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Regular Articles |
From the Laboratories of Molecular Pathology and
Ultrastructure,*
Immunology,
and
Virology,
Regina Elena Cancer Institute; and
the Biotechnology Institute,§
Consiglio
Nazionale delle Ricerche, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated that expression of ET-1 is significantly increased in the majority of ovarian carcinomas compared with normal ovarian tissues. In these tumor cells ET-1 acts as an autocrine growth factor selectively through ETAR, as demonstrated by the inhibitory proliferative effects induced by a specific ETAR antagonist.11-13 Moreover, the presence of ET-1 correlates with tumor vascularity and malignancy in well-vascularized brain tumors,14 in colorectal cancer,15 and ET-1 binding sites have also been characterized in the vessels of pulmonary tumors.16 In addition, because ET-1, predominantly through ETAR, stimulates the synthesis of vascular endothelial growth factor (VEGF) in vascular smooth muscle cells and the VEGF-mediated angiogenic effects,17 we hypothesized that the effect of ET-1 could be mediated by direct actions on tumor vessels and in part by VEGF stimulation.
VEGF a potent and specific mitogen for endothelial cells is also expressed in many tumors, including ovarian carcinoma, where it stimulates the cascade of events required for angiogenesis.18-22 VEGF activity is mediated by two tyrosine kinase receptors, flt-1 expressed predominantly by endothelial cells23,24 and KDR expressed in endothelial cells as well as by ovarian tumor cells.25
To investigate the potential role of ET-1 in ovarian tumor angiogenesis, we performed immunohistochemical analysis of ET-1, VEGF, and their receptors in archival specimens of primary and metastatic human ovarian carcinomas (n = 48). By in situ hybridization and autoradiographic binding studies, we examined the localization of ET-1 receptor expression in ovarian tumor vessels. Furthermore we determined whether expression of ET-1 and its receptors is associated with vessel counts and with VEGF expression. Because ovarian cancer characteristically remains mainly confined to the peritoneal cavity, concentrations of ET-1 were measured in ascitic fluids. We have therefore investigated whether ET-1 released from ovarian carcinoma cells might modulate the production of VEGF and whether it could induce endothelial cell migration, a prerequisite for tumor neovascularization. All these findings, together with the high levels of ET-1 in neoplastic ascitic fluids, are consistent with the hypothesis that ET-1 plays an important role in ovarian cancer related-angiogenesis and represents a potential important target of anti-angiogenic therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human ovarian carcinoma cell line, OVCA 433, a gift from Dr. G. Scambia (Catholic University School of Medicine, Rome, Italy), was cultured in Dulbecco’s modified Eagle’s medium and 10% fetal calf serum. Human endothelial cells were isolated from human umbilical vein (HUVECs) (Promocell, Heidelberg, Germany) and maintained in an endothelial cell growth medium kit containing with 2% fetal calf serum (Promocell). Tumor specimens were obtained with informed consent from 48 patients (age range, 27 to 65 years) undergoing surgery for ovarian carcinomas at the Regina Elena Cancer Institute. Primary tumors included 10 adenocarcinomas and 16 serous, eight mucinous, six endometrioid, and eight omental metastasis derived from five adenocarcinomas and three serous adenocarcinomas. Tissue samples were immediately snap-frozen in liquid nitrogen. From each specimen, 4-µm cryostat sections were obtained and fixed in absolute acetone for 10 minutes. Ascitic fluids were collected from an additional 20 patients with ovarian carcinoma, centrifuged at 4000 x g for 10 minutes, and then stored at -70°C in 1 ml aliquots. Control ascitic samples were obtained from five patients with nonneoplastic diseases.
Immunohistochemistry
Consecutive 4-µm sections were immunostained for VEGF, ET-1, CD31 (specific for endothelial cells), receptors for VEGF (KDR and flt-1), and receptors for ET-1 (ETAR and ETBR). Immunohistochemical staining was performed by the immunoperoxidase technique (Vector Laboratories, Burlingame, CA). Antibodies used were a rabbit polyclonal antibody (Ab) (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:200 dilution for VEGF; a mouse monoclonal Ab (clone TR.E.T.48.5; Affinity Bioreagents, Golden, CO) at 1:200 dilution for ET-1; two rabbit polyclonal antipeptide antibodies (a generous gift from Dr. R. Wu-Wong, Abbott, IL) at 1:20 dilution for ETAR and ETBR; a mouse monoclonal Ab (clone JC/70A; DAKO, Glostrup, Denmark) at a 1:200 dilution for CD31; two rabbit polyclonal antibodies (Santa Cruz Biotechnology) at a 1:100 dilution for KDR and flt-1. The VEGF (147) antibody is a rabbit polyclonal IgG raised against an amino-terminal epitope (1 to 140) common to all splice variants of VEGF. For ETAR, an Ab was raised against a decapeptide (DNPERYSTNL) of the extracellular NH2-terminal domain of ETAR, for ETBR an Ab was raised against a peptide (CGLSRIWGEERGFPPDRTP) of the NH2-terminal domain of ETBR. To ensure specificity, the primary Ab was preabsorbed for 12 hours at 4°C with a 50-fold excess of synthetic ET-1 (Peninsula Laboratories, Belmont, CA) or VEGF (Santa Cruz Biotechnology), omitted, or substituted with preimmune rabbit serum (negative control for ETAR and ETBR) or nonspecific IgG (negative control in all cases). Staining for positive controls was done on tissue from a colon cancer for VEGF; tissue from a breast cancer for ET-1; umbilical vein tissue from a bladder tumor for KDR, flt-1 and ETBR; vascular smooth muscle cells for ETAR. Nuclear counterstaining was performed with hematoxylin. The homogeneous staining for VEGF and ET-1 and their receptors, which was consistently present in all tumor cells, was scored into different grades as intensity of staining on an arbitrary scale of 0 to +3. Grade 0 represented cases in which tumor cells showed no detectable stain or traces (<20% of the tumor cells) of positive staining. Grade 1 represented cases that showed homogeneous reactivity in the majority of the tumor cells but with a weak staining intensity. Grade 2 represented cases that showed diffuse immunoreactivity in the majority of the tumor cells but with an intermediate intensity of staining. Grade 3 was assigned to cases in which the tumor cells homogeneously showed strong positive staining. Two independent observers performed tissue reading. The presence or absence of receptors was evaluated on both tumor endothelial cells and tumor epithelial cells. Vessel count was assessed by light microscopy in areas of the tumor containing the highest numbers of capillaries and small venules at the invasive edge. The highly vascular areas (hot spots) were identified by scanning tumor sections at low power and individual vessel count was performed by two independent observers on a x200 field, according to the criteria of Weidner et al,26 in which vessel lumen was not necessary for a structure to be defined as a vessel. Any immunostained endothelial cell clearly separated from adjacent microvessels, tumor cells, and other stromal elements, was considered a single microvessel.
In Situ Hybridization
Frozen tissue sections (6 µm) were collected onto slides and fixed in 4% paraformaldehyde for 20 minutes at 22°C, dehydrated through alcohols, and stored at -70°C. After rehydration for 15 minutes with phosphate-buffered saline 1x, 50 mmol/L MgCl2 and 15 minutes with 200 mmol/L Tris-HCl, pH 7.5, 100 mmol/L glycine, acetylation with 2x standard saline citrate (SSC), 100 mmol/L triethanolamine, 0.25% acetic anhydride, dehydration with alcohols and 0.6 mol/L ammonioacetate, and air-drying, slides were hybridized with buffer containing 50% formamide, 1 mg/ml salmon sperm DNA, 2x SSC, 10% dextran sulfate, 70 nmol/L dithiothreitol, and 10 to 13 x 103 cpm/µl of probe 35S-labeled CTP (40 mCi/ml; Amersham). Sense and anti-sense riboprobes for ETAR were synthesized from pBluescript SK- vectors with a full-length human ETAR cDNA insert. After hybridization for 16 to 20 hours at 50°C in a humid chamber, slides were washed 20 minutes in 2x SSC at room temperature, twice in 2x SSC-50% formamide at 45°C, once in 1x SSC-50% formamide at the same temperature, and in 0.1x SSC at room temperature. For autoradiography, slides were coated with NTB2 emulsion (Kodak, Rochester, NY) and exposed at 4°C for 2 weeks. After developing in Kodak D19, the sections were fixed and counterstained with hematoxylin.
Autoradiographic Binding Studies
Autoradiography was performed on five human ovarian carcinoma tissues as previously described.27 After washing with 50 nmol/L Tris-HCl buffer, pH 7.4, containing 0.2% bovine serum albumin and 0.005% polyethylenimine (Sigma, St. Louis, MO), consecutive sections (10 µm) were incubated at 25°C for 60 minutes in a buffer supplemented with 40 µg/ml bacitracin (Sigma) in the presence of tracer, 0.1 nmol/L 125I-ET-1 (2,200 Ci/mmol; Dupont New England Nuclear Research Products, Wilmington, DE) in the absence (total binding) and in the presence of 1 µmol/L ET-1 (Peninsula Laboratories) (nonspecific binding). For total ETB binding sections were incubated in the presence of 1 µmol/L BQ 123 (Peninsula) and for total ETA binding with 1 µmol/L sarafotoxin S6c (Peninsula) or 1 µmol/L BQ 788 (Peninsula). After consecutive washes, slides were dipped in NTB2 emulsion and after 15 days developed in Kodak D19, fixed, and counterstained.
Assays of Angiogenic Factors
OVCA 433 cells were seeded at 1 x 106 cells/dish in complete medium and serum-starved for 24 hours. Cells were cultured at 37°C with 95% air/5% CO2 or incubated in aluminum chambers flushed with a gas mixture containing 5% CO2 and 95% N2 (hypoxia). After varying times, cell supernatants were collected, centrifuged, and frozen for subsequent use. Each conditioned media and ascitic sample was subjected to one freeze-thaw cycle only. All assays (ET-1 and VEGF) were performed in duplicate on microtiter plates by an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The working range in the enzyme-linked immunosorbent assay for VEGF assay was 31.2 to 2,000 pg/ml and for ET-1 was 0 to 120 pg/ml. When necessary, dilutions of the ascitic samples were made. Control ascitic samples from five patients with no malignant disease were also assayed.
Chemotaxis Assay
To examine the paracrine chemoattractant effects of the OVCA 433-conditioned medium and of the ascitic fluids on HUVEC migration, a 48-well modified Boyden chamber (Neuroprobe, Pleasanton, CA) and 0.01% gelatin-coated polyvinylpyrrolidone-free polycarbonate filters (8-µm pore, Nucleopore: Costar, New York, NY) were used. The lower compartment of the chamber was filled with chemoattractants or inhibitor (27 µl/well). Cells (5 x 105 cells/ml) were placed in the upper compartment (55 µl/well). Some of the ascitic fluid of OVCA 433-conditioned medium was preabsorbed with antibody (at a dilution of 1:50) to VEGF (Santa Cruz Biotechnology) for 2 hours before use. ET-1 receptor antagonists (BQ 123 and BQ 788; Peninsula Laboratories) were previously added to the HUVECs and preincubated for 15 minutes at 37°C. After 4 hours of incubation at 37°C, filter was removed, and cells on the upper side were scraped off. Migrated cells were fixed, stained with Diff-Quick (Baxter Diagnostics, Miami, FL), and counted in 10 high-power fields. Each experimental point was analyzed in triplicate.
Statistical Analysis
All correlations were examined by the Spearman count correlation coefficient. All statistical analyses were performed by the Inplot software system (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a panel (n = 48) of primary and
metastatic ovarian carcinomas we evaluated the expression of ET-1,
ETAR, and ETBR. The
presence of mature ET-1 was found in 84% of the ovarian carcinomas
examined, including primary and metastatic lesions of different ovarian
cancer histotype. ET-1 was localized in the cytoplasm of tumor and
metastatic cells and in endothelial cells lining the tumor-feeding
vessels (Figure 1A)
. No staining was
observed with anti-ET-1 Ab preabsorbed with an excess of blocking ET-1
(data not shown). Specific immunostaining identifying
ETAR was seen primarily in the majority (89%) of
cell cytoplasms in all areas of the tumors and metastases and in blood
vessels adjacent to neoplastic cells (Figure 1B)
. Immunostaining with
an antipeptide antibody directed against ETBR was
not detected on tumor cells, but positivity for
ETBR occurred significantly on tumor endothelium
(Figure 1C)
. No staining was noted when preimmune rabbit IgG
substituted the anti-ETA or
anti-ETB for the primary Ab, as negative control
in a human serous ovarian carcinoma (data not shown).
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. No specific labeling was
seen with control sense probe (Figure 2, B and D)
. The silver grain
distribution was similar to the distribution of the
ETAR immunostaining.
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in eight ovarian carcinoma specimens.
Autoradiographic localization of ETA binding was
confirmed to be homogeneous within tumors without detectable hot spots.
125I-ET-1 binding was intense in tumor vessels on
both their muscular and endothelial components (Figure 3A)
. The
addition of an excess of cold ET-1 displaced all
125I-ET-1 binding demonstrating the specificity
of the binding (Figure 3B)
. The addition of a potent
ETBR antagonist, BQ 788, did not affect
125I-ET-1 binding to tumor cells, but displaced
it from the vascular endothelium (Figure 3C)
. The BQ 123, a potent
ETAR antagonist, abolished labeling in the tumor
cells, attenuated it in the vascular muscular component and showed only
a decreased labeling related on the presence of
ETB receptors on vascular cells (Figure 3D)
.
Sarafotoxin 6C (S6C), an ETBR agonist, determined
results similar to those obtained with BQ 788 (data not shown). These
results demonstrated the distribution of the ETAR
and ETBR subtypes on tumor cells and vascular
smooth muscle cells and on endothelial cells, respectively. No specific
ETB binding sites were present in ovarian tumor
cells.
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A total of 38 out of 48 (79%) ovarian carcinomas, including
primary and metastatic, showed VEGF immunoreactivity. As previously
reported,25,28,29
the intensity of VEGF staining was
homogeneous within tumors, with no detectable hot-spot, and was mainly
localized in the cytoplasm of all neoplastic cells (Figure 4A)
. Specificity of VEGF immunostaining
was confirmed by a complete loss of staining when the antibody was
preneutralized with the corresponding control antigen (data not shown).
We further characterized the expression of VEGF receptors in
ovarian carcinoma by immunohistochemistry. The vascular cells in the
ovarian carcinomas expressed high levels of KDR and a significant
amount of flt-1 (Figure 4, B and C)
.
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In the same panel of primary (n = 40) and
metastatic (n = 8) ovarian carcinomas, we
performed immunohistochemistry against CD31, a specific marker of
endothelial cells, and the microvessel number was counted in the
regions which included more microvessels than the rest of the tissue
(hot-spot) (Figures 1D and 4D)
. In the total patient population, the
mean microvessel count in the hot-spot was 38.4 ± 23.7. The mean
microvessel numbers of the ET-1-positive group and the ET-1-negative
group were 57.6 ± 31.4 and 25.9 ± 24.0, respectively,
indicating that there was greater neovascularization in the
ET-1-positive tumors (P < 0.01; Table 1
). The expression of
ETAR in tumor blood vessels was higher in
ET-1-positive tumors than in those negative for ET-1, whereas
ETBR expression was similar between the two
groups (Table 1)
.
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, the analysis of the
relation between ET-1 expression and the number of microvessels in the
tumor areas of highest vascularization showed a statistical association
between tumor microvessel count and ET-1 expression
(r = 0.60; P < 0.0002). The
blood vessel numbers (mean ± SD) in the VEGF-negative and
VEGF-positive tumors were 23.8 ± 15.1 and 53 ± 33.2,
respectively. This difference was statistically significant
(P < 0.0001), indicating that there was greater
neovascularization in the VEGF-expressing cancers (Table 2)
. Therefore, a significant correlation
among vessel count and VEGF expression was observed
(r = 0.71; P < 0.0001). KDR was
the predominantly expressed VEGF receptor in ovarian carcinoma vessels,
with no difference between VEGF-positive and -negative tumors. Flt-1
expression was also similar between the two groups. Moreover, because
vessel count was significantly associated with VEGF expression we
analyzed whether ET-1 correlated with VEGF. In our series of 48 ovarian
carcinomas, we found a highly significant correlation between ET-1
staining and VEGF immunoreactivity (r = 0.673;
P < 0.001).
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Because ovarian cancer growth is mainly confined to the peritoneal
cavity with the production of ascitic fluid, we evaluated whether
advanced ovarian carcinoma would be associated with elevated ascitic
concentrations of VEGF and ET-1. In human studies, high levels of VEGF
(>10 pmol/L) were measured in malignant ascites.30
Taking
10 pmol/L as the threshold value for the detection of malignancy, 17 of
the 20 samples of ascitic fluid obtained from patients with ovarian
carcinoma were VEGF-positive (>10 pmol/L). VEGF levels ranged between
25 and 325 pmol/L in ascitic fluids. High concentrations of ET-1 (
10
pmol/L) were measured in the same 17 VEGF-positive ascites of the 20
effusions tested (85%). ET-1 levels in the ascitic fluids ranged
between 10.4 and 78 pmol/L, whereas only very low or undetectable
levels of ET-1 were found in the five patients whose effusions were
without cytological evidence of malignancy (<2 pmol/L). Moreover,
there was a strong correlation (r = 0.717;
P < 0.0026) between ET-1 and VEGF ascitic
concentrations (Figure 6)
suggesting that
ET-1 and VEGF characterize the advanced disease.
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We investigated whether ET-1 might regulate the production of VEGF
as an important mechanism by which ET-1 could modulate angiogenesis.
ET-1 stimulated VEGF production, with detectable production as early as
1 hour, and maximum stimulation (2.0-fold above control levels) at 24
hours (P < 0.005, Figure 7A
). The ability of ET-1 to stimulate
VEGF production was dose-dependent, reaching maximal stimulation at a
concentration of 100 nmol/L (Figure 7B)
. After 24 hour of incubation,
VEGF-stimulation by ET-1 (100 nmol/L) was comparable in magnitude to
that induced by hypoxia, a recognized potent and important stimulus of
VEGF (Figure 8)
. We then investigated the
ET-1 receptor subtype involved in the regulation of VEGF secretion. The
stimulation by ET-1 (100 nmol/L) was completely blocked by the
ETAR antagonist BQ 123, indicating that ET-1
stimulated VEGF production after binding to ETAR
in ovarian carcinoma cells.
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To test whether ET-1 released from ovarian carcinoma cells could
affect endothelial cell mobility, HUVECs were incubated in a chemotaxis
chamber with ascitic fluids obtained from patients with ovarian
carcinoma (Figure 9A)
and with
conditioned medium of OVCA 433 cells (Figure 9B)
. The ascitic fluid and
the OVCA 433 medium contained high levels of ET-1 (38 ± 3.2 pg/ml
and 62.5 ± 5.3 pg/ml, respectively) as measured by enzyme-linked
immunosorbent assay. The ascites and the spent media (used at a
dilution of 1:2) caused a strong increase in endothelial cell migration
above serum-free Dulbecco’s modified Eagle’s medium used as
control. These stimulatory effects were inhibited by 
45% after
preincubation of conditioned medium and ascitic fluid for 2 hours with
an excess of specific antibody to VEGF, which bounds the VEGF secreted.
Preincubation with BQ123 induced a partial decrease (25%), whereas
the addition of BQ 788 determined an higher inhibition (55%) on
endothelial cell migration induced by both ascites and conditioned
media. Interestingly, co-incubation of VEGF Ab and BQ788 with OVCA
433-conditioned medium reduced by 76% HUVEC migration. Endothelial
cell migration induced by 10 nmol/L of basic fibroblast growth factor
was not inhibited by the addition of 1 µmol/L M BQ 788 or BQ 123,
indicating that the inhibitory effect induced by the
ETB and ETA receptor
antagonist was specific and was not because of cytotoxicity (data not
shown). This in vitro model analyzing the potential
paracrine interactions between ovarian tumor cells and endothelial
cells indicates that ET-1 produced by tumor cells, together with VEGF,
was primarily responsible for the increased HUVEC migration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, elevated ET-1 expression was documented by immunohistochemistry in primary and metastatic ovarian carcinoma. ETAR expression, as detected by in situ hybridization and immunohistochemistry, was localized in carcinoma cells as well as in blood vessels, whereas ETBR was confined to vascular endothelial cells. Autoradiographic binding studies confirmed that ovarian carcinoma cells functionally expressed ETAR but not the ETBR, and that blood vessels co-expressed ETAR and ETBR. The vascular smooth muscle cells predominantly expressed ETAR, whereas endothelial cells expressed ETBR. The localization of the ET receptors in blood vessels adjacent to transformed epithelium suggests that ET-1 might exert angiogenic effects providing an ideal microenvironment for tumor growth. VEGF was expressed in the cytoplasm of tumor cells where it is synthesized and stored, whereas VEGF receptors were expressed by endothelial cells. Because VEGF plays a pivotal role in ascites formation both as a potent inducer of vascular permeability and as an angiogenetic factor,34 we hypothesized that advanced ovarian carcinoma may be associated with elevated ascitic concentrations of the angiogenic factors VEGF and ET-1. Our findings indeed demonstrate that in the majority of ovarian carcinomas examined, ascitic concentrations of both factors are concordantly elevated. The unprecedented demonstration of high ET-1 levels in a sizeable percentage (85%) of cases implicates this molecule as relevant in the increased neovascularization and vascular permeability of ovarian carcinoma.
Although microvessel count and VEGF expression have been shown to correlate in several tumors,35-37 to date no such correlation has been found in ovarian carcinoma.33,38 Our results found a strong correlation of VEGF and vessel density in a significant number of cases. In view of the above results, we extended the same analysis to ET-1 demonstrating a significant correlation between ET-1 expression and microvessel density. ET-1 is a potent mitogen for vascular smooth muscle cells as well as for endothelial cells. Previous observations demonstrated that endothelial cells express ETBR and respond to ET-1, which may exert an angiogenic activity.5-8 Thus, we have recently demonstrated that ET-1 induces a pro-angiogenic phenotype in human endothelial cells.39 This phenotype includes both early (ie, increase in cell proliferation, migration, invasion, and MMP-2 production) and late angiogenic events (differentiation into vascular cords). Moreover ET-1 in association with VEGF has a clear angiogenic activity in the Matrigel in vivo assay, comparable to that promoted by bFGF.
Recent studies demonstrated that in vascular smooth muscle cells ET-1, predominantly through ETAR, enhances VEGF secretion and stimulates the VEGF-induced endothelial cell proliferation and invasion.17
Because the regulation of VEGF production is a critical event in tumor angiogenesis, one can envision that in pathological conditions such as cancer, ET-1 may be up-regulated by various stimuli including hypoxia, growth factors, and inflammatory cytokine,40 and that ET-1, in turn, might exert angiogenic effect increasing VEGF production and thereby modifying VEGF-related angiogenic responses. In these studies, we demonstrated that ET-1 stimulates VEGF production in ovarian carcinoma cells and is equipotent to hypoxia, a recognized potent and important stimulus of VEGF production. We also showed that the actions of ET-1 were mediated through the ETAR, because the specific antagonist BQ 123 reversed the stimulation of VEGF production.
Furthermore we showed that elevated levels of ET-1 are released by ovarian carcinoma in ascitic fluids and that this growth factor is primarily responsible for endothelial cell migration, acting through ETBR, as demonstrated by the inhibition induced by ETBR antagonists. The significant inhibition obtained by co-incubating HUVECs with the ETBR antagonist and with VEGF Ab, strongly indicates that ET-1, together with VEGF, play a complementary and coordinated role during neovascularization in ovarian carcinoma. Thus, our hypothesis is that during neovascularization, endothelial cells could be initially stimulated by the ET-1/ETBR interaction to migrate, proliferate, and invade surrounding tissue. Thereafter, vessel maturation could be mediated by ET-1/ETAR binding in part through the stimulation of VEGF in the existing tumor or vasculature, resulting in angiogenesis. This working hypothesis links ET-1 to the early as well as to the late stages of angiogenesis involving both ET-1 receptors.
In conclusion, the present study demonstrates that ET-1 and its receptors are expressed by tumor cells as well as by tumor vessels in ovarian carcinoma. The tumor-promoting activity of ET-1 may occur through an autocrine pathway that stimulates tumor cell proliferation and through a paracrine pathway involving direct angiogenic effects on endothelial cells and in part through the stimulation of VEGF in ovarian carcinoma cells. Among the array of factors that concur to ovarian tumor vascularization, we identified ET-1 and its receptors as angiogenic regulators that could represent novel targets for anti-angiogenic therapies. New therapeutic strategies using specific antagonists provide an additional approach to the treatment of ovarian carcinoma in which ET-1 antagonists would play their anti-tumor role as both anti-angiogenic and anti-mitogenic agents.41,42
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Acknowledgements |
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Footnotes |
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This work was supported by grants from the Associazione Italiana Ricerca sul Cancro, the Ministero della Sanità, and fellowships from the Fondazione Italiana Ricerca sul Cancro (to D. S. and L. R.).
Accepted for publication July 27, 2000.
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L. Rosano, F. Spinella, V. Di Castro, M. R. Nicotra, S. Dedhar, A. G. de Herreros, P. G. Natali, and A. Bagnato Endothelin-1 Promotes Epithelial-to-Mesenchymal Transition in Human Ovarian Cancer Cells Cancer Res., December 15, 2005; 65(24): 11649 - 11657. [Abstract] [Full Text] [PDF]
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A. Bagnato, F. Spinella, and L. Rosano Emerging role of the endothelin axis in ovarian tumor progression Endocr. Relat. Cancer, December 1, 2005; 12(4): 761 - 772. [Abstract] [Full Text] [PDF]
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 V. L. M. Herrera, L. R. B. Ponce, P. D. Bagamasbad, B. D. VanPelt, T. Didishvili, and N. Ruiz-Opazo Embryonic lethality in Dear gene-deficient mice: new player in angiogenesis Physiol Genomics, November 17, 2005; 23(3): 257 - 268. [Abstract] [Full Text] [PDF]
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 H. Kajiyama, K. Shibata, M. Terauchi, T. Morita, K. Ino, S. Mizutani, and F. Kikkawa Neutral Endopeptidase 24.11/CD10 Suppresses Progressive Potential in Ovarian Carcinoma In vitro and In vivo Clin. Cancer Res., March 1, 2005; 11(5): 1798 - 1808. [Abstract] [Full Text] [PDF]
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R. Lahav, M.-L. Suva, D. Rimoldi, P. H. Patterson, and I. Stamenkovic Endothelin Receptor B Inhibition Triggers Apoptosis and Enhances Angiogenesis in Melanomas Cancer Res., December 15, 2004; 64(24): 8945 - 8953. [Abstract] [Full Text] [PDF]
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 F. Spinella, L. Rosano, V. Di Castro, P. G. Natali, and A. Bagnato Endothelin-1-induced Prostaglandin E2-EP2, EP4 Signaling Regulates Vascular Endothelial Growth Factor Production and Ovarian Carcinoma Cell Invasion J. Biol. Chem., November 5, 2004; 279(45): 46700 - 46705. [Abstract] [Full Text] [PDF]
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F. Spinella, L. Rosano, V. Di Castro, M. R. Nicotra, P. G. Natali, and A. Bagnato Inhibition of Cyclooxygenase-1 and -2 Expression by Targeting the Endothelin A Receptor in Human Ovarian Carcinoma Cells Clin. Cancer Res., July 15, 2004; 10(14): 4670 - 4679. [Abstract] [Full Text] [PDF]
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P. Wulfing, C. Kersting, J. Tio, R.-J. Fischer, C. Wulfing, C. Poremba, R. Diallo, W. Bocker, and L. Kiesel Endothelin-1-, Endothelin-A-, and Endothelin-B-Receptor Expression Is Correlated with Vascular Endothelial Growth Factor Expression and Angiogenesis in Breast Cancer Clin. Cancer Res., April 1, 2004; 10(7): 2393 - 2400. [Abstract] [Full Text] [PDF]
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F. Spinella, L. Rosano, V. Di Castro, M. R. Nicotra, P. G. Natali, and A. Bagnato Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells J. Biol. Chem., October 17, 2003; 278(42): 41294 - 41301. [Abstract] [Full Text] [PDF]
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J. J. Yin, K. S. Mohammad, S. M. Kakonen, S. Harris, J. R. Wu-Wong, J. L. Wessale, R. J. Padley, I. R. Garrett, J. M. Chirgwin, and T. A. Guise From the Cover: A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases PNAS, September 16, 2003; 100(19): 10954 - 10959. [Abstract] [Full Text] [PDF]
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P. Wulfing, R. Diallo, C. Kersting, C. Wulfing, C. Poremba, A. Rody, R. R. Greb, W. Bocker, and L. Kiesel Expression of Endothelin-1, Endothelin-A, and Endothelin-B Receptor in Human Breast Cancer and Correlation with Long-Term Follow-Up Clin. Cancer Res., September 15, 2003; 9(11): 4125 - 4131. [Abstract] [Full Text] [PDF]
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 L. Rosano, F. Spinella, V. Di Castro, M. R. Nicotra, A. Albini, P. G. Natali, and A. Bagnato Endothelin Receptor Blockade Inhibits Molecular Effectors of Kaposi's Sarcoma Cell Invasion and Tumor Growth in Vivo Am. J. Pathol., August 1, 2003; 163(2): 753 - 762. [Abstract] [Full Text] [PDF]
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B. A. Zonnenberg, G. Groenewegen, T. J. Janus, T. W. Leahy, R. A. Humerickhouse, J. D. Isaacson, R. A. Carr, and E. Voest Phase I Dose-Escalation Study of the Safety and Pharmacokinetics of Atrasentan: An Endothelin Receptor Antagonist for Refractory Prostate Cancer Clin. Cancer Res., August 1, 2003; 9(8): 2965 - 2972. [Abstract] [Full Text] [PDF]
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L. Rosano, F. Spinella, D. Salani, V. Di Castro, A. Venuti, M. R. Nicotra, P. G. Natali, and A. Bagnato Therapeutic Targeting of the Endothelin A Receptor in Human Ovarian Carcinoma Cancer Res., May 15, 2003; 63(10): 2447 - 2453. [Abstract] [Full Text] [PDF]
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S. Pikkarainen, H. Tokola, R. Kerkela, T. Majalahti-Palviainen, O. Vuolteenaho, and H. Ruskoaho Endothelin-1-specific Activation of B-type Natriuretic Peptide Gene via p38 Mitogen-activated Protein Kinase and Nuclear ETS Factors J. Biol. Chem., January 31, 2003; 278(6): 3969 - 3975. [Abstract] [Full Text] [PDF]
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A. Bagnato, A. Cirilli, D. Salani, P. Simeone, A. Muller, M. R. Nicotra, P. G. Natali, and A. Venuti Growth Inhibition of Cervix Carcinoma Cells in Vivo by Endothelin A Receptor Blockade Cancer Res., November 15, 2002; 62(22): 6381 - 6384. [Abstract] [Full Text] [PDF]
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J. Favier, P.-F. Plouin, P. Corvol, and J.-M. Gasc Angiogenesis and Vascular Architecture in Pheochromocytomas : Distinctive Traits in Malignant Tumors Am. J. Pathol., October 1, 2002; 161(4): 1235 - 1246. [Abstract] [Full Text] [PDF]
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F. Spinella, L. Rosano, V. Di Castro, P. G. Natali, and A. Bagnato Endothelin-1 Induces Vascular Endothelial Growth Factor by Increasing Hypoxia-inducible Factor-1alpha in Ovarian Carcinoma Cells J. Biol. Chem., July 26, 2002; 277(31): 27850 - 27855. [Abstract] [Full Text] [PDF]
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 J. Herrmann, P. J. Best, E. L. Ritman, D. R. Holmes Jr, L. O. Lerman, and A. Lerman Chronic endothelin receptor antagonism prevents coronary vasa vasorum neovascularization in experimental hypercholesterolemia J. Am. Coll. Cardiol., May 1, 2002; 39(9): 1555 - 1561. [Abstract] [Full Text] [PDF]
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 D. Del Bufalo, V. Di Castro, A. Biroccio, M. Varmi, D. Salani, L. Rosano, D. Trisciuoglio, F. Spinella, and A. Bagnato Endothelin-1 Protects Ovarian Carcinoma Cells against Paclitaxel-Induced Apoptosis: Requirement for Akt Activation Mol. Pharmacol., March 1, 2002; 61(3): 524 - 532. [Abstract] [Full Text] [PDF]
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L. Rosano, M. Varmi, D. Salani, V. Di Castro, F. Spinella, P. Giorgio Natali, and A. Bagnato Endothelin-1 Induces Tumor Proteinase Activation and Invasiveness of Ovarian Carcinoma Cells Cancer Res., November 1, 2001; 61(22): 8340 - 8346. [Abstract] [Full Text] [PDF]
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A. Bagnato, L. Rosano, V. Di Castro, A. Albini, D. Salani, M. Varmi, M. R. Nicotra, and P. G. Natali Endothelin Receptor Blockade Inhibits Proliferation of Kaposi's Sarcoma Cells Am. J. Pathol., March 1, 2001; 158(3): 841 - 847. [Abstract] [Full Text] [PDF]
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D. Salani, G. Taraboletti, L. Rosano, V. Di Castro, P. Borsotti, R. Giavazzi, and A. Bagnato Endothelin-1 Induces an Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo Am. J. Pathol., November 1, 2000; 157(5): 1703 - 1711. [Abstract] [Full Text] [PDF]
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