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Tumor Biology |
in the Inhibition of Cancer Growth and Angiogenesis
Department of Biochemistry, Roswell Park Cancer Institute [S. H. A.], State University of New York at Buffalo [S. H. A., A. L. O., D. B., M. B. P., M. J. S., P. D.], Buffalo, New York; Departments of Medicine and Endocrinology [A. L. O.] and Pathology [D. B., M. B. P., M. J. S.], Veterans Administration Medical Center, Buffalo, New York; General Pharmacology, DuPont Pharmaceuticals Company, Wilmington, Delaware 19880-0400 [S. Mou., S. Moh.]; and Endocrine Division, Kaleida Health, Buffalo, New York [S. H. A., P. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER-) is believed to be
favorable in the prognosis and treatment of certain female cancers.
ER- expression in the ER-negative breast cancer cell lines inhibits
their proliferation and invasive, metastatic potential in
vitro. We stably overexpressed the ER- in the human
endometrial cancer cell line Ishikawa and showed that, unlike
estradiol, high levels of ER- significantly inhibit the growth of
tumors xenografted from the Ishikawa cells. Subsequent to ER-
overexpression, in vivo down-regulation of vascular
endothelial growth factor was observed in tumor xenografts. In
addition, these tumors showed an inhibition of vascularization and of
the angiogenic agent, integrin vß3.
Involvement of a switch in the angiogenic pathways during tumorigenesis
has been a recent focus of interest. Our results indicate that a high
level of ER- may be beneficial in the control of female cancers
because of its inhibitory effect on such angiogenic pathways. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
is believed
to be favorable in the control of ovarian (1)
and
endometrial (2)
cancers. In the past, we (10)
and
others have reported the development of cell lines that were stably
transfected with ER- and were used as models to study the mechanisms
involved in the estrogen/ER-mediated control of cellular proliferation
(3)
. In most cases, these cell lines have shown inhibition
in their growth (3)
and metastatic/invasive potential
(4)
, possibly through an effect of ER- on certain
growth-regulating genes, such as c-myc (5)
.
In the ER--overexpressing clone of Ishikawa cell line (ISH-ER)
discussed here, we previously reported an ER--mediated inhibition of
in vitro growth.3
Accompanying this
growth inhibition were a stimulation of NOS activity and VEGF
levels,3
both of
which are critical in determining vascular physiology and mitotic
behavior. Involvement of a switch in the angiogenic pathways during
tumorigenesis has been a recent focus of interest (6)
. The
process of angiogenesis determines the available supply of blood to a
growing cancer and therefore plays an important role in its
progression. Vasoactive factors such as NOS and VEGF may therefore be
involved in the mechanism by which estrogen influences cancer growth.
In this study, we investigate this aspect in vivo. We report
an ER--mediated in vivo growth inhibition of tumors
xenografted from parent and ER-transfected Ishikawa cells. In addition,
we demonstrate an inhibition of VEGF and other angiogenic parameters
after the ER- overexpression in the Ishikawa cells. The present work
indicates that a high level of ER- may be beneficial in controlling
female cancers because of its inhibitory effect on angiogenic pathways.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inoculation of Athymic Mice.
Cells were routinely grown in Medium 199 (Sigma, St. Louis, MO)
supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). On the
day of inoculation, cells were trypsinized and resuspended in Matrigel
(Fisher Scientific Co., Pittsburgh, PA) at 1 million cells per 100
µl. Animals received inoculations containing 100 µl of this
suspension in each flank. Tumors were allowed to grow, and their
dimensions were measured weekly. Tumor weight was determined as follows
(7
, 8)
:
 |
Treatment.
One week after the ovariectomy, animals were inoculated with the cell
suspension and implanted with 60-day time-release pellets of E2, TAM,
OHT, or a placebo (Innovative Research of America, Sarasota,
FL). OHT was obtained from Sigma and was custom-prepared into 60-day
time-release pellets at Innovative Research of America. A 12-gauge
trochar was used to insert the pellet s.c. at the back of each
animal’s neck. Sixty days later, if an animal was still under
observation, it was implanted with a fresh pellet.
The animal protocols described above were approved by the Animal Research Committee at the State University of New York at Buffalo. All experiments on the animals were performed according to the NIH-endorsed guidelines.
Immunostaining for VEGF and Integrin
vß3.
At the end of each experiment, the animals growing tumors were
euthanized, and their tumors were excised and preserved in formalin
until use. The tumors were embedded in paraffin, sectioned, and stained
for either integrin
vß3 (using the
anti-vß3 antibody
LM609; R&D Systems, Inc., Minneapolis, MN) or for VEGF (using an
anti-VEGF antibody; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
The stained slides were then scored for the intensity of stain against
the background of their respective negative controls stained in a
similar fashion, except that the antibody was replaced with pre-immune
serum. The intensity of stain thus measured was expressed as the level
of the VEGF or the integrin
vß3 in tumors.
Staining for Vascularization.
The slides were prepared as for the VEGF and
vß3 staining. This
staining was performed using the Verhoeff elastic stain method
(9)
. Briefly, the slides were stained with a
hematoxylin-ferric chloride iodine solution, which makes the vascular
elastic fibers and the cell nuclei appear black. The slides were then
scored for the magnitude of black-stained fibers, which was expressed
as the level of vascularization in tumors.
In Vitro Assays.
The proliferation assays were performed as described by Ali et
al. (10)
. Briefly, equal numbers of parent,
ER-transfected, and control cells were seeded in Dulbecco’s Minimal
Medium containing 10% fetal bovine serum. The cells were then
counted after trypsinization on alternate days over a period of 10
days. VEGF was also measured as described by Ali et al.
(10)
. Cells were seeded in equal numbers, and VEGF was
measured in the spent medium after 48 h with the Quantikine VEGF
immunoassay (R&D Systems). To assay NOS activity, the cells were
grown in the same way. NOS activity was measured in cell lysates with
the NOSdetect kit (Stratagene). This kit measures the
activity of NOS in an NOS-catalyzed coupled reaction where citrulline
and nitric oxide are produced from arginine in equimolar amounts. The
amount of citrulline thus produced is measured and expressed in terms
of the activity of NOS. The final values for NOS activity were
corrected for total cell protein in each sample.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Ishikawa cells were cotransfected
with a G418 resistance-conferring plasmid and an ER--expressing
vector. The ER-overexpressing clones were selected for G418 resistance
and then screened for high ER- expression. All of the subsequent
experiments, including the ones presented here, were then performed
using the parent Ishikawa cells, an ER-overexpressing clone of Ishikawa
(ISH-ER), and a control-transfected clone of Ishikawa (ISH-NON).
ISH-NON was a G418-resistant clone that was selected during the
screening for ISH-ER. Having gone through the same transfection
process, ISH-NON was deemed a logical control against the phenotypic
changes arising from the transfection protocol alone. In addition,
ISH-NON was also a control against clone-dependent artifacts.
To establish a baseline, each of the cell lines, i.e.,
Ishikawa, ISH-ER, and ISH-NON, was inoculated into the flanks of OVX or
SHAM nude female mice. No visible tumors formed from any of the cell
lines in either SHAM or OVX animals observed over a period of 5 weeks
(Fig. 1A)
.
|
. Tumor
growth was more aggressive in mice inoculated with parent Ishikawa and
control ISH-NON cells, whereas it was significantly slower in the case
of ER-transfected ISH-ER cells. In mice that had received implants of
1.5-mg E2 pellets (producing blood E2 levels of >900 pg/ml), an
insignificant increase in tumor size was observed in the case of
Ishikawa, ISH-NON, and ISH-ER cells. In all groups, therefore, E2 at
levels higher than physiological stimulated tumor growth (Fig. 1, A and C)
. In contrast, ER was found to inhibit
the growth of these tumors.
When OVX mice received implants containing 1.5-mg TAM or OHT pellets
(producing blood levels of 3–4 ng/ml in each case), palpable tumors
from Ishikawa cells developed within 3 weeks. ISH-ER cells,
however, did not form any tumors (Fig. 1B)
. Moreover, the
tumors formed in these groups were significantly smaller than the
tumors in the corresponding groups that had received E2 pellets. This
indicated that the estrogenic effect required for tumor formation was
weaker in the case of antiestrogens compared with E2. In these groups,
therefore, antiestrogens were found to be stimulatory to tumor growth,
but to a lesser extent than E2. ER overexpression was found to
consistently inhibit cell growth.
Effects on Integrin vß3, VEGF, and
Vascularization in Tumors.
To examine the levels of VEGF and
vß3, immunostaining
with an anti-VEGF or an
anti-vß3 antibody,
respectively, was performed on sections from tumors initiated in nude
mice with parent and ER-transfected Ishikawa cells. Slides from these
tumors were also stained by a hematoxylin-ferric chloride-iodine
solution to examine the extent of vascularization. These tumors were
excised from the animals that had received implants of 0.15-mg E2
pellets. The tumors initiated from the ER-transfected cells showed a
significant down-regulation of VEGF (Fig. 2)
and integrin
vß3 (Fig. 3)
. As anticipated, the magnitude
of vascularization was also found to be decreased in the
ER-overexpressing tumors (Fig. 4)
.
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vß3 and VEGF and the
degree of vascularization. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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overexpression in Ishikawa cells was found to be inhibitory
to in vivo tumor growth and to the angiogenic parameters
studied here. We previously reported the development of this
ER--overexpressing clone of Ishikawa cell line, ISH-ER, that has an
ER level 6-fold higher than the parent Ishikawa
cells3
(
50 fmol/mg of WCP in Ishikawa and
300 fmol/mg of WCP in ISH-ER cells, as determined in a
hormone-binding assay). In the previously reported in vitro
assays, each of the three cell lines, i.e., the parent
Ishikawa, the ER-transfected ISH-ER, and the control-transfected
ISH-NON, showed an increase in growth rate when induced with
10-9 M
E2.3
This was expected of a cell line of
endometrial origin. With or without E2 induction, however, the growth
rate of ISH-ER was inhibited significantly compared with the parent or
control-transfected cells. This indicated that although E2 stimulated
the growth of Ishikawa cells, ER inhibited their growth. This growth
inhibition was consistently observed in other ER--overexpressing
clones as well (Table 1)
, indicating that
this effect was not clone-dependent.
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overexpression that was also found to be associated with a
modulation of certain vasoactive/angiogenic factors. The effects of
ER- overexpression were therefore found to be similar in two cell
lines of completely different origins.
As shown in Fig. 1, B and C
, in the OVX or SHAM
animals, no growth was observed from any of the cell lines tested. When
these animals were administered E2, however, each of the three cell
lines, Ishikawa, ISH-ER, and ISH-NON, formed tumors. The tumors formed
by ISH-ER were, however, significantly smaller than the parent or
control cell line. These results clearly indicated that in these
animals, the presence of a higher than the physiological level of E2
was required for tumor growth; E2 was therefore stimulatory for the
growth of these cells in vivo as well. Consistent with the
in vitro results, however, ER was once again found to
inhibit the growth of xenografted tumors. A dissociation in the effects
of ER and its ligand was therefore consistently observed, indicating a
dominant ligand-independent effect of ER after its overexpression. It
is possible that this effect is caused by binding of the overexpressed
ER to ligands other than E2 (11
, 12)
or by binding of ER
to DNA sites in an unliganded conformation (13)
.
The effects of E2 and ER- overexpression observed in the Ishikawa
cells are physiologically consistent. In a physiological milieu,
endometrial cells are, indeed, stimulated by E2. A high level of
ER-, on the other hand, is known to be favorable for controlling the
growth of endometrial and other female cancers (1
, 2)
.
The fact that no tumor growth was observed in SHAM animals indicates that for in vivo growth, Ishikawa cells required a higher level of E2 than the normal physiological level in the nude mouse. Because Ishikawa cells are of human origin, it is quite possible that the E2 blood levels in mice were not high enough to elicit an estrogenic effect in these cells.
The tumors initiated from ER-overexpressing ISH-ER cells showed a
significant down-regulation of VEGF compared with the tumors from
parent Ishikawa cells (Fig. 2)
. We previously showed a similar in
vitro down-regulation of VEGF levels in cultured ER-overexpressing
cells (Ref. 10
and Table 1
). Our in vitro and
in vivo results, therefore, consistently showed inhibition
of VEGF as a result of higher ER- expression. The effect of
estrogen on endometrial growth and endometrial VEGF (14)
is known to be stimulatory. Our results, however, show that the effect
of ER on these two parameters in a cell line of endometrial origin
contrasts with E2, i.e., whereas the hormone is stimulatory,
its receptor is inhibitory.
In case of endometrial cancer, ER-positive tumors exhibit an ER level
of 155–209 fmol/mg of WCP in a hormone-binding assay
(15)
. As reported previously,3
using
a similar technique, we determined that the levels of ER in our
Ishikawa (and control ISH-NON) and ER-overexpressing ISH-ER cells were
50 and 300 fmol/mg of WCP, respectively. Our models, therefore,
cover a broad physiological range of ER expression found in endometrial
cancers.
It has been shown that in women with ovarian (1)
or
endometrial (2
, 16)
cancers, a positive ER status is
predictive of survival. Our results demonstrating a negative
correlation between the growth and ER content of endometrial tumor
xenografts support these earlier findings. We show a possible
involvement of angiogenic factors in ER-related inhibition of tumor
growth. Our in vitro studies with ER-overexpressing clones
of Ishikawa have shown a modulation of growth and of angiogenic
factors, including VEGF (Table 1)
. VEGF is one of the key determinants
of the onset and progression of angiogenesis. It is possible that a
positive correlation between ER levels and the survival rate of ovarian
and endometrial cancer patients involves an ER-mediated, VEGF
associated negative control of angiogenesis in these cancers. In the
present study, the ER-overexpressing tumors that showed down-regulation
of VEGF were indeed found to have a significantly lower level of
vascularization compared with larger tumors expressing basal levels of
ER (Fig. 4)
.
In the ER-mediated inhibition of angiogenesis observed here, it is
possible that other cellular factors that are controlled by angiogenic
cytokines (such as VEGF) are influenced in the downstream pathway,
eventually leading to the inhibition of vascularization. To investigate
this issue, we also examined the effect of ER overexpression on the
expression of integrin
vß3, one of the
adhesion molecules that promotes angiogenesis in certain cancers
(17)
and is positively affected by VEGF (18)
.
We found that the level of integrin
vß3, as anticipated,
was significantly lower in the tumors initiated from ER-overexpressing
cells (Fig. 3)
.
In conclusion, we show here that, in contrast with its ligand E2, high
levels of ER- down-regulate in vivo growth of the
endometrial cancer cell line Ishikawa, and that this growth inhibition
is associated with a down-regulation of the angiogenic factors VEGF and
integrin vß3, leading
to an inhibition of tumor angiogenesis.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Adult Oncology, Dana-Farber Cancer
Institute and Harvard Medical School, 44 Binney Street, Boston, MA
02115. Phone: (617) 632-2109; E-mail: syedhali{at}hotmail.com 
2 The abbreviations used are: ER, estrogen
receptor; NOS, nitric oxide synthase; VEGF, vascular endothelial growth
factor; OVX, ovariectomized; SHAM, sham-operated; E2, 17-ß-estradiol,
TAM, tamoxifen; OHT, 4-hydroxytamoxifen; WCP, whole-cell protein.
3 S. H. Ali, A. L. O’Donnell, and P.
Dandona. Overexpression of estrogen receptor- in the endometrial
carcinoma cell line Ishikawa: inhibition of growth and vascular
endothelial growth factor, stimulation of nitric oxide synthase
activity, manuscript in preparation.
Received 2/10/99. Accepted 10/18/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in ECV304 cells inhibits proliferation and levels of secreted endothelin-1 and vascular endothelial growth factor. Mol. Cell. Endocrinol., 152: 1-9, 1999.[Medline]
vß3 integrin, osteopontin, and thrombin. Am. J. Pathol., 149: 293 1996.[Abstract]
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8). B, groups of OVX
nude animals also received implants of 1.5-mg time-release pellets of
TAM, OHT, or a placebo. The results are expressed as mean;
bars, SE (n 
