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Experimental Therapeutics |
Department of Pharmacology [Y. Y., S. R.], Obstetrics and Gynecology, and Comprehensive Cancer Center [S. R.], University of Minnesota, Minneapolis, Minnesota 55455; Renal Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 [M. D., V. P. S.]; and Tumor Angiogenesis Laboratory, Department of Internal Medicine, University Hospital Maastricht, Maastricht, The Netherlands [A. W. G.]
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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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Etiology of ovarian cancer is not completely understood. Epidemiological studies suggest that ovarian cancer risk is associated with ovulatory cycle (6) and artificial induction of ovulation in infertile patient (7) . A vast majority of ovarian cancers arise from the single layer of epithelium surrounding the ovaries (8 , 9) . Ovarian cancer growth is angiogenesis-dependent (10 , 11) , and secretion of proangiogenic growth factors such as VEGF is of prognostic value (12 , 13) . In addition to inducing tumor angiogenesis, VEGF is also a contributing factor in the formation of malignant ascites in ovarian cancer (14 , 15) . Accumulation of ascites is a characteristic of ovarian cancer. Ascites fluid provides an ideal microenvironment for tumor growth and micrometastasis of the peritoneal wall. After surgical debulking of the primary tumor and ascites drainage, increased growth of metastatic nodules has been observed in ovarian cancer patients (16, 17, 18) . In fact, O’Reilly et al. (19 , 20) discovered angiostatin based on a similar phenomenon in mice bearing a transplantable tumor.
Angiostatin is a proteolytic fragment of plasminogen comprising the first 4-kringle domains. It was first identified as a natural inhibitor of angiogenesis in the serum and urine of tumor-bearing mice (21) . Since then, angiostatin has been used to inhibit growth of many experimental tumors in animals using either human (22, 23, 24, 25) or mouse tumor cell lines (26 , 27) . In parallel to the discovery of angiostatin, O’Reilly et al. (28) also identified endostatin, a proteolytic fragment of collagen type XVIII. Endostatin is a potent inhibitor of angiogenesis and was isolated from a mouse hemangioendothelioma cell line. Recombinant endostatin made in bacteria has been shown to inhibit growth of tumors (29 , 30) and to lead to the regression of transplanted tumors (28) . In addition to angiostatin and endostatin, a number of other endogenous proteins, including cytokines such as interleukin 4 (31) , have been identified to have antiangiogenic activity. Retinal pigment-epithelium derived factor (32) and cleaved form of antithrombin (33) are recently described as potent inhibitors of angiogenesis and tumor growth. Thus far no one has investigated whether any of the angiostatic proteins can be used to treat gynecological malignancies. Using athymic mice transplanted with a human ovarian cancer cell line as an experimental model system, we investigated the relative potency of recombinant mouse angiostatin and endostatin. In this model system, angiostatin was more potent in inhibiting tumor growth than endostatin was. Furthermore, the combined treatment with both angiostatin and endostatin resulted in a synergistic antiangiogenic effect when compared with treatment with either angiostatin or endostatin alone.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of Recombinant Angiostatin and Endostatin.
Mouse angiostatin (kringle 1–4) and endostatin have been cloned and
expressed in Pichia pastoris by Dhanabal et al.
(30)
. Pichia clones were cultured in baffled
shaker flasks and induced by methanol as previously described
(35)
. For large-scale expression, fermentation was used.
Culture supernatants from shaker flasks were precipitated with ammonium
sulfate (50% saturation) and dialyzed against 10
mM Tris-HCl (pH 7.6), 0.5
mM PMSF. Cell-free fermentation product was first
concentrated by ultrafiltration and then dialyzed against 10
mM Tris-HCl buffer (pH 7.6), 0.5
mM PMSF. Further purification was carried out by
heparin affinity column. The heparin column was equilibrated with 10
mM Tris-HCl buffer (pH 7.6), 0.5
mM PMSF. Samples were applied to the column at a
flow rate of 1.0 ml/min on a fast protein liquid chromatography
(Amersham Pharmacia Biotech, Piscataway, NJ). After thorough washing to
remove unbound proteins, the column was eluted with a continuous
gradient of 0–1 M NaCl in 10 mM Tris-HCl (pH
7.6), 0.5 mM PMSF. Endostatin was eluted at about
0.5 M NaCl. Purified endostatin was analyzed on SDS-PAGE (12%
acrylamide gel) under nonreducing conditions by mass spectrometry and
N-terminal sequencing.
A mouse angiostatin expressing Pichia clone was cultured in
baffled shaker flasks. Culture supernatants were precipitated with
ammonium sulfate (50% saturation) and dialyzed against 50
mM phosphate buffer (pH 7.5), 0.5
mM PMSF. Samples were then applied onto a lysine
ceramic column equilibrated with 50 mM phosphate
buffer (pH 7.5), 0.5 mM PMSF. Matrix-bound
proteins were then eluted from the column with a continuous gradient of
0–0.2 mM 
-aminocaproic acid in 50
mM phosphate buffer. Purity of angiostatin was
analyzed by SDS-PAGE (12%) under nonreducing conditions.
Purified materials were dialyzed against PBS [137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4 (pH 7.3)] and stored in aliquots at -70°C.
Endothelial Cell Proliferation Assay.
Essentially, the method described by O’Reilly et al.
(28)
was used. Confluent BCE and HUVE cells were
trypsinized and resuspended in M199 (Life Technologies, Inc.) medium
with 5% FBS. Cells were then seeded into gelatinized, 96-well culture
plates at a density of 5000 cells/well. After 24 h, different
concentrations of angiostatin and/or endostatin were added. Twenty
minutes later, cultures were treated with 5 ng/ml of bFGF (Life
Technologies, Inc.) in the presence of 1 µg/ml heparin. The viability
of the control and the treated cells was determined by the MTT (Sigma
Chemical Co., St. Louis, MO) colorimetric assay (36)
after
72 h of incubation. MTT assay actually determines the metabolic
activity of mitochondria and correlates well with the number of viable
cells (36)
. This assay has been previously used to
evaluate endothelial cell proliferation (37)
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CAM Assay.
The ability of mouse endostatin and angiostatin to inhibit angiogenesis
in vivo was first tested in a CAM assay. Three-day-old
fertilized White Leghorn eggs were incubated at 37°C for 4 days with
rotating everyday. A window (1 x 2 cm) was gently cut
on day 7. On day 9, sterilized silicon rings (1 cm diameter, 1 mm
thickness) were placed on the CAM. Ten micrograms of endostatin or
angiostatin were added inside the rings every day for 3 days. Control
CAMs were treated similarly with sterile saline. At the end of the
experiment, CAMs were fixed with 10% neutral buffered formalin and
photographed using a digital camera.
Tumor Growth Inhibition Studies.
Female athymic nude mice (6–8 weeks old) were purchased from the
National Cancer Institute and allowed to acclimatize to local
conditions for 1 week. Logarithmically growing human ovarian carcinoma
cells were harvested by trypsinization and were suspended in fresh
medium at a density of 2 x 107
cells/ml. One hundred microliters of the single-cell suspension was
then injected s.c. into the flanks of mice. When the tumors became
visible (7 days after inoculation), mice were randomized into four
groups. One group was injected with mouse endostatin s.c. at a dose of
20 mg/kg/day for 30 days. A second group of mice was treated with
angiostatin at the same dose. A third group of mice was treated with a
combination of mouse endostatin (20 mg/kg/day) and angiostatin (20
mg/kg/day) to evaluate the effect of combination therapy. A control
group of mice (fourth) was treated with sterile PBS under similar
conditions. All injections were given s.c. at the neck, which is about
3 cm away from the growing tumor mass. Tumor growth was monitored by
periodic caliper measurements. Tumor volume was calculated by the
following formula: tumor volume (mm3) = (a x b2)/2, where
a = length in mm and b = width in mm.
Statistical significance between control and treated groups was determined by Student’s t test. A minimum of five animals was used in each group and the experiments were repeated at least twice. Data from independent experiments were pooled for statistical analysis.
Determination of Vessel Density and Apoptosis.
To determine the effect of antiangiogenic treatments on vessel density
and apoptosis, residual tumors were surgically resected and snap
frozen. Cryostat sections (4 µm) of tumors were then treated with PBS
containing 0.1% BSA and 5% human serum to block nonspecific binding
(background). Sections were then incubated with 1:50 dilution of an
anti-CD31 (mouse) monoclonal antibody conjugated to phycoerythrin
(Sigma). After 1 h incubation at room temperature, sections were
washed thoroughly with PBS containing 0.1% BSA and 5% human serum and
were then examined under an Olympus (New Hyde Park, NY) BX-60
fluorescence microscope at x10 magnification. Images were captured by
the Metamorph program for analysis. Detection of apoptosis was carried
out by using an In Situ Cell Death Detection Kit (Boehringer
Mannheim, Indianapolis, IN) following the manufacture’s protocol.
Parts of the tumor samples were also fixed in 10% neutral buffered
formalin and processed for histochemistry (H&E staining).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mouse angiostatin was eluted as a
single homogenous peak and contained pure protein with <5%
contamination in SDS-PAGE (Fig. 1C)
. The apparent molecular
weight of the purified angiostatin was about
Mr 42,000 as per the relative
mobility on SDS-PAGE under nonreducing condition. Angiostatin (residues
Val98-Gly458 of
plasminogen) encompassing kringle 1–4 (19
, 24)
has a
total of 361 amino acid residues. Peptide composition analysis
(MacVector, 4.1.5) predicts a theoretical molecular weight of
Mr 41,100, which is very close to the
observed value. Endostatin was eluted from the affinity matrix at 500
mM NaCl as a single peak containing a
Mr 20,000 protein. The preparation of
endostatin showed small but detectable levels of dimers in SDS-PAGE by
silver staining and Western blotting (data not shown). Typically,
angiostatin was expressed in higher quantity than endostatin in shaker
flasks. Yields for angiostatin varied between 15 and 20 mg/L. In
contrast, endostatin was expressed at a lower level (5–8 mg/L). A
batch fermentation run provided about 50–60 mg/L of mouse endostatin
from a working volume of 6 L. Purified endostatin was analyzed by mass
spectrometry. Endostatin showed a molecular weight of
Mr 19,787. In addition, two smaller
peaks corresponding to a molecular weight of
Mr 39,500 and
Mr 59,300 were also observed (Fig. 1D)
. The higher molecular weight peaks correspond to dimeric
and trimeric forms of endostatin. Every batch of endostatin showed a
similar profile. However, the proportion of individual component cannot
be accurately determined from mass spectrometry (inherent limitation).
On the basis of SDS-PAGE, the relative amount of higher molecular
weight endostatin was <5%. Each batch of endostatin was analyzed
for biological activity. For in vivo studies, a
single batch was used for each experiment.
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. Combination of angiostatin and endostatin inhibited
proliferation by 50% at 0.57 µg/ml (IC50).
When compared with IC50 of individual treatment
with angiostatin and endostatin (10 and 8.6 µg/ml, respectively), the
combined treatment showed >16-fold improvement in antiproliferative
activity. We also tested the direct effect of angiostatin and
endostatin on tumor cells such as MA148; SIHA, a human cervical cancer
cell line; and HUFF, a human foreskin fibroblast cell line. Neither
endostatin nor angiostatin inhibited the proliferation of these cell
lines (data not shown). To determine whether the increased
antiproliferative effect was synergistic or additive, isobolographic
analysis was carried out. Different combinations of concentrations of
angiostatin and endostatin were added to BCE cultures either alone or
together. From the dose-response curves, IC40 (a
concentration at which BCE proliferation was inhibited to 40% of
control) values were calculated. These values were then used to
generate isobologram. Data in Fig. 2B
show the effect of
combination treatment. When compared with the theoretical line
(diagonal) representing additive effect, all of the values from
combination treatment are found to be distributed below (to the left
of) the theoretical line. If angiostatin and endostatin acted
additively, the values would have fallen directly on the diagonal line.
On the other hand, a competitive effect between angiostatin and
endostatin would have distributed the values above (to the right) the
theoretical line. The results clearly demonstrate that combination of
angiostatin and endostatin synergistically inhibits proliferation of
endothelial cells.
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show the relative effect of angiostatin and endostatin therapy.
Angiostatin was found to inhibit ovarian cancer growth better when
compared with endostatin in parallel experiments. For example, after 2
weeks of treatment with angiostatin, a mean tumor volume of 200
mm3 was observed. Under similar conditions,
endostatin-treated animals showed a mean tumor volume of 362
mm3, whereas mean tumor volume of control mice
was 589 mm3. Tumor growth was significantly
reduced during the entire treatment period with angiostatin.
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The same frozen
sections were also analyzed for changes in the viability of tumor cells
using a TUNEL assay (Fig. 5. B, E, H).
Serial sections of each tumor
were also stained by H&E to assess necrotic changes (Fig. 5, C, F, and I).
When compared with
control tumor sections, endostatin- and angiostatin-treated tumors
showed pronounced increase in apoptosis. Increased incidence of
apoptosis coincided with increase in calcification and necrosis of
tumor tissue. However, histopathological analysis of normal tissues
from the same animals did not show any increase in apoptosis or
necrosis (data not shown). Collectively, these results show that
antiangiogenic therapy results in reduced tumor angiogenesis leading to
apoptotic death of ovarian cancer cells.
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shows mean tumor volume on day 42. Endostatin and angiostatin alone
showed inhibition of tumor growth by 5% and 57%, respectively, in
this experiment. However, a combination of angiostatin and endostatin
showed better antitumor activity with about 81% inhibition of tumor
growth. Table 1
summarizes relative tumor volume of control and treated groups on three
different time points. Combination therapy showed more than additive
effect on tumor growth inhibition. On day 36, there was 1.34-fold
improvement in antitumor activity in the combination group when
compared with the expected additive effect. At this time point,
endostatin alone inhibited tumor growth by 20% (fractional tumor
volume, 0.797 mm3) when compared with the
control group. With time, there was a progressive improvement in
antitumor activity. On day 42, angiostatin and endostatin combination
group showed a 2-fold higher inhibition of tumor growth over additive
effect (expected fractional tumor volume). In the present study, both
angiostatic proteins were given together in a fixed schedule and dose.
Therefore, the observed synergism can be further improved by modulating
dosage and frequency of administration based on pharmacokinetics,
distribution, and bioavailability.
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DISCUSSION |
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Potency of angiostatic molecules has been found to vary a lot depending on the cell type used. Ji et al. (42) reported that BCE cells are more sensitive than HUVE cells to kringle 5 in a migration assay. Dhanabal et. al. (30) reported that CPAE cells are more sensitive than other endothelial cell lines. Therefore, we compared the effect of angiostatin and endostatin on two different endothelial cells, BCE and HUVE cells. BCE cells were much more sensitive to both angiostatin and endostatin. We also tested CPAE and human microvascular endothelial cells. CPAE cells were as sensitive as BCE cells, and mouse microvascular endothelial cells were similar to HUVE cells (data not shown).
Angiostatin and endostatin effectively inhibited developmental angiogenesis in vivo. We used a modified CAM assay in which test solutions are applied directly onto a localized area of the CAM. In this method, the samples stay inside the rings, and new blood vessel formation inside the rings then can be compared with normal vasculature surrounding the ring. Another advantage of this method is that the blood vessels can be easily fixed by buffered formalin so that it is possible to cut CAMs out and observe them in detail. Direct application of angiostatin and endostatin expressed in yeast clearly inhibited actively growing blood vessels inside the ring. Vasculature outside the ring was not affected by this treatment.
Although angiostatin and endostatin have been tested in many tumor models, the relative potency has not been established in parallel experiments. Angiostatin is found to be effective against Lewis lung carcinoma at doses ranging from 1 mg/kg (24) and 50 mg/kg (44) . Endostatin, on the other hand, is used in the same model in an insoluble form at a dose of 10 and 20 mg/kg (29) . It is difficult to compare the relative potency because the rate of release of endostatin from the insoluble form is not determined. In the present study, we compared the relative antitumor effect of angiostatin and endostatin in soluble form against human ovarian carcinomas established in athymic mice. Both reagents were given at a similar dose and schedule. These studies showed that angiostatin was more potent in inhibiting ovarian cancer growth compared with endostatin. It is possible that the absence of zinc-binding residues could have contributed to the low antitumor activity of endostatin. However, endostatin was equally effective as angiostatin in inhibiting endothelial cell proliferation in vitro and developmental angiogenesis in vivo. Other reasons for the differences in antitumor activity could be due to tumor-dependent variations in the microenvironment affecting endothelial sensitivity. For example, it is possible that different types of tumors can secrete distinct sets of growth factors that can modulate the sensitivity of tumor vasculature to angiostatin and endostatin differently. Differences in pharmacokinetics and tissue distribution can also differentially alter bioavailability of angiostatin and endostatin. Angiostatin is expected to have a longer half-life than endostatin. Endostatin, with a molecular weight of Mr 20,000, will be cleared from the circulation rapidly by renal filtration. Apart from the circulatory half-life, endostatin is observed to bind host vasculature, which can restrict its availability at tumor target site (45) . Interaction with normal blood vessels can affect tissue distribution and will reduce bioavailability of endostatin. It will be possible to improve the efficacy of angiostatic molecules by (a) pharmacological approaches and (b) structural changes to increase half-life/bioavailability.
Angiostatin and endostatin are believed to act on endothelial cells by
different mechanisms. Angiostatin has been recently shown to bind
/ß subunits of a membrane-bound ATP synthase (46)
.
However, endostatin seems to affect levels of antiapoptotic proteins
such as BCL-2 inside the cell (47)
. These studies suggest
that upstream apoptotic signaling cascades of caspases are activated by
endostatin treatment. Due to the nonoverlapping nature of the
inhibitory pathways, treatment of endothelial cells with a combination
of angiostatin and endostatin resulted in synergistic inhibition.
Synergy between the two angiostatic molecules was confirmed by
isobolographic analysis. Improved antiangiogenic activity was also
reflected in vivo when tumor-bearing animals were treated
with a combination of equal doses of angiostatin and endostatin.
Compared with expected additive effects, a 2-fold increase in antitumor
activity was observed when mice were treated with equal doses of
angiostatin and endostatin. The observed synergy between the two
angiostatic proteins can be further improved by optimizing dosage and
schedule of administration. These questions will be addressed in future
studies using genetically redesigned second-generation angiostatic
molecules. Whereas our current studies suggest a potential use of
combination therapy using angiostatic proteins, one could also achieve
better antitumor response by combining antiangiogenic therapy with
other antitumor therapies (e.g., chemo-radiation). For
example, radiation therapy when combined with angiostatin showed
potentiation of antitumor activity (25
, 48)
. In another
related strategy, antibodies to VEGF were used in combination with
radiation therapy to achieve improved antitumor activity
(49)
. Radiation induces elevated expression of VEGF as a
survival factor from tumor cells. Therefore, neutralizing VEGF under
these conditions resulted in better inhibition of tumor growth. In
summary, our studies show for the first time that antiangiogenic
therapy can be used to inhibit the growth of ovarian cancer and that
angiostatin can synergize with endostatin in inhibiting tumor
growth.
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FOOTNOTES |
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1 Supported in part by grants from the United
States Army Medical Research and Materiel Command, Shirley Ann
Sparboe Endowment, Women’s Health Fund, and Gynecological Oncology
Group of America (to S. R.) and by a grant from the Dutch Cancer
Society (to A. G.). 
2 To whom requests for reprints should be
addressed, at 6-120 Jackson Hall, 321 Church Street, S.E., Department
of Pharmacology, University of Minnesota, Minneapolis, MN 55455. Phone:
(612) 624-1461; Fax: (612) 625-8408; E-mail: sunda001{at}maroon.tc.umn.edu
3 The abbreviations used are: FGF, fibroblast
growth factor; bFGF, basic FGF; VEGF, vascular endothelial growth
factor; BCE, bovine adrenal gland capillary endothelial cells; HUVE,
human umbilical vein endothelial; PMSF, phenylmethylsulfonyl fluoride;
MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2,4-tetrazolium bromide;
CAM, chick chorioallantoic membrane; CPAE, bovine pulmonary artery
endothelial; FBS, fetal bovine serum.
Received 9/29/99. Accepted 2/18/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, endostatin; 
, angiostatin;
and 
, combined addition of angiostatin and endostatin. Viability of
cells was determined by MTT assay. One hundred percent inhibition is
equal to complete reduction of bFGF-induced endothelial cell
proliferation to control cells cultured in the absence of bFGF.
B, Combination effect of angiostatin and endostatin on
BCE cells was plotted in an isobologram. The dotted line shows the
theoretical line representing additive effect. Each value is derived
from a dose-response curve and represents a dose required to elicit
40% inhibition (IC40) of BCE proliferation. Statistical
significance between endostatin or angiostatin alone and their
combination was determined using Student’s t test. *,
P < 0.05; **,
P < 0.01.
, Control, PBS; •, mouse
endostatin; 
, mouse angiostatin. Mean tumor volume was determined by
caliper measurements. Data from two independent experiments were pooled
and plotted. Statistical significance was determined using Student’s
t test. *, P < 0.05;
**, P < 0.01. The error bars
indicate the SE.
