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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1038-1044
Antineoplastic Urinary Protein Inhibits Kaposi's Sarcoma and
Angiogenesis In Vitro and In Vivo
By
Rizwan Masood,
Megan E. McGarvey,
Tong Zheng,
Jie Cai,
Naveen Arora,
D. Lynne Smith,
Nathan Sloane, and
Parkash S. Gill
From the University of Southern California School of Medicine, the
Department of Internal Medicine and Pathology, Los Angeles, CA; the
Centre for Biomedical Technology, Delhi, India; and Antitumor Research
Products, Inc, Germantown, TN.
 |
ABSTRACT |
Kaposi's sarcoma (KS) is the most common tumor in human
immunodeficiency virus infection and acquired immune deficiency
syndrome. Recent clinical trials with human chorionic gonadotropin
(hCG) prepared from early pregnancy urine have shown encouraging
results in the resolution of KS lesions. A urinary protein with
antitumor activity, ANUP (antineoplastic urinary protein), a dimer of
32 kD, has previously been shown to inhibit the growth of various tumor
cell lines in vivo. It was thus studied for its activity in KS cell
lines in vitro and in vivo to determine whether it could be a source of
the anti-KS activity observed in hCG preparations. ANUP is a strong
growth inhibitor for KS cell lines, but has little or no effect on
fibroblast, aortic smooth muscle, T- and B-lymphocyte, and monocyte
cell lines. ANUP also inhibited the proliferation of endothelial cell
lines, suggesting that the in vitro effects were endothelial cell
lineage-specific. However, ANUP antibodies did not block the
inhibitory effect of certain commercial preparations of hCG, previously
shown to be active in KS. Thus, the active protein in these commercial
preparations of hCG may be distinct from ANUP. The antitumor activity
of ANUP was further confirmed in a chicken allantoic membrane (CAM)
assay in which vascular endothelial growth factor (VEGF) and beta
fibroblast growth factor (bFGF)-induced angiogenesis was
inhibited by ANUP in a dose-dependent manner. In vivo activity of ANUP
was demonstrated in the murine model of KS, where ANUP inhibited tumor
growth. ANUP is thus a potential candidate for development in the
treatment of KS and other diseases in which angiogenesis plays an
important role.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
KAPOSI'S SARCOMA (KS) is the most common
tumor found in patients with human immunodeficiency virus infection,
with a lifetime risk of up to 30%.1 KS is also reported in
a number of different clinical conditions, including renal
transplantation, chronic use of glucocorticoids, lymphoproliferative
disorders, and autoimmune diseases,2-4 and is endemic in
Central Africa. Epidemiological evidence shows a strong association of
human herpes virus 8/Kaposi's sarcoma-associated herpes virus with
KS, and it is currently considered the causative agent.5
Histologically, KS involves an aberrant proliferation of small vessels
that lack a basement membrane and pericytes and display leaky
behavior.6 In addition, there is proliferation of
spindle-like cells, which are the putative tumor cells. The origin of
KS has been debated to be endothelial cells or vascular smooth muscle
cells, but recent studies showing the expression of endothelial
cell-specific cell-surface tyrosine kinases in KS cell lines and
primary tumor tissues such as Tie-1, Tie-2, Flt-1, Flk-1/KDR,
Flt-47 (R. Masood, unpublished data, May 1998)
suggest that KS originates from endothelial cells. A number of
angiogenic and growth regulatory factors and their receptors such as
vascular endothelial growth factor (VEGF), beta fibroblast growth
factor (bFGF), interleukin-6 (IL-6), and IL-1 are expressed by KS
cells.7-11 Furthermore, these factors regulate KS cell
growth and induce angiogenesis in the tumor lesions. Tumor growth
factor beta is an autocrine growth inhibitory factor and thus appears
to modulate the tumor growth.12
Some intriguing observations and a serendipitous finding have led, over
the past few years, to investigation of pregnancy-related factors as
anti-KS agents. It has been noted that not only does KS develop
predominantly in men, but when it does occur in women, it often
resolves in early pregnancy.1 This, together with the
observation that KS tumor xenografts did not establish in pregnant
mice,13 suggested the presence of tumor inhibitory activity
associated with pregnancy. Human chorionic gonadotropin (hCG) was
considered one of the potential antitumor agents, and several
commercial preparations were tested, of which only one (A.P.L.; Wyeth
Ayerst, Philadelphia, PA) was found to have high tumor inhibitory
activity.14 The most active commercial preparation (A.P.L.)
has been studied in patients with AIDS-KS. Both intralesional and
systemic (subcutaneous) administration induced major tumor responses.14,15 The highly purified hCG (CR127; National
Institutes of Health, Bethesda, MD) and recombinant hCG heterodimer
were inactive in vitro.14 These findings suggested that
either copurified molecule(s) or degradation products of hCG were
responsible for the tumor inhibitory activity. Commercial preparations
of hCG are heterogeneous in protein content.16 Since
commercial preparations of natural hCG are purified from early
pregnancy urine, an attempt has been made to identify the active
factor.17 Anti-KS activity was located in fractions
migrating at 15 to 30 kD and 2 to 4 kD in both pregnancy urine
concentrates and A.P.L. hCG. The hCG-associated fraction (HAF) activity
was not associated with fractions containing hCG heterodimer or
individual subunits,17 which supports the hypothesis that
copurified molecules in A.P.L. hCG are responsible for anti-KS activity.
Antineoplastic urinary protein (ANUP) is a naturally occurring dimer of
32 kD that has previously been characterized for its activity against
various tumor types in vivo.18 ANUP is reported to inhibit
the proliferation of a variety of human tumor cell lines in vitro but
had no effect on a hamster neoplastic cell line, which suggests species
specificity. Normal diploid human cells also were unaffected. Following
the description of ANUP in urine, it was also detected in human
plasma19 and granulocytes,20 where it was
described to have cytokine activity. Because ANUP is a urinary protein
with antineoplastic activity and is of a size consistent with the
anti-KS activity in A.P.L. hCG and urinary concentrates,17
we wished to determine if ANUP has anti-KS activity.
| |
MATERIALS AND METHODS |
Materials.
Electrophoretically homogeneous ANUP was prepared from adult human
urine by absorption onto Florisil (magnesium silicate granules; Florisil Co, Berkely Springs, WV) as previously
described.18 Briefly, urine was absorbed onto Florisil at
pH 3 to 4 at 22°C. ANUP was eluted with cold
acetone:glycerol:H2O (3:6:11 by vol) at pH 9.0 to 9.3. The
eluate was then neutralized and concentrated by sequential treatment in
an Amicon Diaflow UM20 column (Amicon Corp, Danvers, MA) followed by an
Amicon YM30 column to yield proteins in the 20 to 30-kD range.
Chromatography in Sephacryl S-200 yielded electrophoretically pure
ANUP. The monoclonal antibodies to ANUP used here have been previously
described.21
Cell proliferation assay.
The immortalized KS cell lines KS Y-18 and
KS-SLK22 and the nontransformed long-term isolate KS 6-3 (prepared as previously described7,23 were grown in wells
coated with 1.5% gelatin in KS medium consisting of RPMI 1640 (Life
Technologies, Gaithersburg, MD), 100 U/mL penicillin, 100 µg/mL
streptomycin, 2 mmol/L glutamine, 1% essential and
nonessential amino acids, 10% fetal bovine serum. Life Technologies),
and 1% Nutridoma-HU (Boehringer Mannheim, Indianapolis, IN). Human
umbilical vein endothelial cells (HUVECs) (Clonetics, San Diego, CA)
were grown in media containing epidermal growth factor and according to
the instructions of the supplier. Cells were plated at a density of
10,000/well in 48-well gelatin-coated plates on day 0. Similarly, human
aortic smooth muscle cells ([AoSM] Clonetics) and T1, HUT-78, A6876,
and P3HR1 cells (American Type Culture Collection, Rockville, MD) were
seeded in 48-well plates at the same density in their growth media on
day 0. The following day, the cells were treated with various
concentrations of ANUP. After 72 hours of incubation, they were treated
with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) at a final concentration of 0.5 mg/mL. The cells were incubated
for 2 hours, the medium was aspirated, and the cells were then
dissolved in acidic isopropanol (90% isopropanol, 0.5% sodium dodecyl
sulfate, and 40 mmol/L HCl). The developed color was read in an
enzyme-linked immunosorbent assay (ELISA) reader using the isopropanol
as a blank (Molecular Devices, Menlo Park, CA). Experiments were also
performed with ANUP with and without ANUP monoclonal antibodies at
concentrations of 1 mg/mL. In other experiments, a commercial
preparation of hCG (A.P.L.; Wyeth Ayerst) was tested alone and in
combination with ANUP antibodies at the same concentrations.
Cell migration assay.
Cell migration assays were performed in transwells with 8-µm pores
(Costar, Cambridge, MA). Briefly, the wells were coated with
fibronectin (25 µg/mL) overnight, and the endothelial cells or KS
cells were plated in 100 µL Dulbecco's modified Eagle's medium
(DMEM)/0.4% fetal calf serum (FCS) in the upper chamber. Five hundred
microliters of DMEM/0.4% FCS was added to the lower chamber and
incubated at 37°C for 1 hour. The test compounds at various
concentrations were added to the upper chamber and chemotaxis agents
(VEGF or bFGF 20 ng/mL) to the lower chamber. The plates were incubated
for 5 hours at 37°C, and cells that crossed the fibronectin-plated
membrane were quantified after wiping the cells off the upper chamber
with a cotton swab. The cells across the membrane were stained with
Diff-Quik stain set according to the manufacturer's instruction (Dade
Diagnostics Inc, Aguada, PR). The cells were counted at 320×
magnification in four randomly selected fields. The experiments were
performed in duplicate and repeated at least three times.
Chicken chorioallantoic membrane assay.
The chicken chorioallantoic membrane (CAM) assay was used to determine
the effect of test compounds on angiogenesis. Ten-day-old fertilized
chicken eggs were prepared by creating a window in the shell. Filter
paper disks saturated with VEGF or bFGF as positive controls with test
compounds (200 ng per disk) or an equal amount of carrier buffer were
placed on the CAMs. The chicken embryos were incubated at 37°C in a
humidified egg incubator. CAMs were harvested after 48 hours and
analyzed using a stereomicroscope. The number of branching
blood vessels infiltrating under the disks were counted and
photographed. Eight CAMs were studied for the test group and the
experiments were repeated at least twice.
In vivo activity of ANUP.
Nude mice (3- to 4-week-old females) were injected with 2 million
SLK-KS or KS Y-1 cells subcutaneously in a total volume of 100 µL.
After 1 week of tumor development, the mice were injected subcutaneously with either phosphate-buffered saline (PBS) and ANUP (5 mg in 100 µL PBS) or PBS alone at equal volume on days 7, 9, and 11. The tumor size was measured three times per week. The results
represent the median of four mice in each group.
| |
RESULTS |
Activity of urinary proteins in KS cell lines.
ANUP was tested at various dose levels in a cell proliferation assay on
three different KS cell lines, primary endothelial cells grown in the
presence of VEGF or bFGF, vascular smooth muscle cells, and a number of
other tumor cell lines. ANUP inhibited the proliferation of KS cell
lines (KS Y-1, KS-SLK, and KS 6-3) and endothelial cells in a
dose-dependent manner, whereas no significant effect was observed in
the other cell types. The IC50 (50%
inhibitory concentration) for KS Y-1, KS-SLK, and KS 6-3 was 26, 22, and 33 µg/mL, respectively (Fig 1). We also examined the effect of ANUP on endothelial cells and various other cell lines. Endothelial cell (HUVEC) proliferation was also inhibited by ANUP, with an IC50 of 30 µg/mL. Modest inhibition of AoSM was found at
higher doses, with no inhibition of a number of other cell types,
including fibroblast (T1), T-cell leukemia (HuT78), and B-cell lymphoma (P3HR1, A6876, and 23-2) cell lines. The activity was thus highly specific for the endothelial cell lineage (Fig
1B).
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| Fig 1.
Activity of ANUP on (A) 3 different KS cell lines and (B)
control cell lines. All cell lines were plated in 48-well dishes at a
density of 10,000/mL. The following day, a different concentration of
ANUP was added in fresh media. Plates were incubated for an additional
4 days. Cells were stained with MTT and lysed, and the absorbance was
read in an ELISA reader.
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|
ANUP is not the source of antitumor activity in commercial hCG
preparations.
To demonstrate that the effect was observed specifically from the
purified ANUP, the experiments were repeated with and without the
addition of ANUP monoclonal antibodies. The growth inhibitory effect of
ANUP was completely blocked by either antibody and was dependent on the
amount of Igs (Fig 2A). ANUP monoclonal
antibodies block the growth inhibitory effect of ANUP on KS
cells.
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| Fig 2.
ANUP monoclonal antibodies abrogate the effect of ANUP on
KS cell growth inhibition. (A) KS Y-1 cells were seeded at a density of
1 × 104/well on gelatin-coated 48-well plates. Cells were
treated with various concentrations of ANUP alone or with ANUP
antibodies at a concentration of 1 µg/mL. Cell proliferation was
measured after 72 hours. The active preparation of hCG (A.P.L.; Wyeth
Ayerst) inhibited KS cell proliferation (B and C), and this effect was
not blocked by ANUP antibodies. KS cells were treated with hCG (A.P.L.)
at various concentrations alone or in combination with ANUP antibodies
(1 µg/mL each). Cell proliferation was measured at 72 hours. The data
represent the mean ± SD of experiments performed in quadruplicate.
|
|
Certain commercial preparations of hCG have been shown to inhibit KS
cell proliferation; however, the recombinant hCG is inactive, suggesting that another protein may have been copurified with hCG.14 To determine if ANUP was the active factor in hCG,
we tested commercial preparations of hCG alone and in the presence of
ANUP antibodies. ANUP antibodies did not block the growth inhibitory effect of commercial preparations of hCG. Thus, the activity in hCG may
be distinct from ANUP (Fig 2B and C).
ANUP inhibits endothelial cell migration.
Angiogenesis is mediated by complex biochemical processes, which
include degradation of the basement membrane under the existing vessel endothelial cells. This is followed by the proliferation and coordinated migration of endothelial cells to form tubes. In
a final step, vascular smooth muscle cells are recruited to encase the
newly formed vessels.24 We wished to study the effect of
ANUP on the migration of endothelial cells. This was performed in
transwell cultures with endothelial cells in the upper chamber coated
with fibronectin. bFGF or VEGF were added to the lower chamber,
and the migration of endothelial cells across the membrane after
16 hours was quantified. ANUP inhibited endothelial cell migration with
an IC50 of 75 µg/mL (Fig 3A and C). KS cell migration was
affected even more, with an IC50 of 52 µg/mL (Fig 3B and
D). This may be related to the expression
of several chemotaxis factors by KS cells, such as VEGF, bFGF, IL-8,
etc.
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| Fig 3.
ANUP inhibits KS cell migration. Migration assays were
performed in double-chamber wells separated by a fibronectin-coated
membrane. Chemotaxis was induced by addition bFGF (25 ng/mL) to the
lower chamber. Cells (5 × 104/mL) were placed in the
upper chamber in the presence and absence of test compounds. Taxol 10 ng/mL was used as a positive control. Migration of (A) endothelial
cells or (B) KS cells across the membrane was quantified after
overnight incubation. Representative photographs (original
magnification ×360) for controls, various concentrations of ANUP, and
taxol are shown for (C) endothelial cells and (D) KS Y-1 cells (see
page 1039). Cells that migrated to the underside of the transwell
membrane are shown; cells that did not migrate were removed from the
upper surface of the membrane with a cotton swab prior to microscopy.
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ANUP inhibits angiogenesis in CAM assays.
To further test the antiangiogenic activity, CAM assays were performed
with induction of angiogenesis by bFGF with and without the addition of
ANUP. ANUP inhibited angiogenesis in a dose-dependent manner. Complete
inhibition of bFGF-induced angiogenesis was observed at a concentration
of 0.1 µg/mL (Fig 4A and
B). The low concentration of
ANUP required in CAM assays, versus the cell proliferation and
migration assays is attributable to the high local concentration of
ANUP on the assay disks and the fact that most proteins are very stable
on CAMs. Comparable results were obtained for VEGF-induced angiogenesis
(data not shown).
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| Fig 4.
(A) ANUP inhibits angiogenesis in CAM assays. Filter
disks soaked in buffer alone, bFGF alone, or bFGF and test compounds in
a total volume of 10 µL were used on 10-day-old CAMs. CAMs harvested
after 48 hours were resected and photographed, and the number of
branching blood vessels was counted within the area of each disk. The
assay represents the mean ± SE of  8 CAMs per test group. Assays
were repeated at least twice, with similar results. (B) Representative photographs (original
magnification ×40) of control group receiving buffer only, bFGF alone
(200 ng), and bFGF plus ANUP (0.1 µg).
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ANUP is active in vivo.
To determine if ANUP activity can be reproduced in vivo, KS cells were
implanted in immunodeficient mice and treated with either ANUP or
vehicle alone on days 7, 9, and 11 at a total dose of 5 mg
intraperitoneally. The tumor volume was measured three times per week.
Tumor growth was markedly retarded by ANUP compared with the vehicle
control (Fig 5).
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| Fig 5.
Inhibition of KS Y-1 tumor growth in nude mice. Nude mice
were injected with 2 million cells each. After 1 week, mice were
injected intraperitoneally with ANUP (5 mg in 100 µL PBS) or PBS on
days 7, 9, and 11 (arrows). Tumor growth was measured three times per
week. The results represent the median of four mice each.
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| |
DISCUSSION |
We have shown that a urinary protein previously identified for its
ability to inhibit tumor growth18 was highly active in KS
cells in vitro. In contrast to the original report for
ANUP,18 we did not observe growth inhibition in other tumor
cell lines. It should be noted that the anti-KS effect was apparent at
a lower concentration than the previously reported antitumor effect,
which was limited to breast and melanoma cell lines. The growth
inhibition of pancreatic (Capan-1 and SW 1990) and bladder (RT-4) cell
lines previously reported18 was minimal, and is in line
with our results for T-cell leukemia and B-cell lymphoma cell lines. We
did not test the same breast and melanoma cell lines that were
sensitive to ANUP at higher concentrations (>80 µg/mL).
Because KS cells express phenotypic characteristics of endothelial
cells, such as the expression of various tyrosine kinases including
Tie-1, Tie-2, Flk-1/KDR, Flt-1, and Flt-47 (R. Masood,
unpublished data, May 1998), we determined if ANUP also
has activity against the mitogenic and migratory responses of
endothelial cells. Endothelial cell proliferation in response to VEGF
or bFGF was inhibited by ANUP in a dose-dependent manner. Because
migration of endothelial cells is an important element of
angiogenesis,24 we determined that ANUP inhibited cell
migration in response to angiogenic factors such as VEGF and bFGF.
Based on these findings, we determined the angiogenic activity of ANUP by the CAM assay and observed a dose-dependent inhibition of VEGF- and
bFGF-induced angiogenesis. These data show convincingly that one of the
effects of ANUP is to inhibit angiogenesis.
To determine the in vivo activity of ANUP, a KS xenograft model was
used. KS tumor growth was inhibited after only three doses of ANUP.
Other tumor types including HeLa cells and KB (a human laryngeal tumor
cell line) implanted in Balb/C NU+/nu+ mice
have previously been shown to respond to ANUP (N. Sloane, unpublished
data, December 1996), which demonstrates that the effect
is not limited to KS.
A failure of KS tumor growth in pregnant mice suggests that factors
produced during pregnancy, including but not limited to hCG, may
produce this effect. Commercially available hCGs have been tested, and
certain preparations were highly active (A.P.L.; Wyeth Ayerst) in
inhibiting KS cell growth. Highly purified hCG was not active,
suggesting that HAF may be responsible for this activity.17
ANUP could be such a factor. However, ANUP antibodies did not block the
activity of active commercial hCG (A.P.L.). It is thus unlikely that
ANUP and HAF are identical.
The mechanism of action of ANUP is not clear at the present time. It
may be mediated by ANUP binding to a cell surface receptor that has
signaling specificity in the endothelial cell lineage. Since cell
migration was also affected, at least in the presence of fibronectin as
the extracellular matrix, it is possible that ANUP activity inhibits
the binding of endothelial cells to certain extracellular matrix
proteins necessary in angiogenesis. The determination of the effects of
ANUP on endothelial cell migration in the presence of various
extracellular matrix proteins is currently in progress.
The N-terminal sequence of ANUP has been determined.20
Comparison of these 14 amino acids to the database did not reveal any
matches, indicating that ANUP is a novel protein. To further investigate the antiangiogenic properties of this protein, the full-length cDNA is currently being isolated with the intention of
expressing the recombinant protein.
| |
FOOTNOTES |
Submitted May 18, 1998; accepted September 29, 1998.
Supported by the M.P. Aitken Foundation and the Bridges-Larson Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Parkash S. Gill, MD,
Professor of Medicine and Pathology, Norris Cancer Hospital and
Research Institute, 1441 Eastlake Ave, Los Angeles, CA 90033; e-mail:
parkashg{at}hsc.usc.edu.
| |
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Top
Abstract
Introduction