Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
NF-KB activation in endothelial cells is critical for the activity of anαiostatic
agents*
Sebastien P. Tabruyn,1,2 Sylvie Mémet,3,4,5 Patrick Avé,5,6 Catherine Verhaeghe,7 Kevin H. Mayo,9 Ingrid
Struman,8 Joseph A. Martial,8 and Arjan W. Griffioen1,10
1
Angiogenesis Laboratory, Department of Pathology, Research School for Oncology and Developmental Biology, Maastricht University,
Maastricht, The Netherlands; 2Cardiovascular Research Institute, Comprehensive Cancer Center, and Department of Anatomy, University of
California-San Francisco, San Francisco, California; 3Unité de Mycologie Moléculaire, 4URA b CNRS 3012, 5Department of Infection and
Epidemiology, and 6Unité d'Histotechnologie et Pathologie, Institut Pasteur, Paris, France; 7Laboratory of Medical Chemistry and Human
Genetics and 8Molecular Biology and Genetic Engineering Unit, GIGA-Research, University of Liège, Liège, Belgium; 9Department of
Biochemistry, University of Minnesota Health Sciences Center, Minneapolis, Minnesota; and 10Angiogenesis Laboratory, Department of
Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
Abstract
In tumor cells, the transcription factor NF-κB has been described to be antiapoptotic and proproliferative and
involved in the production of angiogenic factors such as vascular endothelial growth factor. From these data, a
protumorigenic role of NF-κB has emerged. Here, we examined in endothelial cells whether NF-κB signaling
pathway is involved in mediating the angiostatic properties of angiogenesis inhibitors. The current report
describes that biochemically unrelated agents with direct angiostatic effect induced NF-κB activation in
endothelial cells. Our data showed that endostatin, anginex, angiostatin, and the 16-kDa N-terminal fragment of
human prolactin induced NF-KB activation in endothelial cells in both cultured human endothelial cells and in
vivo in a mouse tumor model. It was also found that NF-κB activity was required for the angiostatic activity,
because inhibition of NF-κB in endothelial cells impaired the ability of angiostatic agents to block sprouting of
endothelial cells and to overcome endothelial cell anergy. Therefore, activation of NF-κB in endothelial cells can
result in an unexpected antitumor outcome. Based on these data, the current approach of systemic treatment with
NF-KB inhibitors may therefore be revisited because NF-κB activation specifically targeted to endothelial cells
might represent an efficient strategy for the treatment of cancer.
Introduction
Inhibition of angiogenesis is a promising therapeutic strategy in the fight against cancer. Many angiostatic
approaches are based on neutralizing angiogenic proteins, such as vascular endothelial growth factor, basic
fibroblast growth factor (bFGF), and platelet growth factor, or blocking of their receptors by tyrosine kinase
inhibitors (1, 2). Other approaches work through direct targeting of endothelial cells, resulting in inhibition of
migration, proliferation, survival, or maturation of endothelial cells (3). Although the best developed angiostatic
strategies are based on vascular endothelial growth factor targeting, considerable progress has been made in the
discovery and development of agents that directly interfere with tumor endothelial cells. However, a clear and
precise view on the mechanisms of action of these angiostatic agents is still lacking and necessary for the design
of improved angiostatic compounds.
The NF-κB/Rel family of transcription factor includes five proteins, p50, p52, p65 (or RelA), RelB, and c-Rel,
which exist as homodimers or heterodimers. The most common NF-κB heterodimer is composed of p50 and p65.
In resting cells, NF-κB is sequestered in the cytoplasm by its association with proteins belonging to the IκB
inhibitor family. Stimuli such as the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin-1
or bacterial products such as lipo-polysaccharide can activate NF-κB. In the canonical pathway, these stimuli
trigger activation of an IκB kinase complex, which in turn phosphorylate NF-κB inhibitors (IκB; ref. 4). This
*
Grant support: SENTER/NOVEM (A.W. Griffioen), Belgian American Educational Foundation fellowship (S.P. Tabruyn and C.
Verhaeghe), "Centre Anticancéreux" (University of Liège) fellowship (S.P. Tabruyn), and Fonds National de la Recherche Scientifique and
"Région Wallonne" program NEOANGIO (J.A. Martial and I. Struman).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
phosphorylation step leads to the ubiquitination and subsequent degradation of IκBS by the proteasome. The NFκB complex translocates into the nucleus where it binds to κB enhancers present in the regulatory regions of
target genes and where it activates transcription (5). Many proteins encoded by NF-κB target genes participate in
the immune response. However, NF-κB is also involved in the activation of genes involved in proliferation,
apoptosis, and angiogenesis. For this reason, NF-κB is considered to be connected with multiple aspects of
oncogenesis (6).
In tumor cells, NF-κB is generally seen as an antiapoptotic factor because it induces antiapoptotic genes that
block the caspase cascade or antiapoptotic members of the Bcl family, described to inhibit cytochrome c release.
Therefore, activation of NF-κB in cancer cells by chemotherapy or radiation therapy is often associated with the
acquisition of resistance to apoptosis. This has emerged as a significant impediment to effective cancer
treatment. In combination with chemotherapy, inhibitors of the NF-κB pathway were recently successfully used
as treatment against cancer (7). However, in contrast to the negative effects of NF-κB activation, recent reports
suggest that, in certain situations, NF-κB can promote apoptosis and may be viewed as a tumor suppressor gene
(8). NF-κB has been described to induce expression of several proapoptotic molecules such as Bcl-Xs, Bax, Fas,
or Fas ligand (9) and blockade of NF-κB predisposes murine skin to squamous cell carcinoma (10). It has been
shown that the dual activity of NF-κB, as an inhibitor or activator of apoptosis, may depend on the relative levels
of p65 and c-Rel subunits; the later has been described to induce apoptotic signal in response to TNF-related
apoptosis-inducing ligand (11). These data clearly illustrate the dual activity of NF-κB and the complexity of the
systemic application of broad-spectrum NF-κB inhibitors for the treatment of cancer.
The current report describes the role of NF-κB in angio-static signaling in endothelial cells. We show that
various angiostatic agents induce NF-κB activation in endothelial cells in both cultured endothelial cells in vitro
and tumors in vivo. In addition, we show that NF-κB inhibition impairs the ability of angiostatic agents to block
sprouting of endothelial cells and to overcome endothelial cell anergy Therefore, our data illustrate that
activation of NF-κB is a common pathway activated by angiostatic agents in endothelial cells and show that NFκB activation in endothelial cells contributes to a nondirect antitumor activity via angiogenesis inhibition and to
overcoming endothelial cell anergy. Moreover, they suggest that selective NF-κB activation in endothelial cells
might be an efficient strategy for the treatment of cancer, thereby raising a cautionary note against the broad and
systemic use of pharmaceutical agents that block NF-κB for cancer therapy.
Materials and Methods Cell Cultures and Reagents
Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described previously (20). Cells
between passages 1 and 3 were used for experiments. Anginex and recombinant 16K hPRL were produced and
purified as described previously (12, 13). Anginex is a 33-mer designer peptide with antiangiogenesis and
antitumor activity. The structure of it is based on the three-dimensional structure of the chemokines platelet
factor-4 and interleukin-8 (14).
NF-κB Binding Activity
HUVECs were stimulated with the different compounds as indicated. The p65 DNA-binding activity of NF-B
was quantified by ELISA by means of the EZ Transcription Factor kit NF-κB p65 (Pierce, Thermo-Scientific)
according to the manufacturer's instructions on nuclear extract. Nuclear protein cell extracts were obtained as
described previously (15). Protein concentrations were determined by the Bradford method using the Bio-Rad
protein assay reagent. NF-B binding to the target oligonucleotide was detected by incubation with primary
antibody specific for the activated form of p65, visualized by anti-IgG horseradish peroxidase conjugate and
developing solution, and quantified by chemilumines-cence with a luminometer. Specificity was established by
incubation with a wild-type consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'), which
competes with the substrate for binding. All samples were analyzed in duplicate.
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays were done as described previously (16). Nuclear extract samples containing
5 µg protein were mixed the 32P-labeled probe (sequence available upon request). The complexes were separated
on a polyacrylamide gel. To confirm specificity, competition assays were done with a 50-fold excess of
unlabeled wild-type and nonspecific probes. For supershift-ing experiments, 1 µg of each antibody (p50 or p65;
both from Santa Cruz Biotechnology) was incubated with the extracts for 30 min on ice before the addition of
the 32P-labeled probe.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
Mouse Tumor Model and lacZ Staining
C57BL/6 κB-dependent lacZ transgenic mice, line 189-4 (17), 10 to 15 weeks old, were s.c. inoculated on the
right flank with 105 B16F10 melanoma cells. These transgenic mice contain the pl05 promoter upstream of the
lacZ reporter gene, which causes production of β-galactosidase in response to the activation of NF-κB. At day
11, these mice were treated with anginex (10 mg/kg), endostatin (10 mg/kg), or TNF-α (5 µg/kg). Twenty-four
hours after treatment, mice were sacrificed and tumors were resected and snap frozen immediately.
Tissue sections were processed for β-galactosidase activity detection as described previously (18). In short,
tissues were fixed in ethanol, dehydrated in xylene, and then embedded by three passages at 44°C in low melting
point paraffin (Merck, VWR). For β-galactosidase activity detection, paraffin sections (7 µm) were incubated in
X-Gal solution at 32°C for 16 h as described previously.
Dual staining for PECAM-1 and β-galactosidase has been done with a rat anti-mouse PECAM-1 (Becton
Dickinson) and an anti-rat secondary antibody coupled to the peroxidase (blue staining) and a rabbit anti-βgalactosidase (kindly given by J.F. Nicolas, Institut Pasteur) identified by an anti-rabbit secondary antibody
coupled to the alkaline phosphatase (red staining).
RNA Extraction and Quantitative Reverse Transcription-PCR
Total RNA was extracted from tumor using the RNeasy kit (Qiagen) according to the manufacturer's
instructions. Synthesis of cDNA was done starting with 1 µg total RNA, which was reverse transcribed with the
Transcriptor First-Strand cDNA Synthesis kit (Roche Clinical Laboratories) according to the manufacturer's
instructions. The resulting cDNA (300 pg) was used for real-time PCR with the iQSYBR Green PCR Supermix
(Bio-Rad). Thermal cycling was done on an ABI PRISM 7700 Sequence Detection System apparatus (Applied
Biosystems). For all reactions, negative controls were run with no template present, and random RNA
preparations were also subjected to sham quantitative reverse transcription-PCR (no reverse transcription) to
verify lack of genomic DNA amplification. Relative transcript levels for each gene were obtained using the
relative standard curve method and normalized with respect to the housekeeping gene cyclophilin B. Primers, the
sequences of which are available upon request, were designed using the Primer Express software and selected to
span exon-exon junctions to avoid detection of genomic DNA.
Proliferation/Viability Assay
HUVECs were seeded at 5,000 per well in fibronectin-coated flat-bottomed 96-well tissue culture plates in
culture medium supplemented with 10% FCS. After pretreatment of 1 h with BAY1170-82 (10 µmol/L), cells
were treated with 6.5 µmol/L anginex. After 3 days, viability was assessed by adding 10 µL WST-1 reagent
(Roche) and by measuring absorbance at 450 nm according to the manufacturer's recommendations.
Migration Assay
Migration assays were carried on as described previously (19). Briefly, HUVEC cultures were grown to
confluency in gelatin-coated 24-well tissue culture plates. A cross-shaped wound was made by scratching the
monolayer with a plastic tip. Wounded monolayers were washed with PBS and incubated with complete culture
medium supplemented with bFGF (10 ng/mL). The wound width was measured microscopically at four different
places 0, 2, 4, 6, and 8 h after wounding the culture.
Sprouting Assay
Sprouting assays were carried on as described previously (20). HUVECs were suspended in culture medium and
seeded on nonadherent plates. The suspended cells integrate into a single spheroid of defined size and cell
number. After 24 h of incubation at 37°C, the spheroids were embedded in a collagen gel and treated with the
different factors. Quantification was done by digital imaging and calculating length of the spheroid sprouts after
8 h of incubation. At least 10 spheroids per group were analyzed.
Leukocyte Adhesion Assay
Human peripheral blood leukocytes were isolated by Ficoll gradient centrifugation (Amersham) and labeled with
10 µmol/L 5-(6)-carboxyfluorescein diacetate succinimidyl ester (Molecular Probes) for 30 min at 37°C. Cells
were washed twice with PBS and subsequently allowed to adhere to confluent endothelial cells for 1 h at room
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
temperature. After removal of the nonadhered cells, the wells were trypsinized and harvested cells were fixed for
30 min with 1% paraformaldehyde at room temperature. Leukocyte-endothelial cell adhesion was quantified by
flow cytometry analysis by detecting the number of 5-(6)-carboxyfluorescein diacetate succinimidyl esterlabeled leukocytes versus endothelial cells (21).
Fluorescence-Activated Cell Sorting Analysis
Expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface has been analyzed by
flow cytometry as described previously (22). In short, cells were fixed with 1% paraformaldehyde and ICAM-1
was detected using a mouse anti-human ICAM-1 monoclonal antibody (MEM111; Monosan).
Statistics
All values are expressed as means ± SD. All experiments were done in triplicate and repeated at least three
times. Comparisons between different treatments were assessed using the Student's t test.
Results
Angiostatic Agents Induce NF-κB Activation in Endothelial Cells
We have shown previously that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent
angiostatic and antitumor factor, acting by an apoptosis-inducing mechanism that requires activation of NF-κB in
endothelial cells (16, 23, 24). To investigate whether this activity is a common feature of angiostatic agents, we
tested several other angiostatic compounds with a direct effect on endothelial cells, anginex (12, 14), endostatin
(25), and angiostatin (26), along with 16K hPRL. These agents specifically target endothelial cells but are
otherwise biochemically unrelated. Cultured human endothelial cells (HUVEC) were treated for 1 and 4 h with
these inhibitors. The p65 DNA-binding activity of NF-κB was quantified by ELISA by immobilization of a
double-stranded DNA consensus sequence for p65. TNF-α, used as a positive control, induced a significant 2.7fold up-regulation of NF-κB activity in HUVEC after 1 h as shown by a higher nuclear p65 DNA-binding
activity (Fig. 1A). Interestingly, all four angiostatic molecules significantly activated NF-κB in endothelial cells
after 1 and 4 h of stimulation (Fig. 1B-E). The level of activation for all four angiostatic agents was found to be
comparable with that of TNF-α (2-to 3-fold induction). In contrast, bevacizumab/Avastin (1), a humanized
monoclonal vascular endothelial growth factor neutralizing antibody, failed to activate NF-κB (Fig. 1F). All four
angiostatic agents activated NF-κB at concentrations described previously to block angiogenesis in vitro (data
not shown). To confirm these results, RF-24 immortalized human endothelial cells were treated with TNF-α and
the four angiostatic molecules for 1 h. ELISA revealed that TNF induced a significant 4.0-fold up-regulation of
NF-κB activity (Fig. 1G). As observed previously in HUVEC, all four angiostatic agents significantly activated
NF-κB in RF-24.
We then focused on the dissection of the molecular action of anginex as a model angiostatic compound. We first
quantified the activation of NF-κB in HUVEC by a concentration range of anginex. Anginex induced NF-κB
activity in a concentration-dependent manner starting at 1.25 µmol/L as shown by ELISA (Fig. 2A). A timecourse experiment was then done with HUVEC exposed to 6.5 µmol/L anginex, a concentration known to block
proliferation, migration, and sprouting of endothelial cells. Anginex already activated p65 DNA binding after 30
min of stimulation (Fig. 2B). Peak levels of activation were observed at 60 min, after which NF-κB activation
decreased to basal levels over a period of ~2 h. A significant activation was then observed after4 h of
stimulation, suggesting a biphasic NF-κB activation. Interestingly, significant activation of NF-κB was still
observed 24 h after stimulation with anginex. A similar kinetic of activation was observed for endostatin,
angiostatin, and 16K hPRL (data not shown). These results suggest that NF-κB activation is a proximal event in
the pathway activated by angiostatic agent in endothelial cells.
To further identify the NF-κB dimer activated by anginex, electrophoretic mobility shift assays were done.
Nuclear extracts from HUVEC, stimulated with 6.5 µmol/L anginex for 60 min, were incubated with a 32Plabeled κB DNA sequence and subsequently run on acrylamide gel. Only one specific band corresponding to the
κB/NF-κB complex was found to increase on treatment with anginex (Fig. 2C, lane 2) compared with the band
obtained from untreated cells (lane 1). The presence of only band suggested that only one NF-κB dimer was
activated by anginex. The specificity of the NF-κB complex detected was shown by its disappearance in the
presence of 100-fold molar excess of unlabeled KB probe (lane 5) and its steadiness in the presence of an
unlabeled mutated probe (lane 6). Furthermore, use of p50- and p65-specific antibodies (lanes 3 and 4)
completely shifted the NF-κB complex induced by anginex. Further experimentation may be required to analyze
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
if other NF-κB subunits may also be activated by anginex. From these experiments, we concluded that anginex
mainly activates the p50/p65 het-erodimer in cultured endothelial cells.
Angiostatic Agents Activate NF-κB in Tumor Endothelial Cells
We next investigated whether NF-κB is activated in vivo in tumor endothelial cells on treatment with angiostatic
agents. To that end, we used transgenic mice in which a NF-κB-responsive promoter element drives a lacZ
reporter gene containing a nuclear localization sequence at its 5' terminus (17). B16F10 melanoma cells were
injected s.c, which gave rise after 11 days to a tumor of 500 to 1,000 mm3. Mice were then treated with the
angiostatic agents anginex or en-dostatin (10 mg/kg) or injected with TNF-α (5 µg/kg) as a positive control of
NF-κB activation. Anginex and endostatin has been found previously to reduce B16F10 melanona tumor growth
and microvessel density with a maximum of effect between 5 and 20 mg/kg/d (22, 27). Twenty-four hours later,
tumors were resected and the level of NF-κB activation in tumor tissues was assessed by X-Gal staining of tissue
sections. Interestingly, β-galactosidase activity was observed exclusively in tumors of treated mice with anginex
or endostatin as well as in tumors of mice that were injected with the positive control TNF-α (Fig. 3A). βGalactosidase immunoreactivity colocalized with the endothelial cell marker PECAM-1. Endothelial cells with
β-galactosidase activity were distributed throughout the tumor with most activity observed in the outer rim of the
tumor. Even if we cannot rule out the possibility that TNF-α activated NF-κB in tumor cells, no tumor cells were
positive for β-galactosidase after TNF-α treatment because tumor cells did not harbor the κB-lacZ reporter
construct.
To sensitively quantify the level of NF-κB activation observed in tumor endothelial cells, quantitative real-time
reverse transcription-PCR experiments using primers complementary to the lacZ mRNA sequence were done on
mRNA extracted from treated and untreated tumors. A significant and strong increase of the lacZ mRNA was
observed in tumors treated with anginex (mean 4.8-fold) and TNF-α (mean 4.4-fold), whereas endostatin led to a
significant but lower enhancement of this mRNA (mean 2.0-fold; Fig. 3B). We then confirmed the NF-κB
activation by analyzing the expression of a natural target of NF-κB: IκB-α (Fig. 3C). IκBa is a well-described
target gene of NF-κB, which is involved in a negative feedback loop. Consistently with the lacZ results, we
found a significant increase of IκBα mRNA in tumor treated with anginex (mean 1.9-fold) and TNF-α (mean 1.3fold). For endostatin, the increase was very moderate and not significant. This moderate increase of IκBα
expression compared with the one observed for lacZ could reflect that basal expression of IκBα is ubiquitous (in
tumor and nontumor cells) compared with the lacZ expression, which is restricted to the nontumor cells.
Therefore, the increase of IKBα expression may have been masked by the basal level of IκBα expression.
Together, these results show that NF-κB-dependent transcription is significantly induced in tumor endothelial
cells after treatment with angiostatic agents.
Implication of NF-κB Activation in the Angiostatic Signaling
To assess the importance of NF-κB in the angiostatic signaling, we blocked NF-κB activation with BAY117082, a chemical inhibitor known to interfere with IκB kinase (28, 29). In HUVEC, 10 µmol/L BAY1170-82
efficiently inhibited activation of NF-κB treated with anginex (Fig. 4A). As reported previously, and as shown in
Fig. 4B, anginex strongly reduced the proliferation of HUVEC (~80%). Pretreatment for 1 h with the NF-κB
inhibitor did not affect this decrease, suggesting a NF-κB-independent effect of anginex on endothelial cell
proliferation. Endothelial cell migration was then investigated using the wound assay. As shown in Fig. 4C,
anginex inhibited the wound closure by endothelial cells. This effect was already significant after 4 h of
stimulation. Pretreatment of endothelial cells with the NF-κB inhibitor did not modify the anginex antimigratory
feature. Finally, the role of NF-κB in the sprouting of endothelial cells was investigated. In the absence of
growth factors (e.g., bFGF), HUVEC spheroids embedded in a collagen gel showed very little sprouts.
Stimulation of spheroids with bFGF significantly increased the sprout lengths (Fig. 5A). Interestingly, NF-κB
inhibitor BAY11 70-82 tended to increase the sprout length on nonstimulated spheroids. Anginex treatment
completely reduced the sprouting of endothelial spheroids induced by bFGF (Fig. 5B). After treatment with
anginex, the sprout length was similar to that observed in the absence of bFGF. Pretreatment of endothelial cells
with the NF-κB inhibitor for 1 h before anginex treatment strongly abrogated the anginex-induced inhibition of
sprouting. These results showed the role of NF-κB in the control of endothelial cell sprouting and suggested the
NF-κB dependency of anginex inhibition of endothelial cell sprouting.
NF-κB Is Required for Angiostatic-lnduced Reversal of Endothelial Cell Anergy
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
Angiogenesis inhibitors are also shown to display an indirect antitumor activity through reversal of endothelial
unresponsiveness to inflammatory signals, a process called endothelial cell anergy. It has been shown that this
activity, restoration of adhesion molecules such as IC AM-1 to normal levels, is present in many, if not all,
angiostatic agents (22,30). To determine whether NF-κB activation is involved in this process, we monitored
peripheral blood leukocyte adherence on a surface of cultured endothelial cells. HUVECs treated for 24 h with
anginex showed an increased capacity to bind leukocytes. Pretreatment with the NF-κB inhibitor BAY1170-82
blocked ~80% of this activity (Fig. 6A). Fluorescence-activated cell sorting analysis showed that whereas
anginex up-regulated ICAM-1 expression on the endothelial cell surface, NF-κB inhibitor abolished this upregulation significantly (Fig. 6B). Altogether, these results show that the NF-κB pathway in endothelial cells is
involved in overcoming endothelial cell anergy induced by ongoing angiogenesis.
Discussion
Angiostatic agents are increasingly important in the clinical management of cancer. Many angiostatic agents
interfere with the angiogenic process by blocking the proliferation/ survival, the migration, or the maturation of
endothelial cells. The molecular targets of these agents have only been partially discovered (3, 31, 32). A
thorough understanding of the mechanism of action of angiostatic agents, mainly at the level of proximal
signaling events, is still required for the development of efficient antitumor therapies. The current report
describes that angiostatic agents with a direct activity on endothelial cells, such as 16K hPRL, anginex,
endostatin, and angiostatin, activate NF-κB in endothelial cells.
NF-κB activation was observed in endothelial cells already after 30 min of stimulation with angiostatic agents.
This time-course activation is comparable with what has been described previously for TNF-α in endothelial
cells (33). These results suggest that the NF-κB pathway is an early event in response to binding of angiostatic
agents to their receptors. The transgenic mice harboring a NF-κB-responsive promoter element driving a lacZ
reporter gene provided the opportunity to evaluate NF-κB activation in vivo after treatment with angiostatic
agents in tumor endothelial cells both spatially and quantitatively.
Twenty-four hours after treatment with anginex or endostatin, NF-κB was activated in tumor endothelial cells as
measured by β-galactosidase activity and expression of lacZ and IKBα mRNA. To our knowledge, this is the first
report illustrating activation of NF-κB in tumor endothelial cells after treatment with angiostatic agents. In this
context, it is interesting to note that NF-κB is known to be activated by proinflammatory cytokines such as TNFα, interleukin-1, and IFN-γ. These cytokines have antiangiogenesis activity as well. This is most well known for
TNF-α (34) and IFN-γ(35), which, depending on the dose, can even induce apoptosis in endothelial cells.
Interleukin-1 is mostly known as an angiogenesis stimulator, but some reports identified inhibitory activities as
well (36).
The role of NF-κB in endothelial cells is poorly understood (37). We found previously that, in bovine endothelial
cells, 16K hPRL induces caspase-dependent apoptosis by a mechanism that requires activation of NF-κB (30).
Interestingly, endostatin and anginex have also been described to induce apoptosis in endothelial cells (12). We
here showed that NF-κB is induced in human and mouse endothelial cells after exposure to any of these
inhibitors. However, specific inhibition of NF-κB does not counteract anginex-induced inhibition of
proliferation/viability but abrogate anginex-induced inhibition of sprouting. These results suggest that even if
NF-κB activation is a common pathway activated by angiostatic agents, the role of NF-κB is agent-specific
and/or cell-specific.
Even if BAYll 70-82 has been shown previously to target specifically the NF-κB pathway at concentrations <20
µmol/L, we cannot rule out the possibility that this compound may have off-target effects in endothelial cells.
Therefore, p65 silencing would be an optimal strategy to confirm our results. However, transfection of primary
endothelial cells such as HUVEC is a challenging option. In our hands, optimized transfection protocols gave an
efficiency rate of ~30%, indicating that the NF-κB pathway could be blocked in a maximum of 30% of the cells
and therefore making the data interpretation difficult. In addition, the toxicity we observed following transfection
was not negligible, as is not unusual with primary endothelial cells, suggesting that many stress-related pathways
would be activated by the transfection process and confuse the results about the role of NF-κB in angiogenesis.
Very recently, NF-κB signaling has been found to regulate endothelial cell integrity and vascular homeostasis in
vivo. Treatment of zebrafish embryos with NF-κB inhibitors provoked vascular leakage and altered vessel
morphology (38). Tie2 promoter/enhancer-IκBS32A_S36A transgenic mice, in which the endothelial-specific Tie2
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
promoter drives the expression of a trans-dominant-negative mutant of NF-κB and therefore present an inhibition
of endothelial NF-κB signaling, developed normally and displayed, in particular, a normal pattern of vascular
development (39). However, inoculated tumors grew faster in these mice, and histologic analysis revealed a
striking increase in tumor vascularization. The molecular mechanisms accounting for such increase are still
unknown. Our data are in accordance with these observations and furthermore suggest that NF-κB activation is
mainly involved in the spatial organization of endothelial cells such as sprouting.
The activation of NF-κB can also be connected with an indirect antitumor activity through reversal of endothelial
unresponsiveness to inflammatory signals, a process called endothelial cell anergy. The latter is defined by us as
the absence of leukocyte-binding adhesion molecules on endothelial cells and the unresponsiveness to
inflammatory cytokines (40). This phenomenon occurs due to exposure of endothelial cells to angiogenic growth
factors. NF-κB is best known for its regulation of proinflammatory genes. We have reported previously that
angiogenic factors (e.g., bFGF) down-regulate endothelial adhesion molecules such as ICAM-1, vascular cell
adhesion molecule-1, and E-selectin through NF-κB inhibition (41). In addition, tumor endothelial cells
displayed a reduced expression of adhesion molecules compared with that of normal endothelial cells,
accounting for the observation of a reduced number of infiltrated leukocytes within the tumor (42). Accordingly,
we found in the current study that NF-κB activation by angiostatic agents is correlated with ICAM-1 upregulation at the endothelial cell surface. This expression further results in an enhanced NF-κB-dependent
leukocyte-endothelial cell interaction. Together, these data show that anginex, and possibly other angiogenesis
inhibitors as well, overcome endothelial cell anergy by activation of the NF-κB pathway. This activity may be
shared with the abovementioned proinflammatory cytokines that can have intrinsic antiangiogenesis activities as
well (34-36). Therefore, activation of NF-κB in endothelial cells resulting in stimulation of antitumor immunity
can clearly result in an antitumor outcome.
The fact that different biochemically and biologically unrelated angiostatic agents activate the NF-κB pathway is
intriguing. Recently, galectin-1 has been identified as the anginex receptor at the surface of endothelial cells
(43). The importance of gal-1 in angiogenesis has been further illustrated in the zebrafish model, where
expression knockdown results in impaired vascular guidance and growth of dysfunctional vessels. However, thus
far, no direct evidence has linked galectin-1 and the NF-κB pathway. Endo-statin may exert its antiangiogenic
effect through its binding to α5β1 integrin or to binding to glypican (44, 45). However, it is not obvious which
endostatin receptor conveys the effective antiangiogenic signals. Our data showing that NF-κB pathway is
activated by different angiostatic agents already after 1 h of treatment suggest that a common receptor/
coreceptor exist at the surface of endothelial cells. Further studies will be required to decipher the pathway
upstream NF-κB activation up to the receptor.
We report for the first time that activation of NF-κB is a common pathway used by multiple and biochemically
unrelated angiostatic agents. NF-κB activation has been observed both in vitro and in vivo in tumor endothelial
cells. Furthermore, we showed that NF-κB inhibition impairs the ability of angiostatic agents to block sprouting
of endothelial cells and to overcome endothelial cell anergy. This is of special interest because, in tumor cells,
NF-κB activation has been associated to inhibition of apoptosis. Therefore, currently novel treatment strategies
are being developed based on inhibition of NF-κB. Although this would cause an antitumor effect in tumor cells,
it is suggested to coincide with proangiogenic activity. Based on our results, the antitumor activity of angiostatic
agents is linked to activation of NF-κB in endothelial cells. This activation could result in stimulation of
antitumor immunity and in inhibition of angiogenesis. It is attractive to speculate that optimal therapeutic results
could be achieved by either (or both) targeting inhibitors of NF-κB to tumor cells and/or by specific activation of
NF-κB in endothelial cells.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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Published in: Molecular Cancer Therapeutics (2009)
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Figure 1. Angiostatic agents induce NF-κB activation in endothelial cells. HUVECs were stimulated with TNFct (A; 10 ng/mL), anginex (B; 6.5 µmol/L), endostatin (C; 5 µg/mL), angiostatin (D; 5 µg/mL), 16K hPRL (E; 20
nmol/L), and bevacizu-mab (F; 10 µg/mL) or for 1 and 4 h. G, RF-24 cells were treated with TNF-α (10 ng/mL),
anginex (6.5 µmol/L), endostatin (5 µg/mL), angiostatin (5 µg/ mL), or 1 6KhPRLfor 1 h. Nuclear extracts were
evaluated by ELISA for the presence of p65. Results are relative mean values of at least three experiments. *, P
< 0.05, compared with control.
Figure 2. Characterization of the NF-κB activation by anginex. A, dose response of NF-κB activation in HUVEC
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
treated by anginex for 1 h. B, kinetics of NF-κB activity in HUVEC stimulated for a time range with 6.5 µmol/L
anginex. In A and B, nuclear extracts were evaluated for the presence of NF-κB p65 by ELISA. Results are
relative mean values of at least three experiments. *,P < 0.05, compared with control. C, electrophoretic
mobility shift assay analysis in HUVEC stimulated with 6.5 µmol/L anginex for 1 h. Electrophoretic mobility
shift assays were done on nuclear extracts preincubated either without (lanes 1 and 2) or with addition of p50specific antibody (lane 3) or p65-specific antibody (lane 4). In competition assays, extracts were preincubated
with a 100-fold molar excess of unlabeled oligonucleotide containing either wild-type (wt; lane 5) or mutated
(mut; lane 6) NF-κB binding sites, ns, nonspecific band. Representative result of at least three independent
experiments.
Figure 3. Angiostatic agents activate NF-κB in tumor endothelial cells in vivo. A, B16F10 mouse melanoma
tumor cells were injected s.c. in mice carrying a KB-dependent lacZ reporter gene. At day 11 after inoculation,
when tumors reached the size of 500 to 1,000 mm3, mice were treated with anginex, endosta-tin (both 10 mg/kg),
or TNF-ct (5 µg/kg). Twenty-four hours later, mice were sacrificed. Tumor sections were stained for NF-KBdependent nuclear β-galactosi-dase activity and counterstained with H&E or stained for PECAM-1 and β-galactosidase. B and C, RNA extracted from the tumors was analyzed by quantitative reverse transcription-PCR
with primers for lacZ (B) or lκBα (C) mRNAs. Results are relative mean values of at least five tumors. *,P <
0.05; **, P < 0.01, compared with untreated controls.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
Figure 4. Implication of NF-κB activation in the angiostatic signaling. A, NF-κB activity in HUVEC pretreated
or not with BAY1170-82 (10 µmol/L) and stimulated for 48 h with anginex (6.5 µmol/L). Nuclear extracts were
evaluated for the presence of NF-κBp65 by ELISA. Results are relative mean values of at least three
experiments. *, P < 0.05, compared with untreated plus vehicle; #,P< 0.05, compared with anginex plus vehicle.
B, HUVECs were pretreated or not with BAY1170-82 (10 µmol/L), stimulated for 48 h with anginex (6.5
µmol/L), and assessed for their ability to incorporate bromodeoxyuridine. Bromodeoxyuridine incorporation
was measured by ELISA and presented as relative mean values of at least three experiments. *,P< 0.01,
compared with untreated plus vehicle. C, migration of endothelial cells was determined by wound assays.
Confluent HUVEC cultures were pretreated or not with BAY1070-82 (10 µmol/L), wounded, and then treated
with anginex (6.5 µmol/L). Wound width is plotted as relative mean values of at least three experiments. *, P <
0.05; **, P < 0.01, compared with untreated plus vehicle.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
Figure 5. Implication of NF-κB activation in the endothelial cell sprouting. A, HUVEC spheroids were treated
with bFGF (10ng/mL)or BAY1170-82 (10 µmol/L) for 8 h. *, P < 0.05, compared with untreated. B, HUVEC
spheroids were cultured in the presence of bFGF (10 ng/mL) and pre-treated or not with BAY1170-82 ( 10
µmol/L) for 1 h. Subsequently, they were treated with anginex (6.5 µmol/L) for 8 h. For each experiment, at least
10 spheroids per group were analyzed and cumulative spheroids length was measured. Results are relative mean
values of at least three experiments. *,P < 0.05, compared with untreated plus vehicle; #,P < 0.05, compared
with anginex plus vehicle.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)
Figure 6. NF-κB is required to overcome endothelial cell anergy. A, flow cytometry assessment of adhesion of 5(6)-carboxyfluorescein diacetate succinimidyl ester-labeled human peripheral blood leukocytes to monolayers of
HUVEC pretreated or not for 1 h with 10 µmol/L BAY11-7082 and then treated or not with anginex for 16 h.
Results are relative mean values of at least three experiments. *,P < 0.05, compared with untreated plus vehicle;
#P < 0.05, compared with anginex plus vehicle. B, flow cytometry analysis of ICAM-1 expression on HUVEC.
Results are relative mean values of at least three experiments. *,P < 0.01, compared with untreated plus vehicle;
#,P < 0.01, compared with anginex plus vehicle.
Published in: Molecular Cancer Therapeutics (2009)
Status: Postprint (Author’s version)