| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
1Correspondence: Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1–5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: mshichiri.cme{at}tmd.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: endostatin • angiogenesis • angiostatin • migration • c-myc
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3)
. Systemic therapy with
recombinant endostatin results in tumor regression via a complete
inhibition of angiogenesis (3)
. Presently, the
antiangiogenesis signals triggered by endostatin are unknown.
Endostatin is an endogenous 20 kDa carboxyl-terminal fragment of
collagen XVIII, and systemic therapy is not associated with acquired
resistance, as mouse tumors do not develop resistance to multiple
cycles of endostatin therapy (3
, 4)
. Tumors produce
angiogenesis factors that promote vascularity in areas of new growth.
Therefore, angiogenesis inhibitors must elicit potent and profound
signals to overcome the combined effects of these factors. In reduced
serum conditions, endostatin induces endothelial apoptosis and blocks
proliferation and migration induced by vascular endothelial growth
factor (VEGF) (5
6
7
8)
. In this study, we present evidence
that endostatin potently down-regulates many growth- and
apoptosis-related genes in exponentially growing endothelial cells, the
spectrum of which is similar to that following serum deprivation.
However, in serum-supplemented conditions that represent maximal growth
stimulation, endostatin is not pro-apoptotic and only slightly reduces
proliferation and cell cycle transition. Endostatin appears to trigger
gene suppression signals specifically in growing endothelial cells,
resulting in a potent antimigratory effect. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasmids
The c-myc expression plasmid pSPT-myc cDNA,
originally developed by Dr. N. Nomura, was obtained from the Human
Science Research Resources Bank (Osaka, Japan). pSP-Max and pSP-Mad
plasmids were kindly supplied by Dr. R. N. Eisenman, of the Fred
Hutchinson Cancer Research Center (Seattle, Wash.). All recombinant DNA
manipulations were performed by standard procedures.
Cell culture
Cells were maintained in a 5% CO2
atmosphere at 37°C. Endothelial cells were prepared from either the
aorta (rAE cells) or the pulmonary artery (rPAE cells) of male Wistar
rats by collagenase and elastase digestion (9)
. Aortic
vascular smooth muscle (rVSM) cells were prepared by the explant method
(10)
. These primary cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS). The following panel of fibroblast cell lines was
generously provided by Dr. John M. Sedivy (Brown University,
Providence, R. I.) and cultured in DMEM supplemented with
glutamine and 10% calf serum: TGR-1, a nontransformed diploid cell
line originally derived from Rat-1 fibroblasts (11)
; two
homozygous c-myc knockout cell lines, HO15.19 and HO16C
(12)
, derived from their heterozygous parental cells,
HET15 and HET16, respectively (13)
; and LACO3 and LACO16,
each containing a stable c-myc transgene introduced into
HET15 and HET16, respectively (13
, 14)
. Human stomach
adenocarcinoma cells, MKN-1 (JCRB0252), obtained from the Human Science
Research Resources Bank (Osaka), were cultured in RPMI 1640 with 10%
FBS. Human umbilical vein endothelial cells (HUV-EC-C,
CRL-1730) obtained from American Type Culture Collection (Manassas,
Va.) were cultured in Ham’s F12K medium with 2 ml of glutamine, 0.1
mg/ml heparin, 0.05 mg/ml endothelial cell growth supplement, and 10%
FBS. Human adult dermal microvascular endothelial (hADM) cells were
purchased from Cell Systems (Kirkland, Wash.) and cultured with the
CS-C Medium Kit supplemented with VEGF, basic fibroblast growth factor
(bFGF), heparin, endothelial cell growth supplement, and 20% FBS.
Endothelial origin was confirmed by cobblestone morphology and by
immunocytochemistry with antibodies to factor VIII-related antigen and
rat endothelial cell antigen (Sanbio BV, Uden, The Netherlands).
Quantification of mRNAs by using two reverse transcriptase
(RT)-PCR methods
For quantification of rat c-myc,
max, integrin 
v,
-tubulin, and
ß-tubulin mRNA, we used the following two
methods: 1) a real-time quantitative RT-PCR method based on the TaqMan
fluorescence method with the ABI Prism 7700 Sequence Detection System
(PE Applied Biosystems, Foster City, Calif.) and 2) a LightCycler
(Roche Molecular Biochemicals, Mannheim, Germany) PCR protocol in which
fluorescence emission resulting from binding of SYBR Green I dye to
amplified products could be detected and measured. Both the ABI Prism
7700 Sequence Detection System and the LightCycler showed statistically
identical results when we quantified transcripts of c-myc,
max, integrin v,
-tubulin, and
ß-tubulin. Therefore, subsequent analyses of
genes regulated by endostatin and angiostatin were performed with the
LightCycler. Total RNA was extracted by using RNA zol B (TEL-TEST,
Friendswood, Tex.) and cDNA synthesized with a First-Strand cDNA
Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, N.J.).
Quantification of c-myc and max using the TaqMan
fluorescence method was performed as described previously
(9)
. Rat integrin v,
-tubulin, and
ß-tubulin were amplified (integrin
v: forward primer
5'-GCAAGACTGTCCTGTGCATTTAAG-3', reverse primer
5'-AGTTGAGTTCCAGCCTTCTTCG-3', and TaqMan probe
6FAM-AGAAAACCAAACCCGGCAGGTGGT-TAMRA;
-tubulin: forward primer
5'-TGTGGTCCCCAAAGATGTCA-3', reverse primer 5'-AAACTGGATGCTGCGCTTG-3',
and TaqMan probe 6FAM-TGCTGCCATTGCCACCATCAAGA-TAMRA;
ß-tubulin: forward primer
5'-GCAGATGCTCAACGTGCAGA-3', reverse primer 5'-GGCCGTCTTCACATTGTTGG-3',
and TaqMan probe 6FAM-CAAGAACAGCAGCTACTTCGTGGAATGGA-TAMRA) and the
reaction produced 88 bp, 45 bp, and 53 bp products, respectively.
LightCycler was also used to quantify mRNA. Each amplification reaction
(DNA Master SYBR Green I: Roche Diagnostics) contained 50 nM template
cDNA, 0.5 µM primer DNA, and 4 mM MgCl2.
TaqStart antibody (CLONTECH, Palo Alto, Calif.) was used to prevent
generation of nonspecific amplification products. The sequences of each
set of primers are listed in Table 1
. Quantification of c-myc using the LightCycler was performed
by using two sets of primers, each amplifying either exon 2 or exon 3.
After the completion of each extension step (72°C), the fluorescence
of each sample was measured at 82°C to exclude any possible
nonspecific reactions. After the amplification was finished, the
products were subjected to a temperature gradient from 65 to 95°C at
0.2°C/s with continuous fluorescence monitoring to produce a melting
profile of the products. The fluorescence data were quantitatively
analyzed by using serial dilution of control samples included in each
reaction to produce a standard curve. For verification of the melting
curve results, the PCR reactions were examined by 1.5% agarose gel
electrophoresis.
|
Western blot analyses
Quantitative Western blotting was performed as previously
described (9)
. Confluent cells (5
x106 cells/dish) were treated with and without
10-6 M endostatin for 16 h. Extracted
proteins were separated on an 8% SDS-polyacrylamide gel and
transferred to Hybond ECL nitrocellulose membranes (Amersham). Primary
incubations were with the following primary antibodies at 4°C
overnight: rabbit polyclonal antibody against human Max p21/p22 (1:500;
Santa Cruz Biotechnology, Santa Cruz, Calif.), mouse monoclonal
antibody against -tubulin (1:1,000; Sanbio BV), mouse monoclonal
antibody against ß-tubulin (1:1,000; Chemicon International,
Temecula, Calif.), mouse monoclonal antibody against Bcl-2 (1:500;
Transduction Laboratories, Lexington, Ky.), mouse monoclonal antibody
against mitogen-activated protein kinase MAPK-1 (1:5000; Transduction
Laboratories), rabbit polyclonal antibody against integrin v
(1:100; Chemicon International), and mouse monoclonal antibody against
integrin ß3 (1:250; Transduction Laboratories). The secondary
antibody (donkey anti-rabbit immunoglobulin G or sheep anti-mouse
immunoglobulin horseradish peroxidase, 1:500; Amersham) was incubated
for 1 h, and exposure was performed by using an ECL kit
(Amersham).
Secretion of endothelin-1 from endothelial cells
rAE cells plated in 3.5 cm dishes were pretreated with
endostatin, or were not pretreated with endostatin, for 16 h,
after which replacement fresh serum-free DMEM was used for 4 h.
Endothelin-1 concentrations in the conditioned media were measured by
using an Endothelin-1 ELISA system (Amersham) according to the
manufacturer’s protocol.
Cell cycle analysis and cell proliferation assay
Exponentially growing endothelial cells that received treatment
with or without endostatin, or were deprived of serum for 14 h,
were harvested by trypsinization and stained with propidium iodide (50
µg/ml in 0.1% Triton X-100/0.1% sodium citrate, pH 7.0) in subdued
light for 15 min at 4°C. Cellular fluorescence signals were recorded
with an FACS Calibur flow cytometer (Becton Dickinson, Mountain View,
Calif.) using the CELL QUEST software program. The 72 h cell
proliferation assays were performed as previously described
(3)
without reducing serum or growth factor
supplementation. Cells were plated in 12-well dishes at a density of
1.5 x 104 cells/well and were incubated for
72 h in the presence or absence of endostatin. Cells were released
from culture dishes by trypsinization and were counted by using a
Sysmex CDA-500 Autoanalyzer (Toa Medical Electronics, Kobe, Japan).
Detection of apoptosis
To detect nucleosomal ladders, fragmented cellular DNA was
extracted by using Nonidet P-40 lysis, fractionated on a 1.6% agarose
gel and stained with ethidium bromide (14
15
16)
. Cells were
also stained for free 3'-hydroxyl ends of DNA fragments with
dUTP-fluorescein isothiocyanate by using an APO-DIRECT apoptosis
detection kit (PharMingen, San Diego, Calif.) and counterstained
for total DNA content with propidium iodide. Stained cells were then
analyzed with an FACS Calibur flow cytometer as described previously
(10)
. Caspase-3 activity was assayed by using the CaspACE
Assay System, Colorimetric (Promega, Madison, Wis.) as described
earlier (10)
.
Determination of intracellular free Ca2+
concentrations ([Ca2+]i)
Cells were dispersed with 0.25% trypsin/0.02% EDTA and were
incubated with 5 µM Fura-2 acetoxymethyl ester (Molecular Probes,
Eugene, Ore.) at 37°C for 20 min in HBBS. Fluorescence of
Fura-2-loaded cells was measured by using continuous rapid alternating
excitation from dual monochromators (340 and 380 nm) and emission at
510 nm (CAF-100, Japan Spectroscopic, Tokyo). The readings were
converted to [Ca2+]i as
previously described (17)
.
Determination of kinase activities and measurement of cAMP
PathDetect Trans-Reporting Systems (Stratagene, La Jolla,
Calif.) were used to measure p42/p44 MAPK, p38 kinase, c-Jun N-terminal
kinase (JNK), and cyclic AMP-dependent protein kinase (PKA) activities.
The system includes the fusion activator plasmids that consist of the
DNA binding domain of the yeast GAL4 fusion activator (pFR-Luc) and the
activation domain of the transcription factor, Elk1, CHOP, c-Jun, and
CREB (pFA2-Elk1, pFA-CHOP, pFA2-cJun, pFA2-CREB). Cells plated in
96-well plates were cotransfected with each pFA2 plasmid (50 ng
each/well), pFR-Luc reporter plasmid (1 µg each/well), and pRL-TK
vector (1 µg each/well) (Promega), which expresses renilla luciferase
as an internal control. Transient transfections were performed with the
synthetic cationic liposome (+)-N,N-bis
(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl]ammonium
iodide according to instructions provided by the supplier (Promega) and
were modified to use transferrin-receptor operated transfer as
described earlier(17
, 18)
. The cells were incubated for
48 h after transfection in DMEM containing 10% FBS and were
further incubated with endostatin for 6 h, after which firefly and
renilla luciferase activities were measured via the Dual Luciferase
Reporter Assay System (Promega) in a single-tube assay format using
MicroLumatPlus (EG&G Berthold, Wildbad, Germany).
The firefly luciferase activity of each sample was normalized to an
internal reference standard of renilla luciferase activity. The
intracellular cAMP concentration of endothelial cells before and 15 min
after endostatin treatment was measured by using a cAMP enzyme
immunoassay (EIA) system (Amersham).
Monolayer-denuding cell migration assay
Cells were cultured in collagen-coated 3.5 cm dishes (Iwaki,
Chiba, Japan) to confluence, incubated for 3 days, and then received
pretreatment with or without endostatin for 16 h. Several areas
300 µm wide were denuded with a sterile single-edged surgical
blade (Surgical Blade No. 11, Stainless Steel, Feather Safety Razor,
Osaka, Japan). Phase-contrast photomicroscopy at 100 x
magnification was performed immediately and 4, 6, 8, 16, and 24 h
after denudement at four distinct sites, with the center of the wound
(marked with a scratch) in the middle of each frame. The mean distance
from a defined line in the middle of the wound to the nuclei of 20
furthest-migrating cells in a particular frame was measured by Adobe
Photoshop 5.0 software after incorporation of photographs using GT8000
(Epson, Tokyo, Japan) and then normalized to a standard field width.
Distance migrated was calculated from the mean distances in successive
frames of a particular field. Repeated measurements of the same area
generated standard deviations in the range of 1–4%.
Statistical analysis
All values were given as means ± SE.
Statistical analysis was performed with Student’s t test or
Wilcoxon’s rank sum test. P values less than 0.05 were
considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Endostatin treatment down-regulated mxi1,
bcl-2, bcl-X(L), and bad but did not
alter the expression of other genes such as bax,
p53, cdc25A (Fig. 1)
, or p38 kinase (data not
shown). Endostatin also down-regulated cdc25B,
MAPK-1 and MAPK-2,
-tubulin, ß-tubulin,
integrin v, integrin
ß3, AT1 and
AT2 (angiotensin II receptors),
ETB (endothelin receptor type B), and
preproendothelin-1. Endostatin did not alter gene expression
in quiescent rAE cells, exponentially growing rVSM cells, or TGR-1
fibroblasts (data not shown). The results of angiostatin treatment were
in marked contrast to those for endostatin. Angiostatin treatment
down-regulated c-fos, MAPK-2, and
bcl-2 and up-regulated mxi1, bad,
bax, p53, ETB,
and preproendothelin-1 (Fig. 2
).
|
|
The ability of endostatin to down-regulate gene expression is not
restricted to rAE cells but was also observed in hADM cells (Fig. 3A
) as well as in rPAE cells and HUV-EC cells (data not
shown). In hADM cells, endostatin treatment down-regulated the
expression of c-myc, max, mxi1,
integrin v, integrin
ß3, ETB,
preproendothelin-1, PECAM-1, FAK
(focal adhesion kinase), and cadherin-5. The magnitude of
down-regulation of most of these genes was more pronounced in hADM
cells than in rAE cells, even when the culture media were supplemented
with VEGF, bFGF, and 20% FBS.
|
Despite rapid mRNA down-regulation, endostatin treatment did not
significantly reduce the protein levels of many constitutively
expressed genes in hADM cells. Quantitative Western blot analyses
revealed a minimal inhibitory effect on Max p21/p22, whereas there was
no appreciable change in p42/p44 MAPK-1, -tubulin, ß-tubulin, or
Bcl-2. However, endostatin treatment suppressed the protein levels of
integrin v and integrin ß3 (Fig. 3B
). Pretreatment with
endostatin decreased the amount of endothelin-1 secreted by rAE cells
into culture media in a dose-dependent manner
(10-8-10-6 M) (Fig. 3C
).
Endostatin increased [Ca2+]i and cAMP
activity
Endostatin induced a sustained increase in the
[Ca2+]i in a
dose-dependent manner
(10-8-10-6 M) (Fig. 4A
). The magnitude of increase in
[Ca2+]i was greater after
VEGF treatment. The increase in
[Ca2+]i by endostatin was
blocked by pretreatment with either nicardipine or EGTA. However,
neither nicardipine nor W7 (a Ca2+/calmodulin
inhibitor) blocked the ability of endostatin to down-regulate gene
expression. Endostatin treatment activated cAMP-dependent protein
kinase (Fig. 4B
, left panel) and increased intracellular
cAMP in a dose-dependent manner (Fig. 4B
, right panel).
Endostatin treatment did not alter the activity of p42/p44 ERK/MAP
kinase, p38 kinase, or JNK.
|
Endostatin had a weak growth-inhibitory influence on
endothelial cells
Flow cytometry, caspase 3 determination, and TUNEL staining showed
that endostatin did not induce apoptosis in rAE cells or hADM cells
cultured in 10% FBS. Yet endostatin treatment did reduce the
percentage of cells in S phase (Fig. 4C
). The 72 h
proliferation assays (3)
revealed the weak
growth-inhibitory effect of endostatin on exponentially growing rAE,
HUV-EC, and hADM cells but not on rPAE (Fig. 4D
), TGR-1, or
rVSM cells or a human gastric cancer cell line, MKN-1.
Endostatin arrested endothelial cell migration
The antimigratory effect of endostatin and angiostatin
was evaluated with a monolayer-wounding protocol in which cells migrate
from a confluent area onto a denuded area. Cell proliferation assays
demonstrated that endostatin did not affect the growth rate of any
cells during the initial 24 h.
[3H]Thymidine incorporation revealed that
confluent cultures of hADM or rAE cells that are denuded do not
proliferate during the first 8 h. Therefore, repopulation within
8 h is due solely to cell migration. Endostatin inhibited the
migration of rAE cells (Fig. 5A
) and rPAE cells (data not shown) in a dose-dependent
manner. After endostatin pretreatment, rAE cells and rPAE cells
migrated more slowly than did controls. A minority of treated cells
migrated, whereas a majority of control cells migrated rapidly until
the denuded area was confluent (Fig. 5B
). Arrested cells
regained the ability to migrate when endostatin was removed from the
culture medium. Endostatin did not inhibit the migration of
nonendothelial cells such as TGR-1 (Fig. 6B
) or rVSM cells (data not shown). hADM cells cultured with
complete growth supplementation migrated faster than did rAE cells yet
were still subject to migration arrest by endostatin treatment (Fig. 5C
). When pretreated with 10-6 M
endostatin, hADM cells appeared almost unable to migrate (Fig. 5D
). In contrast, angiostatin did not show an antimigratory
effect on endothelial cells.
|
|
c-myc down-regulation correlated with the arrest
of cell migration
A c-myc-expressing plasmid (pSPT-Myc) was transiently
transfected into hADM cells by using transferrin receptor-operated
transfer (9)
, and the resulting elevation in
c-myc expression was not suppressed by endostatin treatment.
Transfection of pSPT-Myc resulted in an increased migratory rate, and
endostatin treatment failed to arrest migration of hADM cells
transfected with pSPT-Myc (Fig. 6A
). Overexpression of
max or mad down-regulated E-box transcription
because Max-Mad heterodimers competitively inhibited Myc-Max
heterodimers. Transfection of pSP-Max or pSP-Mad resulted in a
significant decrease in the migration rate of hADM cells (Fig. 6A
).
A panel of isogenic fibroblast cell lines with differential
c-myc expression levels was used to investigate the role of
endogenous c-myc in cell migration. We compared two
c-myc null cell lines obtained by gene targeting of the
endogenous c-myc copies
(myc-/-) (12)
to their
isogenic diploid parental cell line TGR-1
(myc+/+) (11)
, and two
derivatives, LACO3 and LACO16, which stably overexpress
c-myc (myc-/+,++) (13
, 19)
. The two myc-/- cell lines
showed a reduced migratory rate compared with TGR-1 cells (Fig. 6B
). The c-myc transgene-expressing cells
(myc-/+,++) displayed an increased
migratory rate (Fig. 6B
).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, down-regulates a variety of
genes in growing endothelial cells. Immediate early genes, such as
c-fos and c-myc, were down-regulated
simultaneously with max. Other genes closely related to cell
growth, such as cdc25B, MAPK-1,
MAPK-2, preproendothelin-1,
ETB receptor, and
AT1 receptor, were also
down-regulated. Endostatin down-regulated genes encoding for cell
structure components such as - and ß-tubulin, which polymerize
into microtubules that form a network of essential structures including
mitotic spindles (20)
. Because the downstream consequences
of endostatin treatment on gene expression are similar to those
following serum deprivation, we initially speculated that endostatin
exerts an antiangiogenesis effect by eliciting quiescence signals,
rapidly shifting growing endothelial cells into
G0. However, our results show that endostatin has
only a weak growth-inhibitory effect on endothelial cells. Quiescence
is therefore unlikely to be the mechanism for the potent in
vivo antiangiogenesis activity of endostatin.
Several explanations may account for the inability of endostatin to
arrest endothelial cell proliferation. First, the rapid mRNA
down-regulation of constitutively expressed genes such as
max or MAPK-1 was not immediately followed by a
decrease in their protein levels. Second, growth signals may be
mediated by changes in protein phosphorylation and therefore not
influenced by changes in protein levels. For example, our results show
that endostatin did not alter p42/p44 ERK/MAPK or p38 kinase activity.
Third, reduced c-myc levels may not markedly inhibit cell
growth because c-myc heterozygous knockout cells
(myc-/+) exhibit only a 2 h delay in
the G1 to S transition (13)
, and
c-myc null cell lines (myc-/-)
still grow (12)
. Fourth, c-myc suppression
itself may not cause down-regulation of other growth-associated genes.
We have confirmed a previous report (21)
that the
expression of proposed "c-myc target" genes, such as
cdc25A and p53 (22
, 23)
, that are
up-regulated by c-myc overexpression are unchanged in
c-myc null cells.
Endostatin dramatically arrested the migration of endothelial cells.
This effect was especially profound in hADM cells, which normally
migrate faster than do endothelial cells derived from larger vessels.
An ideal angiogenesis inhibitor must offset the combined effects of
growth factors and other angiogenesis mediators that are vigorously
produced by tumors. In the present study, the ability of endostatin to
arrest endothelial cell migration was undiminished even when cells were
maximally stimulated with nutrients and growth factors. Endostatin also
inhibited genes associated with cell migration and angiogenesis, such
as endothelin-1, ETBreceptor, integrin v,
integrin ß3, FAK,
cadherin-5, and PECAM-1. Endothelin-1, for
example, is an endothelial cell growth factor (18)
that
mediates migratory and angiogenic effects via the
ETB receptor (24)
. The cell adhesion
molecules integrin vß3 and integrin vß5, expressed on the
surface of endothelial cells, mediate interactions with the
extracellular matrix, an important process during angiogenesis.
Although the arrest of endothelial cell migration is certainly
multifactorial, our results suggest that one intracellular factor is
the down-regulation of c-myc expression. Transfection of a
c-myc-expressing vector completely abrogated the
antimigratory effect of endostatin, whereas antagonism of c-Myc
function at E-box promoters by overexpressing max or
mad resulted in a reduced endothelial cell migration rate.
In addition, c-myc null fibroblasts showed a reduced
migration rate compared with their isogenic parental cells, again
suggesting a role for c-myc in cell migration. It should be
noted, however, that neither overexpression of max and
mad nor c-myc gene knockouts completely arrested
migration, suggesting that cells can still migrate in the absence of
c-myc and that c-myc is not uniquely responsible
for endostatin-induced migration arrest. Taken together, the results
indicate that c-myc is involved in both baseline cell
migration and endostatin-induced inhibition of endothelial migration,
although down-regulation of other migration-associated genes may likely
contribute to the potent antimigratory effect of endostatin.
The intracellular events immediately following endostatin stimulation
leading to the down-regulation of genes remain unknown. Because
endostatin increased
[Ca2+]i via the influx of
extracellular Ca2+ and activated PKA, signals
triggered by endostatin may be transmitted via an as yet unidentified
cell surface receptor. Limited mechanistic insights into the action of
endostatin have been previously described. Endostatin has been shown to
antagonize both the cell cycle progression and the migration of
quiescent human umbilical endothelial cells induced by bFGF (5
, 8)
and to cause apoptosis of FGF-treated endothelial cells by
suppressing bcl-2 without affecting bax
(6)
or by inducing tyrosine kinase activity
(7)
. Although these data are consistent with ours,
down-regulation of apoptosis inhibitors would not cause the massive
apoptotic elimination of tumor vascular beds in in vivo
endostatin therapy. Endostatin-induced apoptosis and bcl-2
suppression were blocked by the addition of serum or
endothelium-derived survival factors to the culture medium (data not
shown). Repeated use of any agent that induces massive endothelial
apoptosis or complete growth arrest could result in systemic vascular
diseases, including ischemic heart disease. Such side effects have not
been previously reported with endostatin. Arrest of endothelial cell
migration more clearly explains the mechanism of tumor regression. In
contrast to endostatin, angiostatin showed a limited gene suppression
profile and was devoid of an ability to arrest endothelial cell
migration. This difference suggests a different mechanism of
antiangiogenesis for the two factors. Angiostatin has been shown to
bind to the ß subunits of ATP synthase on the cell surface, which
may result in tumor regression by depleting the ATP energy required for
angiogenesis (25)
. Therefore, the action of angiostatin
may not require the down-regulation of the number of genes as seen with
endostatin. To date, no bioactive substance is known to exert such a
novel antimigratory signal.
|
ACKNOWLEDGMENTS |
|---|
Received for publication December 22, 1999.
Revision received October 5, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Yuan, Y. Fu, X. Wang, H. Shi, Y. Huang, X. Song, L. Li, N. Song, and Y. Luo Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis FASEB J, August 1, 2008; 22(8): 2809 - 2820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Sodha, M. Boodhwani, R. T. Clements, S.-H. Xu, K. R. Khabbaz, and F. W. Sellke Increased Antiangiogenic Protein Expression in the Skeletal Muscle of Diabetic Swine and Patients Arch Surg, May 1, 2008; 143(5): 463 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.R. Dass, T.M.N. Tran, and P.F.M. Choong Angiogenesis Inhibitors and the Need for Anti-angiogenic Therapeutics J. Dent. Res., October 1, 2007; 86(10): 927 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Suhr, K. Brixius, M. de Marees, B. Bolck, H. Kleinoder, S. Achtzehn, W. Bloch, and J. Mester Effects of short-term vibration and hypoxia during high-intensity cycling exercise on circulating levels of angiogenic regulators in humans J Appl Physiol, August 1, 2007; 103(2): 474 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Schaffhauser, T. Veikkola, K. Strittmatter, H. Antoniadis, K. Alitalo, and G. Christofori Moderate antiangiogenic activity by local, transgenic expression of endostatin in Rip1Tag2 transgenic mice J. Leukoc. Biol., October 1, 2006; 80(4): 669 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Celik, O. Surucu, C. Dietz, J. V. Heymach, J. Force, I. Hoschele, C. M. Becker, J. Folkman, and O. Kisker Therapeutic Efficacy of Endostatin Exhibits a Biphasic Dose-Response Curve Cancer Res., December 1, 2005; 65(23): 11044 - 11050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Sugiyama, T. Yoshimoto, K. Tsuchiya, N. Gochou, Y. Hirono, T. Tateno, N. Fukai, M. Shichiri, and Y. Hirata Aldosterone Induces Angiotensin Converting Enzyme Gene Expression via a JAK2-Dependent Pathway in Rat Endothelial Cells Endocrinology, September 1, 2005; 146(9): 3900 - 3906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Nyberg, L. Xie, and R. Kalluri Endogenous Inhibitors of Angiogenesis Cancer Res., May 15, 2005; 65(10): 3967 - 3979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Marneros and B. R. Olsen Physiological role of collagen XVIII and endostatin FASEB J, May 1, 2005; 19(7): 716 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Tjin Tham Sjin, R. Satchi-Fainaro, A. E. Birsner, V.M. S. Ramanujam, J. Folkman, and K. Javaherian A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity Cancer Res., May 1, 2005; 65(9): 3656 - 3663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Zhang, E. G. Teggatz, A.-P. Zou, W. B. Campbell, and P.-L. Li Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2-{middle dot} production in the intact coronary endothelium Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H686 - H694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. Skovseth, M. J. T. Veuger, D. R. Sorensen, P. M. De Angelis, and G. Haraldsen Endostatin dramatically inhibits endothelial cell migration, vascular morphogenesis, and perivascular cell recruitment in vivo Blood, February 1, 2005; 105(3): 1044 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Fukai, T. Yoshimoto, T. Sugiyama, N. Ozawa, R. Sato, M. Shichiri, and Y. Hirata Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E56 - E62. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Ozawa, M. Shichiri, N. Fukai, T. Yoshimoto, and Y. Hirata Regulation of Adrenomedullin Gene Transcription and Degradation by the c-myc Gene Endocrinology, September 1, 2004; 145(9): 4244 - 4250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yoshimoto, N. Fukai, R. Sato, T. Sugiyama, N. Ozawa, M. Shichiri, and Y. Hirata Antioxidant Effect of Adrenomedullin on Angiotensin II-Induced Reactive Oxygen Species Generation in Vascular Smooth Muscle Cells Endocrinology, July 1, 2004; 145(7): 3331 - 3337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Davis, Y. Shen, N. A. Mullani, S. Wen, R. S. Herbst, M. O'Reilly, J. L. Abbruzzese, and D. J. McConkey Quantitative Analysis of Biomarkers Defines an Optimal Biological Dose for Recombinant Human Endostatin in Primary Human Tumors Clin. Cancer Res., January 1, 2004; 10(1): 33 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Macpherson, S. S.W. Ng, S. L. Forbes, G. Melillo, T. Karpova, J. McNally, T. P. Conrads, T. D. Veenstra, A. Martinez, F. Cuttitta, et al. Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay Mol. Cancer Ther., September 1, 2003; 2(9): 845 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. DEININGER, W. A. WYBRANIETZ, F. T.C. GRAEPLER, U. M. LAUER, R. MEYERMANN, and H. J. SCHLUESENER Endothelial endostatin release is induced by general cell stress and modulated by the nitric oxide/cGMP pathway FASEB J, July 1, 2003; 17(10): 1267 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Nyberg, P. Heikkila, T. Sorsa, J. Luostarinen, R. Heljasvaara, U.-H. Stenman, T. Pihlajaniemi, and T. Salo Endostatin Inhibits Human Tongue Carcinoma Cell Invasion and Intravasation and Blocks the Activation of Matrix Metalloprotease-2, -9, and -13 J. Biol. Chem., June 13, 2003; 278(25): 22404 - 22411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
) or had no pretreatment with (
) 10-6 M human
endostatin and of representative myc-/-
(
) and myc-/+,++ (
) cells.