not
found in the center of the tumor.
B Gastric tumor-bearing KKLS/
VEGF-C cells on day 28. Lymphatic
vessels are rich in the periphery of
the tumor, and are also found in the
central part of the tumor. C Normal
skin stained for 5-Nase. Lymphatic
vessels are found (asterisks). D Subcutaneous tumor stained for 5-Nase.
Widespread lymphangiogenesis was
detected in the subcutaneous tumorbearing KKLS VEGF-D cells on day
28. E ALP-ase-5-Nase double-staining of normal mouse stomach. Lymphatic vessels are stained brown
(arrows in E, F, G). E, F, G Two
slices from serial sections of KKLS/
VEGF-C gastric tumor. Lymphatic
vessel (hatch symbol in F) connects
with an intratumoral lymphatic
vessel (hatch symbol in G) and a collecting lymphatic vessel in the muscularis mucosa (mm; asterisk in G).
lp, lamina propria; ss, subserosa;
BV, blood vessel; L, lymphatic
vessel; mp, proper muscle
E
A
Fig. 8. Detection of human b-globin gene (576 bp) in the livers and lungs of chick embryos by polymerase chain reaction (PCR), using a specific
primer pair (Hub-1 and Hub-8). Six embryos (lanes 16) for each group were studied for the metastatic ability of cancer cells. M, marker; HT,
HT-1080
326
Conclusion
The present study demonstrated an intimate relationship
between VEGF-C/VEGF-D and lymphangiogenesis in ex-
327
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