| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
vß3 and
vß5 Are Expressed by Endothelium of High-Risk Neuroblastoma and Their Inhibition Is Associated with Increased Endogenous Ceramide1
Divisions of Hematology-Oncology [A. E-E., M. L., L. S. M., R. C. S., D. L. D.] and Infectious Diseases [K. S. K., M. F. S.], Neil Bogart Memorial Laboratories, and Departments of Pediatrics and Pathology [H. S.], Childrens Hospital Los Angeles, University of Southern California School of Medicine, Los Angeles, California 90027; and Department of Preventive Medicine [S. G.], University of Southern California School of Medicine and the Childrens Cancer Group, Arcadia, California 91066
| ABSTRACT |
|---|
|
|
|---|
vß3 and
vß5,
prevents endothelial cell anchorage and induces endothelial apoptosis,
which results in disruption of tumor angiogenesis and inhibition of
tumor growth in animal models. In this study, we demonstrate by
immunohistochemical analysis that integrin
vß3 was expressed by 61% (mean) of
microvessels in high-risk neuroblastomas (stage IV and MYCN-amplified
stage III; n = 28) but only by 18%
(mean) of microvessels in low-risk tumors (stages I and II and
non-MYCN-amplified stage III; n = 12).
Integrin
vß5 was found on 60% (mean) of
microvessels in 21 Stage IV tumors. These data suggest that
neuroblastomas may be targeted for antiangiogenic treatment directed
against endothelial integrins
vß3 and
vß5. In cell culture, inhibition of
integrin-dependent endothelial cell anchorage to vitronectin by RGDfV,
an RGD function-blocking cyclic peptide, induced apoptosis in bovine
brain endothelial cells compared with the control peptide, RADfV
(37.5% versus 8.7%, respectively), as detected by
chromatin condensation and nuclear fragmentation. Treatment with RGDfV
but not with RADfV, which prevented attachment of endothelial cells to
vitronectin or fibronectin, was associated with up to a 50% increase
in endogenous ceramide, a lipid second messenger that can mediate cell
death. Furthermore, exogenous C2-ceramide was cytotoxic to
bovine brain endothelial cells and induced activation of
C-jun N-terminal kinase (JNK), a MAP kinase that can be
activated in stress-induced apoptosis pathways. This suggests that
ceramide may function in detachment-induced endothelial cell apoptosis,
originating from inhibition of vitronectin binding to integrins such as
vß3 and
vß5.
This is the first report to demonstrate expression of integrins
vß3 and
vß5
by microvascular endothelium of a childhood tumor and association of
their expression with neuroblastoma aggressiveness. Furthermore, our
data provide the first suggestion that inhibition of endothelial cell
anchorage, resulting from specific blockade of RGD-binding integrins,
increases endogenous ceramide, which may contribute to endothelial cell
death. | INTRODUCTION |
|---|
|
|
|---|
/ß heterodimeric cell-surface receptors that
link the extracellular matrix with the cell cytoskeleton and generate
intracellular signals to regulate cell survival and growth, cell
differentiation, and cell motility. Vitronectin, a matrix protein
comprising two hemopexin domains, a somatomedin B domain and a
cell-binding region, binds integrins
vß1,
vß3,
vß5,
vß6,
vß8,
IIbß3, and
8ß1 via the RGD
sequence in the cell-binding region at its
NH2-terminus (1, 2, 3, 4)
. Integrin
binding to vitronectin can be inhibited by specific function-blocking
antibodies or by synthetic RGD peptides, which mimic the conformation
of the RGD sequence (5, 6, 7, 8)
. Of the synthetic RGD peptides,
some of the most active in inhibiting cell attachment to vitronectin
are the cyclic pentapeptides (5
, 6)
. To date, the only
vitronectin-binding integrins that have been described on the surface
of endothelial cells are
vß3,
vß5
(7, 8, 9)
, and to a lesser extent
vß6 (10)
.
The angiogenic integrins,
vß3 and
vß5, which are
expressed on endothelial cells, are crucial for their survival
(8
, 11, 12, 13, 14)
. Survival signals transmitted by integrin
vß3 lead to inhibition
of p53 activity, decreased expression of p21WAF1/CIP1, and suppression
of the bax cell death pathway in endothelial cells (15)
.
When plated on osteopontin, the
vß3-dependent signals
for endothelial cell survival are mediated via
NF
B3
(16)
. These survival pathways can be blocked either by
function-blocking antibodies or by peptide analogues, which block the
active RGD binding site on integrins
vß3 and
vß5, thus inducing
apoptosis of the angiogenic endothelial cells (8
, 11
, 12)
.
Endothelial apoptosis results in inhibition of new blood vessel
formation, disruption of existing angiogenic vasculature, inhibition of
tumor growth, and tumor regression (8
, 11
, 12
, 14, 15, 16)
.
Apoptosis of endothelial cells can occur in response to a variety of
stress stimuli such as LPS (17)
, TNF
(18)
,
ionizing radiation (19)
, growth factor withdrawal
(20)
, and disruption of matrix binding or specific
integrin ligation (11
, 21)
. Ceramide, a lipid second
messenger that is synthesized de novo or derived from
membrane sphingomyelin, is implicated in apoptotic signaling pathways
induced by stimuli such as irradiation, TNF
, and LPS
(22, 23, 24)
and has been linked to activation of
pro-apoptotic MAPKs (e.g., JNK/SAPK; Ref. 23
).
The delicate balance between cellular levels of the survival-promoting,
sphingosine-1-phosphate and the apoptosis-linked ceramide, both
metabolites of sphingomyelin, provides a means for sensitive regulation
of pro- and antiapoptotic MAPKs (e.g., JNK/SAPK, p38, or
extracellular regulated kinases), thus contributing to regulation of
cell survival and death (25)
. Interestingly, ceramide
enhances expression of MMP-13 in fibroblasts
(26)
, suggesting that it may mediate signals for matrix
remodeling and possibly for angiogenesis. Although ceramide has been
implicated in endothelial apoptosis induced by a variety of stimuli
both in vivo (17)
and in vitro
(19
, 23)
, it is not known whether ceramide plays a role in
signaling of apoptosis resulting from inhibition of integrin-mediated
endothelial cell anchorage.
Treatment of NB, the second most common solid tumor in childhood, is successful in less than half of patients with high-risk (stage IV and MYCN-amplified stage III) disease (27, 28, 29) . A high vascular index in NB correlates with poor prognosis (30) , suggesting dependence of aggressive tumor growth on active angiogenesis, the sprouting of new capillaries from existing blood vessels. The matrix-degrading enzymes MMP-2 and MMP-9 are elevated in high-risk NB compared with low-risk disease (31) and are expressed in NB cell lines, which themselves induce proliferation of endothelial cells (32) , supporting the angiogenic phenotype of this tumor. Moreover, several antiangiogenic approaches have demonstrated activity in animal models of NB (33, 34, 35) . Thus, utilization of antiangiogenic approaches, by themselves or in combination with other therapies, could potentially improve the outcome in children with NB and may warrant further study.
In view of the in vivo efficacy of antiangiogenic treatment
directed against integrins
vß3 and
vß5 in animal models
(8
, 11 , 12
, 35)
and the correlation of a high vascular
index with poor prognosis in NB (30)
, we hypothesized that
tumor endothelium in NB may present a potential target for
antiangiogenic treatment directed against integrins
vß3 and/or
vß5. Furthermore, we
hypothesized that ceramide, which can contribute to apoptotic signaling
in endothelial cells, may be involved in apoptosis induced by
inhibition of integrin-mediated anchorage in these cells. In this
study, we report that inhibition of endothelial cell attachment and
spreading and the resultant apoptosis, originating from blockade of
RGD-binding integrins such as
vß3 and
vß5, are associated
with the generation of endogenous ceramide. In addition, we show by
immunohistochemical analysis that integrins
vß3 and
vß5 are expressed on
average by 61% of microvessels in the high-risk NB (stage IV and
MYCN-amplified stage III; n = 28) but
vß3 is only expressed in 18% of low-risk
tumors (stages I and II and non-MYCN-amplified stage III;
n = 12). These data suggest that NB
endothelium may be targeted for antiangiogenic treatment directed
against integrins
vß3
and
vß5.
| MATERIALS AND METHODS |
|---|
|
|
|---|
vß3 on their surface
(mean, 92.4%, SD, 5.8%, by fluorescence-activated cell-sorting
analysis using mAb LM609 in 11 separate determinations over a period of
at least 4 months in culture). Primary HUV-EC-C were purchased from the
American Type Culture Collection (passage 16; Manassas, VA), maintained
according to the American Type Culture Collection recommendations, and
used between passages 16 and 24. Previously characterized
affinity-purified mAb to integrin
vß3 (LM609) and the
v integrin subunit (LM142) were a generous
gift from D. Cheresh (7
, 12
, 14)
. Monoclonal antibody
clone P1F6 against integrin
vß5 was purchased from
Life Technologies (Gaithersburg, MD). Isotype-specific mouse IgG1
(control for the mAb stains) and polyclonal anti-Factor VIII were from
Dako (Carpinteria, CA). Secondary antibodies for the double
immunofluorescent stains were FITC-conjugated goat
F(ab')2 anti-rabbit IgG and rhodamine-conjugated
goat F(ab')2 anti-mouse IgG (BioSource
International, Camarillo, CA). Human fibronectin (domains 211,
constituting the 110-kDa cell-binding fragment containing the RGD site
but not the Hep-2/CS1 region) was purchased from Upstate Biotechnology
(Lake Placid, NY), and human vitronectin was purchased from Promega
(Madison, WI) or prepared as described by Yatohgo et al.
(37)
. The cyclic pentapeptides RGDfV and RADfV were
generous gifts from Merck (Darmstadt, Germany) via Alfred Jonczyk. All
cell culture experiments were repeated at least three times.
Patients and Tumor Specimens.
The NB specimens included in this study were resected at institutions
of the CCG (Arcadia, CA) between 1986 and 1992. Clinical staging
was performed according to standard criteria used by the CCG
(38)
. Neuroblastoma tumor tissue was obtained surgically,
snap-frozen, and shipped on dry ice to the CCG Neuroblastoma Biology
Reference Laboratory. Upon receipt, a portion was removed without
thawing, placed in Tissue-Tek OCT embedding compound (Miles, Elkart,
IN), and maintained at -70°C. Another part of each tumor was fixed
in formalin and embedded for light microscopic examination.
Histological sections of tumors were evaluated for stromal cells,
neuroblastic differentiation, and mitotic/karyorrhectic cell number and
categorized as having favorable or unfavorable histology
(39)
. Forty-two tumors were analyzed. Two of the forty-two
samples processed were excluded from the analysis after processing
because during the pathological analysis one sample was found to be a
lymph node metastasis rather than a sample from the primary tumor and
one OCT block was inadequate (it contained only tumor capsule). Patient
and tumor characteristics for the remaining 40 patients are summarized
in Table 1
. Thirty-five of the samples were obtained at the time of diagnosis,
before the patient had received any chemotherapy or radiation; five of
the samples were obtained from tumors resected from patients who had
already received chemotherapy (i.e., at the time of delayed
resection or after relapse).
|
vß3)
only recognizes the nondenatured functional conformation of integrin
vß3 (40)
,
it was not possible to use paraffin blocks for this study. All washes
and dilutions of reagents were done in Tris-buffered saline (10
mM Tris-HCl (pH 7.6) and 130 mM NaCl) at room
temperature. After three washes the sections were blocked for 5 min in
2% goat serum (Life Technologies); reacted with primary antibodies for
2 h (LM609, 1:500; P1F6, 1:400; or Anti-Factor VIII, 1:1000);
washed three times; incubated for 60 min with multilink (swine)
anti-goat, -mouse, and -rabbit immunoglobulins (1:50; Dako); washed
again; incubated for 30 min with avidin-biotin-peroxidase (Vector
Laboratories, Burlingame, CA); reacted for 10 min with
3,3-diaminobenzidine (0.4 mg/ml); rinsed; counter-stained with Mayers
hematoxylin; mounted; and read. Mouse IgG1, used as a negative control,
was negative in all cases.
Immunofluorescence Analysis.
Immunofluorescent staining was performed as previously described with
minor modifications (12)
. Four-µm cryostat NB sections
in OCT were fixed in cold acetone for 30 s, air dried, and
subsequently blocked with 2.5% BSA in PBS (pH 7.4) for 12 h at room
temperature. Sections were then washed 58 times in PBS and incubated
with monoclonal
anti-
vß3 (LM609 1:50,
60 µg/ml) and polyclonal anti-Factor VIII (1:1000) for 1 h at
room temperature. Sections were washed and then incubated for 1.5 h at room temperature with rhodamine-conjugated anti-mouse IgG (1:300)
and FITC-conjugated anti-rabbit IgG (1:300). Tissue sections were then
washed, mounted, and photographed using an Olympus AX70 compound
microscope (Olympus America, Melville, NY) at x200.
Cell Radiolabeling and Analysis of Ceramide.
Ceramide metabolism was studied as described (41, 42, 43)
.
Briefly, endothelial cells were plated the day before the experiment
(3 x 106 cells/100-mm plate, in
their usual growth medium) and metabolically labeled with 9,10
[3H]-(N)-palmitic acid (1 µCi/ml;
NEN, Boston, MA) overnight at 37°C. The next day the medium was
collected, and cells were briefly trypsinized, washed, and replated on
non-tissue culture-treated 6-well plates that were precoated with
fibronectin (10 µg/ml), vitronectin (2 µg/ml), or BSA (0.5%) and
blocked with BSA (0.5%) in the medium in which they were incubated
overnight (106 cells/well). Where indicated, the
cells were replated in the presence or absence of the cyclic
RGD-blocking peptide RGDfV or control RADfV. At the end of incubation
the cells were briefly trypsinized, combined with the nonadherent cells
of the same well, washed (at 4°C), and lipid was extracted
using equal volumes of methanol/2% acetic acid (v/v), water, and
chloroform. The dried lipid was solubilized in chloroform:methanol (2:1
v/v) and analyzed using TLC plates with a solvent system consisting of
chloroform:acetic acid (9:1 v/v) and using lipid standards as markers
as described (41, 42, 43)
. Ceramide and total cellular lipids
were quantitated in a liquid scintillation counter (cpm), and ceramide
was calculated and expressed as percent [
3H]ceramide of total
[3H]-labeled cellular lipids extracted.
Cell Density and Viability.
Cell viability was assessed by the uptake of MTT (Thiazolyl blue;
Sigma), a process which is dependent on intact mitochondrial function,
as described by Mosmann (44)
. This method yields results
similar to those of other methods of assessing endothelial cell
viability (45)
. Briefly, to assess viable cell density,
MTT (250 µg/ml) was added to the cells and incubated for 2 h at
37°C. Subsequently the medium was carefully removed in such a way
that any detached or loosely adherent cells were not removed, and the
cells containing the trapped MTT crystals were solubilized in 200 µl
DMSO at 37°C for 15 min. Absorbance was determined in a microtiter
plate reader (Molecular Devices, Menlo Park, CA) at 550 nm and
subtracted from the A650 nm.
Cell Adhesion.
Cell adhesion was assayed by plating 100,000 trypsinized cells onto
wells of a 48-well non-tissue culture-treated plate coated overnight
with vitronectin (2 µg/ml) or the 110-kDa RGD-containing fragment of
fibronectin (10 µg/ml), which were washed and blocked with 0.5% BSA
in PBS. After 1 h, the wells were washed 3 times to remove
nonadherent cells, and the remaining cells were quantitated using the
MTT assay as described above.
Hoechst Stain.
BBEC were stained with Hoechst-bisbenzamide 33258 (Sigma) according to
the manufacturers instructions to assess chromatin condensation and
nuclear fragmentation. Approximately 300 cells in each sample were
counted under UV filter at magnification x400.
JNK Activation.
Lysates from 5 x 106 BBEC that
have been exposed to C2-ceramide were prepared in
Triton X-100 extraction buffer [EB buffer: 1% Triton X-100, 10
mM Tris (pH 7.6), 50 mM NaCl, 0.1% BSA, 1
mM PMSF, 1% aprotinin, 5 mM EDTA, 50
mM NaF, 0.1% 2-ß mercaptoethanol, 5 µM
phenylarsine oxide, and 100 µM vanadate],
precleared (15,000 x g for 45 min at 4°C),
and anti-SAPK/JNK immunoprecipitation was performed using
anti-SAPK1/JNK1 rabbit polyclonal antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA) as previously described (46)
.
Immunoprecipitates were washed several times with buffer [20
mM PIPES (pH 7.0), 0.1 M NaCl, and 20 µg/ml
aprotinin], and kinase activity of JNK was quantitated using a slight
modification of an in vitro kinase assay as described
(47)
. JNK immunoprecipitates were incubated in a solution
containing 20 mM PIPES (pH 7.0), 10
mM MnCl2, 5 µCi
[32P]ATP (3000 Ci/mmol), 20 µg/ml
aprotinin, and 1 µg of GST-c-jun fusion protein
(NH2 terminus, residues 1154) in final volume
of 20 µl. After an incubation of 30 min at 30°C, the reactions were
terminated by the addition of 20 µl of 2x SDS-sample buffer and
heated at 98°C for 5 min. The JNK kinase and GST-c-jun substrates
were resolved by SDS-PAGE, phosphorylated GST-jun protein was
visualized by autoradiography, and the amount of JNK kinase protein
immunoprecipitated was verified by anti-JNK Western blotting.
Statistical Analyses.
All laboratory and histopathological analyses were performed
independently and without knowledge of clinical data.
Immunohistochemical slides prepared from serial sections were analyzed
by two observers (A. E-E. and H. S.), who visually
determined the proportion of microvessels in the whole section in which
the microvascular endothelium specifically stained with the LM609 or
P1F6 mAb compared with the total Factor VIII-positive vessels in the
adjacent section. The observers repeated the analysis of the sections
on two separate occasions and were blinded to the clinical
characteristics of the patients and to their previous readings. The
variation for integrin
vß3 expression between
the first and second reading in the 40 tumors examined was 15% in 2
tumors, 10% in 9 tumors, and 05% in the remaining 29 tumors. The
variation for integrin
vß5 between the first
and second readings in the 21 tumors evaluated was 15% in 1 tumor,
10% in 6 tumors, and 05% in 13 tumors. The value used for
statistical analysis and for plotting the figures was the average of
the readings for each tumor. A simple linear regression analysis was
used to evaluate the association of age (as a continuous variable and
also dichotomized as
12 months versus >12 months), MKI
(low + intermediate versus high; Ref.
3
), pathology (favorable versus unfavorable),
MYCN (not amplified versus amplified), and whether or not
the patient had received treatment prior to tumor sampling, with
expression of
Vß3 and
Vß5 as measured by the
percent of microvessels that stained with the antibodies LM609 (for
Vß3) and P1F6 (for
Vß5). The P
values reported are based on the F test from the regression
analysis and are all two-sided (Table 1)
. Means and 95% confidence
intervals based on the individual SD were calculated. To summarize the
association between expression of
Vß3 and
Vß5, scatter plots
were drawn and Pearsons correlation coefficient was calculated with
its associated P value (Fig. 4)
. To test whether the
observed correlation could be explained by MYCN amplification, a
partial correlation was calculated. To evaluate whether the association
between
Vß3 and
Vß5 was similar or
different in patients whose tumors expressed MYCN amplification or not,
separate regression models were fit for the two subgroups of patients
and were compared with the single model fit for all 21 patients.
|
| RESULTS |
|---|
|
|
|---|
vß3 Is Expressed on
Angiogenic Endothelial Cells of High-Risk NB.
vß3 treatment, we
examined whether the tumor vasculature expressed this integrin. Serial
sections from 40 NB tumors were immunohistochemically stained with
anti-Factor VIII or the anti-integrin
vß3 (LM609) antibodies
and analyzed for microvascular expression of integrin
vß3. Fig. 1
vß3. In the
low-risk NB, there was no endothelial expression of integrin
vß3 (Fig. 1B)
v chain (LM142) confirmed an
endothelial expression similar to that of integrin
vß3 (data not shown).
|
vß3 was further
demonstrated by simultaneous immunostaining of the same section for
both integrin
vß3 and
Factor VIII using a mAb to integrin
vß3 (LM609) and a
rabbit polyclonal antibody to Factor VIII (Fig. 2)
vß3 (red
fluorescence, mAb LM609, Fig. 2B
vß3 to the endothelium
was further verified by double exposure of the same frame to the green
and red fluorescence, resulting in a yellow fluorescence in the regions
where both Factor VIII and integrin
vß3 were colocalized
(Fig. 2C)
vß3 was limited to the
endothelial cells in all cases examined and was not detected on the
tumor cells themselves (Fig. 1, A and C)
|
vß3 is highly
expressed on most microvessels in high-risk but not low-risk NB.
Univariate analysis demonstrates that the high-risk tumors (stage III,
MYCN-amplified, and stage IV) expressed endothelial integrin
vß3 on the majority of
their microvessels (mean, 61%; 95% confidence interval, 5469%;
n = 28; Table 1
vß3 in high-risk NBs
was found both in tumor specimens obtained at the time of diagnosis
(mean, 59%; 95% confidence interval, 5067%; n = 23) and in tumors procured at the time of delayed resection or
tumor relapse (mean, 72%; 95% confidence interval, 5688%;
n = 5). In comparison, a significantly lower
proportion of microvessels expressed endothelial integrin
vß3 in low-risk tumors
(stages I and II and stage III non-MYCN-amplified; mean, 18%; 95%
confidence interval, 1324%; n = 12; Fig. 3
vß3 between the
MYCN-amplified/unfavorable histology and MYCN non-amplified/favorable
histology tumors (mean, 87% and n = 3
versus mean, 20% and n = 2,
respectively; Figs. 1
vß3 expression
(i.e., LM609 staining) on the tumor neovasculature
(P < 0.001), whereas MKI and pathology were
not (P = 0.15 for each). We conclude that
integrin
vß3 is highly
expressed on microvessels of high-risk NBs.
|
vß3 and
vß5
in High-Risk Neuroblastomas.
v integrin,
vß5, is also expressed
on endothelium of angiogenic vessels and, like integrin
vß3, is crucial to
angiogenesis (8)
. We asked whether integrin
vß5 also is expressed
in angiogenic endothelium in NBs. Twenty-one of the 40 NBs (all stage
IV) were evaluated for expression of integrin
vß5 (Fig. 4)
vß5 on 50% or more of
their angiogenic vessels (Fig. 4)
vß5 was 60% (95%
confidence interval, 5367%; n = 21). The
correlation between expression of integrin
vß3 and
vß5 was high
(r = 0.75; P < 0.001). When controlling (i.e., stratifying) for MYCN
status, the partial correlation coefficient became rp = 0.74 (P < 0.001), suggesting that both integrins play a role in tumor
angiogenesis independent of MYCN amplification. Like integrin
vß3, integrin
vß5 was only expressed
on the angiogenic endothelium and not by tumor cells in any of the
cases we examined (data not shown). These data demonstrate that the
angiogenic integrins
vß3 and
vß5 are strongly
associated and are expressed on most angiogenic microvessels in
high-risk NBs. This suggests that both integrins may present a useful
target for antiangiogenic treatment of high-risk NB.
Inhibition of Integrin-mediated Endothelial Cell Anchorage Is
Associated with Elevation of Endogenous Ceramide.
Integrin-mediated signals are required for anchorage-dependent
survival of endothelial and epithelial cells (21
, 48
, 49)
.
Inhibition of integrins
vß3 and
vß5 on endothelial
cells lining tumor neovasculature in animal models induces endothelial
cell apoptosis, disruption of tumor angiogenesis, and inhibition of
tumor growth (8
, 11
, 14)
. Endothelial cells also require
integrin-mediated attachment and spreading on specific matrix proteins
to survive in culture (14
, 21
, 48)
. Because we showed that
endothelium in high-risk NBs expressed integrins
vß3 and
vß5 (Figs. 1
2
3
4)
, we
sought to further study the signaling pathways that may contribute to
endothelial apoptosis mediated via inhibition of these integrins.
On the basis of in vitro and in vivo experiments
demonstrating involvement of ceramide in UV irradiation, LPS, and
TNF
-induced endothelial apoptosis (17
, 19
, 23)
, we
hypothesized that ceramide could be involved in endothelial cell death
resulting from inhibition of integrin-mediated anchorage. Because NB is
a neural crest-derived tumor, in testing this hypothesis we chose to
use BBEC immortalized (large-T transfected) endothelial cells derived
from a neural microenvironment (the brain). Like other endothelial
cells, BBEC spontaneously express high levels of integrin
vß3 on their surface.
Cellular lipids of attached, log-phase growth BBEC were labeled with
[3H]palmitic acid, detached briefly by
trypsin/EDTA, and then replated on BSA, fibronectin, or vitronectin for
4 h. Lipids were extracted and separated by TLC as detailed in
"Materials and Methods." Prevention of attachment and spreading of
endothelial cells onto extracellular matrix by plating them on a
BSA-coated surface induced elevation of ceramide of up to 50% above
that observed in cells plated on fibronectin or vitronectin (Fig. 5A)
. The mean increase in endogenous ceramide in BBEC plated
on BSA was 35% (SD, 15%; n = 5 experiments)
compared with vitronectin and 29% (SD, 9%; n = 3 experiments) compared with fibronectin. These results were
replicated in primary HUV-EC-C. An increase of 54% in endogenous
ceramide was observed in the HUV-EC-C when they were plated on BSA
compared with vitronectin (SD, 1.33 ± 0.1%
[3H]ceramide on BSA compared with
0.87 ± 0.01% on vitronectin).
|
vß3 and
vß5 using
function-blocking antibodies or the cyclic pentapeptide RGDfV, which
block the RGD-binding site on these integrins, has been reported to
prevent endothelial cell attachment to matrix and to induce apoptosis
(5
, 8 , 11
, 12)
. Exposure of BBEC to the RGD-blocking
cyclic pentapeptide RGDfV but not to the control peptide RADfV
prevented BBEC attachment and spreading on vitronectin in a
dose-dependent pattern, with complete inhibition achieved with as low
as 5 µg/ml RGDfV (Fig. 5B
Ceramide-induced Cytotoxicity Is Associated with the Activation of
JNK.
Some stress signals that lead to apoptosis in endothelial cells induce
endogenous ceramide, which is associated with activation of JNK,
a MAPK that functions in apoptosis (23)
. We showed that
inhibition of integrin-mediated endothelial cell anchorage, which
results in apoptosis, induces elevation of endogenous ceramide. We
asked whether ceramide itself could induce JNK activation and BBEC
death. C2-ceramide was cytotoxic to BBEC with a mean
concentration which induces 50% cell death (LC50)
of 31.7 µM (SD, 10.5 µM) at 24 h, as
determined in eight separate experiments (each done in replicates of
510 points for each ceramide concentration). The death response was
proportional to the concentration of ceramide added (Fig. 6A)
and to the length of time BBEC were exposed to the lipid
(Fig. 6B)
. The inactive ceramide analogue dihydroceramide
elicited only a limited amount of cell death, which was constant and
not dose dependent between 20 and 100 µM
dihydroceramide (data not shown). Exposure of BBEC to increasing
concentrations (0.140 µM) of exogenous
C2-ceramide for 2 h induced activation of
JNK, as assayed by measurement of phosphorylation of GST-c-jun fusion
protein by JNK immunoprecipitated from the cells (Fig. 7)
. The increase in JNK phosphorylation of the GST-c-jun fusion protein
was observed as early as 1 h after the addition of
C2-ceramide, with the largest increment in the
increase in JNK activation occurring between 1 and 2 h or later
(data not shown). Thus, exogenous C2-ceramide,
which induces apoptotic cell death (45)
, also induces the
activation of JNK in endothelial cells. From these data we conclude
that ceramide is a potential participant in detachment-induced
apoptotic signaling in endothelial cells and, specifically, that it
may mediate some of the signals associated with integrin blockade.
|
|
| DISCUSSION |
|---|
|
|
|---|
vß3 and
vß5 by microvascular
endothelium of a pediatric tumor and association of this expression
with tumor aggressiveness. Because these integrins are markers of
active angiogenesis (8
, 14) , this finding extends the data
of Meitar et al. (30)
, who reported that a high
vascular index in NB correlates with metastatic disease, MYCN
amplification, and poor outcome. In invasive breast cancer, endothelial
integrin
vß3
expression and microvessel density are both prognostic indicators
(50
, 51) , suggesting that this correlation also may be
found in other tumors. Our ongoing analysis of NBs will reveal whether
endothelial integrin
vß3 expression is a
prognostic factor independent from MYCN and pathology in NB.
Demonstration that angiogenic endothelium in high-risk NBs highly
expressed integrins
vß3 and
vß5 suggests that
these integrins could be therapeutic targets.
Our data showing that microvessels in MYCN-amplified NB express high
levels of integrins
vß3 and
vß5 point to a
potential role for the MYCN oncogene in angiogenesis. Because
MYCN itself is a transcription factor, it could regulate angiogenic
factors originating in the tumor cells, such as vascular endothelial
growth factor or matrix-degrading proteins, as has been shown for
mutant K-ras and polyoma middle T antigen, respectively
(52
, 53)
. Supporting this, NB tumors and their surrounding
stromal cells express matrix metalloproteinases (31)
.
Moreover, MYCN is required for motility of NB cell lines and for their
proteolytic activity in vitro, suggesting that MYCN may
contribute to NB aggressiveness by enhancing angiogenesis
(54)
. Thus, it is possible that overexpression of MYCN may
contribute to NB aggressiveness by promoting angiogenesis.
The immunohistochemical stains of the NBs in our series showed that
integrin
vß3 and
vß5 expression was
confined to the endothelium of tumor microvessels and did not appear on
the tumor cells themselves (Figs. 1
and 2
and data not shown). Gladson
et al. (55)
examined paraffin sections of NBs
for expression of the individual
v,
ß3, and ß5 integrin
subunits. Our immunostaining with the mAb against
vß5 (P1F6) are in
agreement with their description that the ß5
integrin subunit is not expressed at the protein level in neuroblasts
of undifferentiated tumors. However, Gladson et al.
(55)
observed that the
v and
ß3 subunits were associated with most of the
undifferentiated neuroblasts, a finding interpreted to imply that
integrin
vß3 is
expressed on those cells. It is conceivable that the binding partner(s)
for the
v and ß3
integrin chains seen in that work did not belong to integrin
vß3 but may have
originated from different integrin partners (i.e.,
vß6 or
vß8; Refs.
56, 57, 58
), some of which are highly expressed in the
mammalian brain (58)
. This could explain the specific
microvascular endothelial expression of integrin
vß3 we found using mAb
LM609, which specifically recognizes the native conformation of the
functional RGD-binding domain on integrin
vß3 (7
, 59) .
We demonstrate here for the first time that ceramide is elevated in
endothelial cells that are prevented from attaching and spreading on
matrix, either by plating on BSA or by inhibition with the RGDfV
inhibitory peptide (Fig. 5)
. The increase in endogenous ceramide
induced by RGDfV and plating on BSA are in a range comparable with
increases observed following exposure to stressors such as TNF
,
sorbitol, H2O2, LPS, UV
irradiation, or
irradiation (17
, 60)
. Our data suggest
that ceramide may be involved in signaling pathways originating from
specific inhibition of the RGD binding site on vitronectin-binding
integrins such as
vß3
and
vß5. Although we
cannot exclude possible involvement of multiple vitronectin-binding
integrins under these conditions, it is likely that inhibition of
adhesion and spreading on vitronectin by RGDfV was mediated mostly by
inhibition of integrins
vß3 and
vß5, both of which are
highly expressed on endothelial cells (7
, 9)
.
Alternatively, increased ceramide may be the result of inhibition of
cell anchorage and/or of the associated cytoskeletal signals resulting
from shape change per se (61)
, may be unrelated
to the alteration of integrin signaling, or possibly may be related to
direct activation of apoptosis by an RGD-blocking peptide
(62)
. To implicate specific signaling intermediates that
may link integrins, ceramide, and regulation of cell survival
(i.e., phosphatases such as protein tyrosine
phosphatase-
or kinases such as AKT/PKB) and to
determine the role of cytoskeletal shape change versus
integrin blockade in cell survival, it will be necessary to explore
these pathways in detail by using specific dominant-negative/positive
intermediates or anti-integrin antibodies that block signaling without
inhibiting cell attachment (63)
. Although there have been
no prior reports of ceramide as a second messenger in integrin
signaling, recent work suggests that selectins, another family of cell
adhesion molecules, may use ceramide signaling in the activation of
lymphocytes (64)
. Increased endogenous ceramide may
contribute to endothelial apoptosis, as shown by the fact that
incubation of endothelial cells with C2-ceramide
is associated with DNA laddering (data not shown and Ref.
45
). Our results compare favorably with those described by
Xu et al. (45)
, as can be seen in the fact that
we achieved a mean
LC50 of 31.7
µM ± 10.5 SD
C2-ceramide in our experiments compared with
their report of 50% endothelial cell kill at 50
µM C2-ceramide. Our data
provide the first evidence that signaling pathways originating from
inhibition of endothelial cell anchorage are associated with increased
endogenous ceramide, which may contribute to apoptosis.
In several cell types including endothelial cells, JNK is thought to
lie downstream of ceramide in apoptotic pathways induced by stress
(23
, 65)
. In epithelial cells, detachment from matrix
induces JNK activation, which can be inhibited by the interleukin 1ß
converting enzyme protease inhibitor crmA or by overexpression
of Bcl-2 (66)
. Our experiments, in which exogenous
C2-ceramide induced activation of JNK in
endothelial cells, suggest that the increased endogenous ceramide we
observed after inhibition of integrin-mediated cell anchorage (Fig. 5)
may contribute to JNK activation in signaling of apoptosis (Fig. 7)
.
However, the ceramide-JNK-apoptosis link is not universal, and several
reports describe the dissociation of ceramide production from JNK and
other death signals (67, 68, 69, 70)
. Recent work by Ameyar
et al. (67)
described lack of correlation
between JNK activation and ceramide production in breast carcinoma
cells, suggesting that the ceramide-JNK link may be specific to
individual cell types. Adding to the complexity, Khwaja and Downward
(68)
show that, although JNK is activated by detachment of
normal MDCK epithelial cells, it is unlikely that it plays a direct
role in detachment-induced apoptosis in these cells because activated
phosphatidylinositol 3'-kinase and AKT prevented apoptosis but did not
prevent the associated JNK activation. Activated Raf and dominant
negative SAPK/ERK kinase-1, which inhibited the
detachment-induced activation of JNK, did not prevent apoptosis,
suggesting induction of apoptosis by elements which are independent of
JNK activation in the MDCK epithelial cells (68)
. Clearly
the specificity of cellular responses to ceramide and other
apoptosis-mediating signals depends upon many factors including the
nature of the stimulus, costimulatory signals, the cell type involved,
and (most likely) the surrounding matrix proteins. Thus, although
inhibition of endothelial cell anchorage induced elevation of ceramide
in our studies and exogenous ceramide induced JNK activation and
apoptotic cell death, a direct causal link between the three needs to
be investigated further.
Cell survival is dictated by a delicate dynamic balance between pro-
and anti-apoptotic signals, which in turn depend upon a complex
interplay of factors such as integrins and growth factors. For example,
in fibroblasts interleukin-1-mediated inflammatory responses, which
induce activation of the proapoptotic JNK/SAPK, and the
survival-mediating NF
B are both modulated by integrin binding to RGD
motifs on fibronectin (71)
. Both the JNK/SAPK and NF
B
pathways, as well as phosphatidylinositol 3'-kinase/AKT, are tightly
controlled, and several reports suggest that ceramide may help regulate
them to tip the balance in favor of apoptosis (60
, 72, 73, 74, 75)
. These reports support our finding that ceramide may be
involved in regulation of anchorage-induced signaling in endothelial
cells. Furthermore, NF
B mediates integrin
vß3-dependent
endothelial cell survival (16)
, whereas ceramide levels
increase in endothelial cells upon loss of integrin-dependent matrix
anchorage (Fig. 5)
. It is possible that under conditions of inhibition
of cell anchorage ceramide mediates signals to down-regulate NF
B
activation in the endothelial cells, thus promoting their apoptosis
through activation of JNK/SAPK (Fig. 7
and Refs. 23
, 66
, and 72
). It is also possible, that stress conditions which augment
ceramide and/or JNK may synergistically increase apoptosis associated
with loss of anchorage-specific survival signals, thereby increasing
any antiangiogenic activity of the RGDfV peptide.
The relationship between ceramide generation and caspase activation is dependent on cell type and the apoptotic stimulus used. At present, the relationship between ceramide metabolism and regulation of caspase activation in our system has been only preliminarily defined. Both nuclear fragmentation of BBEC plated on vitronectin in the presence of RGDfV and DNA laddering in BBEC plated on BSA-coated plates are inhibited by the pan-caspase inhibitor, BOC-D-FMK (data not shown). In preliminary experiments BOC-D-FMK did not prevent the increase in endogenous ceramide in BBEC plated on vitronectin for 4 h in the presence of RGDfV, suggesting that ceramide may be upstream of the BOC-D-FMK-inhibitable caspases (data not shown). Consistent with this, initial experiments show that DNA laddering induced by exogenous C2-ceramide can be inhibited by BOC-D-FMK (data not shown). This suggests a situation similar to that found in apoptosis induced by low-dose okadaic acid in human neuroepithelioma cells in which endogenous ceramide accumulation is insensitive to caspase inhibitors and precedes caspase activation, whereas exogenous C6-ceramide-induced apoptosis was inhibited by the caspase inhibitors (76 , 77) . In other types of apoptotic signals, such as those mediated by TNF superfamily receptors, the initiator caspases are thought to be upstream of ceramide generation, whereas effector caspases are downstream of it (78, 79, 80, 81) . Because specific signaling cascades may differ depending on the stimulus and the cell type (78) , this needs to be examined individually for each cell type and experimental model. Ongoing investigations using BBEC as a model seek to determine the direct relationship between ceramide and endothelial anoikis.
In summary, this is the first description that suggests that
integrins
vß3 and
vß5 are highly
expressed on the microvascular endothelium in a pediatric malignant
tumor, high-risk neuroblastoma. Also novel is our finding that
endothelial cell apoptosis, induced by inhibition of
vitronectin-binding integrins and the prevention of endothelial cell
anchorage on matrix, is associated with increased endogenous ceramide.
Taken together, these data suggest that RGD-dependent,
vitronectin-binding integrins on endothelial cells, such as
vß3 and
vß5, may be potential
targets for antiangiogenic treatment of NB.
| ACKNOWLEDGMENTS |
|---|
| FOOTNOTES |
|---|
1 This work was supported by Grant CA75637 to
D.L.D. from the National Institutes of Health and by grants to A.E-E.
from the Childrens Cancer Research Fund and the Concern Foundation.
This work was also supported in part by Grant UO1, CA70903-02 from NIH
(to D. L. D.); Grants CA60104 and CA02649 from the National
Cancer Institute, Department of Health and Human Services (to
R. C. S.); Grant CA14089 (to S. G.); Grants NS26310 and HL61951 (to
K. S. K.); the Neil Bogart Memorial Fund of the T.J. Martell
Foundation for Leukemia, Cancer, and AIDS Research; "My Brother Joey
Foundation" founded by Judi and Art Partridge, and the Michael
Hoefflin Foundation for Childrens Cancer (to A. E-E.); and by the
STOP Cancer Award (to D. L. D.). ![]()
2 To whom requests for reprints should be
addressed, at the Department of Pediatrics, Herman B. Wells Center for
Pediatric Research, Cancer Research Institute, 1044 West Walnut Street,
Room 468, Indiana University School of Medicine, Indianapolis, IN
46202. Phone: (317) 278-3718; Fax: (317) 274-8679; E-mail: ddurden{at}iupui.edu ![]()
3 The abbreviations used are: NF
B, nuclear
factor
B; LPS, lipopolysaccharide; TNF
, tumor necrosis factor
; MAPK, mitogen-activated protein kinase; JNK, C-jun
N-terminal kinase; SAPK, stress-activated protein kinase; MMP, matrix
metalloproteinase; NB, neuroblastoma; BBEC, bovine brain endothelial
cells; mAb, monoclonal antibody; HUV-EC-C, human umbilical vein
endothelial cells; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST,
glutathione S-transferase; MKI, mitosis-karyorrhexis
index; AKT/PKB, protein kinase B; BOC-D-FMK,
BOC-aspartyl(OMe)-fluoromethylketone. ![]()
Received 4/19/99. Accepted 12/ 2/99.
| REFERENCES |
|---|
|
|
|---|
vß6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin. J. Cell Sci., 111: 2189-2195, 1998.[Abstract]
vß8: interaction with vitronectin and functional divergence of the ß8 cytoplasmic domain. J. Biol. Chem., 269: 28708-28715, 1994.
8ß1 functions as a receptor for tenascin, fibronectin, and vitronectin. J. Biol. Chem., 270: 23196-23202, 1995.
IIbß3,
Vß3, and
5ß1 integrins. J. Biol. Chem., 269: 20233-20238, 1994.
v integrins. Science (Washington DC), 270: 1500-1502, 1995.
v integrin receptors in wound-induced human angiogenesis in human skin/SCID mice chimeras. Am. J. Pathol., 151: 975-983, 1997.[Abstract]
vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79: 1157-1164, 1994.[Medline]
vß3 blocks human breast cancer growth and angiogenesis in human skin. J. Clin. Invest., 96: 1815-1822, 1995.
vß3 and
vß5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. USA, 93: 9764-9769, 1996.
vß3 for angiogenesis. Science (Washington DC), 264: 569-571, 1994.
Vß3 during angiogenesis. J. Clin. Invest., 98: 426-433, 1996.[Medline]
B mediates
vß3 integrin-induced endothelial cell survival. J. Cell Biol., 141: 1083-1093, 1998.
v antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases. Proc. Natl. Acad. Sci. USA, 96: 1591-1596, 1999.
vß3 integrin: adhesion mechanism for transformed glial cells. J. Clin. Invest., 88: 1924-1932, 1991.
/cycloheximide-induced cerebral endothelial cell death. J. Biol. Chem., 273: 16521-16526, 1998.
RI-induced myeloid oxidant signaling. Exp. Cell Res., 237: 288-295, 1997.[Medline]
RI receptor signals through the activation of hck and MAP kinase. J. Immunol., 154: 4039-4047, 1995.[Abstract]
vß3: a new prognostic indicator in breast cancer. Clin. Cancer Res., 4: 2625-2634, 1998.[Abstract]
vß3 in neuroblastic tumors. Am. J. Pathol., 148: 1423-1434, 1996.[Abstract]
V Integrins on HT-29 colon carcinoma cells: adhesion to fibronectin is mediated solely by small amounts of
Vß6, and
Vß5 is codistributed with actin fibers. Exp. Cell Res., 234: 156-164, 1997.[Medline]
vß3,
vß5, and
vß6. Cancer Res., 54: 2102-2107, 1994.
vß8 in mouse and rat brain. Brain Res., 791: 271-282, 1998.[Medline]
vß3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J., 18: 882-892, 1999.[Medline]
: ceramide-dependent and -independent mitogen-activated protein kinase cascades. J. Biol. Chem., 271: 13094-13102, 1996.
B and stress kinase pathways. Biochem. J., 330: 975-981, 1998.
(TNF-
) signal transduction through ceramide: dissociation of growth inhibitory effects of TNF-
from activation of nuclear factor-
B. J. Biol. Chem., 268: 17762-17766, 1993.
B by inducing the processing of p105. J. Biol. Chem., 273: 15494-15500, 1998.
: CrmA and Bcl-2 target distinct components in the apoptotic pathway. J. Exp. Med., 185: 481-490, 1997.This article has been cited by other articles:
![]() |
W. Wrasidlo, A. Mielgo, V. A. Torres, S. Barbero, K. Stoletov, T. L. Suyama, R. L. Klemke, W. H. Gerwick, D. A. Carson, and D. G. Stupack The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8 PNAS, February 19, 2008; 105(7): 2313 - 2318. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Peddinti, R. Zeine, D. Luca, R. Seshadri, A. Chlenski, K. Cole, B. Pawel, H. R. Salwen, J. M. Maris, and S. L. Cohn Prominent Microvascular Proliferation in Clinically Aggressive Neuroblastoma Clin. Cancer Res., June 15, 2007; 13(12): 3499 - 3506. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Erdreich-Epstein, L. B. Tran, O. T. Cox, E. Y. Huang, W. E. Laug, H. Shimada, and M. Millard Endothelial apoptosis induced by inhibition of integrins {alpha}v{beta}3 and {alpha}v{beta}5 involves ceramide metabolic pathways Blood, June 1, 2005; 105(11): 4353 - 4361. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. J. Morowitz, R. Barr, Q. Wang, R. King, N. Rhodin, B. Pawel, H. Zhao, S. A. Erickson, G. S. Sheppard, J. Wang, et al. Methionine Aminopeptidase 2 Inhibition Is an Effective Treatment Strategy for Neuroblastoma in Preclinical Models Clin. Cancer Res., April 1, 2005; 11(7): 2680 - 2685. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Kitlinska, K. Abe, L. Kuo, J. Pons, M. Yu, L. Li, J. Tilan, L. Everhart, E. W. Lee, Z. Zukowska, et al. Differential Effects of Neuropeptide Y on the Growth and Vascularization of Neural Crest-Derived Tumors Cancer Res., March 1, 2005; 65(5): 1719 - 1728. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. S. Aguzzi, C. Giampietri, F. De Marchis, F. Padula, R. Gaeta, G. Ragone, M. C. Capogrossi, and A. Facchiano RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells Blood, June 1, 2004; 103(11): 4180 - 4187. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. L. Wahl, T. L. Moser, and S. V. Pizzo Angiostatin and Anti-angiogenic Therapy in Human Disease Recent Prog. Horm. Res., January 1, 2004; 59(1): 73 - 104. [Abstract] [Full Text] |
||||
![]() |
D. Pradip, X. Peng, and D. L. Durden Rac2 Specificity in Macrophage Integrin Signaling: POTENTIAL ROLE FOR Syk KINASE J. Biol. Chem., October 24, 2003; 278(43): 41661 - 41669. [Abstract] [Full Text] [PDF] |
||||
![]() |
Q.-W. Yang, S. Liu, Y. Tian, H. R. Salwen, A. Chlenski, J. Weinstein, and S. L. Cohn Methylation-associated Silencing of the Thrombospondin-1 Gene in Human Neuroblastoma Cancer Res., October 1, 2003; 63(19): 6299 - 6310. [Abstract] [Full Text] [PDF] |
||||
![]() |
G. P. Pidgeon, K. Tang, Y. L. Cai, E. Piasentin, and K. V. Honn Overexpression of Platelet-type 12-Lipoxygenase Promotes Tumor Cell Survival by Enhancing {alpha}v{beta}3 and {alpha}v{beta}5 Integrin Expression Cancer Res., July 15, 2003; 63(14): 4258 - 4267. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. L. Weinstein, H. M. Katzenstein, and S. L. Cohn Advances in the Diagnosis and Treatment of Neuroblastoma Oncologist, June 1, 2003; 8(3): 278 - 292. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Pankov, E. Cukierman, K. Clark, K. Matsumoto, C. Hahn, B. Poulin, and K. M. Yamada Specific beta 1 Integrin Site Selectively Regulates Akt/Protein Kinase B Signaling via Local Activation of Protein Phosphatase 2A J. Biol. Chem., May 9, 2003; 278(20): 18671 - 18681. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Chlenski, S. Liu, S. E. Crawford, O. V. Volpert, G. H. DeVries, A. Evangelista, Q. Yang, H. R. Salwen, R. Farrer, J. Bray, et al. SPARC Is a Key Schwannian-derived Inhibitor Controlling Neuroblastoma Tumor Angiogenesis Cancer Res., December 15, 2002; 62(24): 7357 - 7363. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Erdreich-Epstein, L. B. Tran, N. N. Bowman, H. Wang, M. C. Cabot, D. L. Durden, J. Vlckova, C. P. Reynolds, M. F. Stins, S. Groshen, et al. Ceramide Signaling in Fenretinide-induced Endothelial Cell Apoptosis J. Biol. Chem., December 13, 2002; 277(51): 49531 - 49537. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Chavakis and S. Dimmeler Regulation of Endothelial Cell Survival and Apoptosis During Angiogenesis Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 887 - 893. [Abstract] [Full Text] [PDF] |
||||
![]() |
F. TOSETTI, N. FERRARI, S. DE FLORA, and A. ALBINI Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents FASEB J, January 1, 2002; 16(1): 2 - 14. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Isogai, W. E. Laug, H. Shimada, P. J. Declerck, M. F. Stins, D. L. Durden, A. Erdreich-Epstein, and Y. A. DeClerck Plasminogen Activator Inhibitor-1 Promotes Angiogenesis by Stimulating Endothelial Cell Migration toward Fibronectin Cancer Res., July 1, 2001; 61(14): 5587 - 5594. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Shusterman, S. A. Grupp, R. Barr, D. Carpentieri, H. Zhao, and J. M. Maris The Angiogenesis Inhibitor TNP-470 Effectively Inhibits Human Neuroblastoma Xenograft Growth, Especially in the Setting of Subclinical Disease Clin. Cancer Res., April 1, 2001; 7(4): 977 - 984. [Abstract] [Full Text] |
||||
![]() |
C. C. Kumar, M. Malkowski, Z. Yin, E. Tanghetti, B. Yaremko, T. Nechuta, J. Varner, M. Liu, E. M. Smith, B. Neustadt, et al. Inhibition of Angiogenesis and Tumor Growth by SCH221153, a Dual {{alpha}}v{beta}3 and {{alpha}}v{beta}5 Integrin Receptor Antagonist Cancer Res., March 1, 2001; 61(5): 2232 - 2238. [Abstract] [Full Text] |
||||
![]() |
D. Huang, J. L. Rutkowski, G. M. Brodeur, P. M. Chou, J. L. Kwiatkowski, A. Babbo, and S. L. Cohn Schwann Cell-conditioned Medium Inhibits Angiogenesis Cancer Res., November 1, 2000; 60(21): 5966 - 5971. [Abstract] [Full Text] |
||||
![]() |
H. M. Katzenstein, S. L. Cohn, S. Crawford, D. Meitar, A. Canete, V. Castel, and S. Navarro Angiogenesis in Neuroblastoma J. Clin. Oncol., July 14, 2000; 18(14): 2789 - 2791. [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |