a-fetoprotein derived from a human hepatoma prevents...
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Vol. 4, 2877-2884, November 1998 Clinical Cancer Research 2877
a-Fetoprotein Derived from a Human Hepatoma Prevents
Growth of Estrogen-dependent Human
Breast Cancer Xenografts’
James A. Bennett,2 ShuJi Zhu,
Andrea Pagano-Mirarchi, Theresa A. Kellom, and
Herbert I. Jacobson
Albany Medical College, Albany, New York 12208
ABSTRACTa-Fetoprotein (AFP) is a transport protein that has
growth-regulatory properties in many different tissues. It
is known to interfere with responses stimulated by estro-
gen. The purpose of this study was to determine whether
human AFP would inhibit the growth of human breast
cancer. AFP was isolated from the culture supernatant of
human hepatoma cells (HepG2) grown in serum-free me-
dium and was purified by immunoaffinity chromatogra-
phy. Human breast cancers were grown as xenografts
under the kidney capsule of severe combined immunod-
eficient mice. The minimum inhibitory dose of AFP
against estradiol (E2)-stimulated growth of human
MCF-7 breast cancer xenografts was 10 pg/mouse/day,
and maximum inhibition (no growth) was achieved with100 p�g/mouse/day. Daily treatment was required to sus-
tam inhibition. This 100-pg dose of AFP also inhibited
xenograft growth of E2-dependent T47 human breast car-cinoma. Estrogen receptor-negative MDA MB 231 and
BT2O human breast carcinoma xenografts were not inhib-ited by AFP (100 �i�mouse/day). Elevation in serum E2occurred during AFP treatment. AFP did not compete
with agonists for the estrogen receptor. These laboratory
results are consistent with the findings of a literature
search, which consistently showed an association between
elevated pregnancy levels of AFP and subsequent reduced
risk for breast cancer later in life. We conclude that AFP
can inhibit growth of estrogen-dependent breast cancer
and warrants further development as an agent for thetreatment and perhaps even the prevention of human
breast cancer.
Received 5/22/98; revised 8/17/98; accepted 8/21/98.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by Department of the Army Grant DAMD
l7-94-J-4278.
2 To whom requests for reprints should be addressed, at ME-S 14, MailCode 62, Albany Medical College, 47 New Scotland Avenue, Albany.
NY 12208. Phone: (518) 262-5247; Fax: (518) 262-5999.
INTRODUCTIONAFP3 is a glycoprotein produced during gestation initially
by the fetal yolk sac and then by the fetal liver and is a major
protein constituent of the fetal plasma throughout gestation ( 1).
It has 39% primary structure homology to albumin, and like
albumin, it has a molecular weight OfMr �69,000 and is divided
into a three-domain structure based on its disulfide bonding
pattern (2, 3). However, unlike albumin, upon parturition the
gene for AFP is repressed, which diminishes the serum concen-
tration of AFP to a negligible level. The restriction of the
physiological presence of this protein to embryonic life suggests
a unique role for AlP in cell growth and differentiation, which
are the hallmarks of embryonic life. Evidence for this role has
been obtained in a variety of studies showing that AFP can
regulate the growth or secretory activity of certain tissues such
as liver (4), lymphocytes (5), placenta (6), ovaries (7), and
uterus (8) and can interact with certain ligands such as arachi-
donic acid (9), docosahexanoic acid (9), and retinoic acid (9), all
of which influence differentiation. However, a complete under-
standing of the physiological role of AFP is not yet available.
Previous studies have suggested a possible role for AFP in
the regulation of breast cancer growth. Sonnenschein et a!. ( I 0)
have shown that estrogen-dependent rat mammary tumors re-
gressed in rats bearing hepatomas that were secreting milligram
quantities of AlP into the serum. This serum also inhibited in a
dose-dependent manner the estrogen-stimulated induction of
progestin receptor in cultures of rat pituitary tumor cells (I I).
Our own studies have shown that injecting mice with nanogram
quantities of human cord sera AFP that had been preincubated
with a molar excess of E2 inhibited the estrogen-stimulated
growth of the uterus ( 12) and of estrogen receptor-positive
human breast cancer xenografts (13). In the absence of prein-
cubation with E2, this material was inactive when given in
nanogram amounts ( 12, 1 3), suggesting that the molar excess of
E2 constituted a chemical environment that fixed more of the
AFP molecules in their active form (14, 15). The data of
Sonnenschein et a!. taken together with our data suggested that
a small proportion of the AFP molecules were already in their
active form, and if we were to then raise the dose of AFP, we
could achieve antiestrotrophic activity with human AFP alone,
abrogating the need for prior activation with higand. To test this
possibility, we needed a uniform source from which we could
readily and reproducibly obtain milligram quantities of human
AFP. Pooled human cord serum did not seem practical because
of its heterogeneity and its large content of albumin relative to
3 The abbreviations used are: AFP, cx-fetoprotein: E2. estradiol: ATCC,
American Type Culture Collection: SCID. severe combined immunod-
eficient: MS. maternal serum.
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220
2878 Inhibition of Breast Cancer Growth by AFP
40
.� 30
� 20
10
0 10 20 30 40 5095 100
Days in Serum.Free Medium
Fig. I Effect of serum-free medium on the secretion of AFP by HepG2
cells. HepG2 cells were grown to confluence in serum-containing me-
dium. After cells reached confluence, the medium was changed to a
serum-free medium. Cells were then maintained in serum-free medium
with a medium replenishment schedule of every 3-4 days. AFP con-
centration was measured in spent medium by enzyme immunoassay
using the Abbott IMx automated immunofluorescence system as de-
scribed previously (13).
AFP (12, 16), making purification of AFP difficult because of
its 39% homology to albumin (2). The recombinant systems
available to us also yielded insufficient quantities of human
AFP. Tecce and Terrana (17) had reported that the human
hepatoma cell line HepG2 secreted significant quantities of AFP
into the culture supernatant when grown in serum-free medium
and that AFP was the main protein secreted under these culture
conditions. This seemed to be a reasonable source of AlP for
the purpose of our study, which was to determine whether
human AFP alone and in a dose-dependent manner could inhibit
the growth of human breast cancers grown as xenografts in
immunodeficient mice.
MATERIALS AND METHODS
Culture Conditions for HepG2 Cells. HepG2 cells
were maintained and grown as a monolayer in aMEM (Life
Technologies, Inc., Gaithersburg, MD) supplemented with 5%
serum (40% calf serum, 60% FCS), penicillin G (100 units/mi),
and streptomycin (100 �i.g/ml). Cells were released from mono-
layer with 0.25% trypsin and 0.25% EDTA. Subculturing into
additional flasks was carried out by fivefold dilution of cells in
the above maintenance medium. Confluent flasks were switched
to serum-free medium to up-regulate production of AFP as
described by Tecce and Terrana (17). Serum-free medium was
composed of 3 parts aMEM and 1 part Waymouth’s MB 752/1
containing 3 X lO_8 M sodium selenite, 2 mrvt L-glutamine, and
1 .5% antibiotic/antimycotic mixture from Life Technologies.
Cells were refed with serum-free medium every 3 days.
Purification of AFP from Culture Supernatants of
HepG2 Cells. HepG2 culture supernatants were pooled and
concentrated using P- 10 Centriprep concentrators (Amicon,
Inc., Beverly, MA). Concentrate (10 ml) containing -3 mg of
AFP were loaded onto an 18 cm X 2.5 cm immunoaffinity
column [rabbit anti-human AFP (DAKO, Carpinteria, CA) con-
jugated to cyanogen bromide-activated Sepharose 4B1 in a load-
ing buffer of 100 mM NaCI and 10 msi sodium phosphate (pH
7.4). The concentrate was incubated on the column at room
temperature for 30 mm. Non-AFP proteins were ehuted with
-200 ml of loading buffer until no protein was detectable in the
eluate by UV absorbance (280 nm). AFP was eluted with -200
Crude Purified
Fig. 2 Purity of AFP after chromatography of HepG2 crude supernatant
on an immunoaffinity Sepharose 4B column. Shown in reducing SDS-
PAGE gels stained with Coomassie Blue. Four p.g of protein were loaded
onto a 12.5% gel. Molecular weight standards are shown in Lane M.
ml of 1.8 M MgC12 and dialyzed immediately against an excess
of 10 mM sodium phosphate buffer (pH 7.2). This material was
washed and concentrated in a buffer composed of 100 mrvi
sodium chloride and 10 m�i sodium phosphate (pH 7.2).
Uterine Growth Bioassay. The immature mouse uterine
growth bioassay is based on the finding that i.p. injection of 0.5
� l7�3-estradiol (E2) into 14- to 18-day-old female Swiss mice
results in a 70% increase in uterine wet weight, with a corre-
sponding parallel increase in mitotic index in 22 h (8). This
provides a relatively quick in vivo bioassay for assessing agents
that interfere with estrogen-stimulated growth. Immature female
(15- to 18-day-old) Swiss mice (Taconic Farms, Germantown,
NY) received i.p. injections of various quantities of AlP ob-
tamed from HepG2 cells, whereas control mice received injec-
tions of saline. One h after this first injection, test mice and
positive-control mice received injections of 0.5 jig E2, and
negative control mice received injections of 0. 1 ml of saline.
Twenty-two h after the second injection, the uteri were dis-
sected, trimmed free of mesenteries, and immediately weighed.
The uterine weights were normalized to mouse body weights
(mg uterine weightlg of body weight) to compensate for differ-
ences in body weight among hitters of the same age. Each
experiment was performed with five to eight mice per group,
and the mean normalized uterine weights ± SE for each group
were calculated. The percentage of growth inhibition was cal-
culated by the following relationships of normalized uterine wet
weights:
positive control - test group.. . Xl00%
positive control - negative control
Human Breast Cancer Xenografts. The MCF-7 and
MDA-MB-23 1 human breast cancer cell lines were obtained
from ATCC, Rockville, MD and were grown in DMEM sup-
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50
40
30
20
10
0
�D U)
WEoEC-
20
.0
0.1 1 10 100 1000
Clinical Cancer Research 2879
Table I Inhibition of E,-stimulated mouse uterin e growth by AFP derived fr om HepG2 cells
Test material” Dose”
% inhibition of E2-stimulated
growth of mouse uterus’
Mean ± SE
Crude supernatant
Fraction of crude supernatant non-adherent to anti-AFP column
Fraction of crude supernatant adherent to anti-AlP column
100 p.g AFP
100 p.g Protein
100 �.tg AFP
45 ± 4
045 ± 3
‘I Three-day supernatant from HepG2 cells that had been grown in serum-free medium for 10 days.1� One hundred p.g of material were injected i.p. in a volume of 0.5 ml 1 h before challenge of mice with an i.p. dose of 0.5 p.g of E2 in a volume
of 0.2 ml. Negative-control mice received two injections of saline. Positive-control mice received one injection of saline followed I h later by an
injection of 0.5 pg of E2.CMice were sacrificed 22 h after injection of E2; uteri were excised and weighed immediately. Uterine wet weight (mg) was normalized to mouse
body weight (g). Percent inhibition was calculated by dividing the mean E,-induced increase in uterine weight in the presence of test material by themean E,-induced increase in uterine weight in the absence of test material. There were five to eight mice per treatment group.
pg AFP
Fig. 3 Antiuterotrophic activity of AFP. Various doses of HepG2-
derived crude (#{149})and purified (0) AFP were injected i.p. into immaturefemale Swiss mice. One h later, 0.5 jig of E2 were injected i.p. into thesemice. Twenty-two h later. uteri were dissected and weighed. The per-centages of growth inhibition calculated for each dose of AFP arepresented as mean values; SE (bars). There were five to eight replicatemice per treatment group.
plemented with 5% FCS, 1% nonessential amino acids, 10
ng/ml insulin, 2 mM L-glutamine, 100 units/ml penicillin, and
100 p�g/ml streptomycin. The BT2O human breast cancer cell
line (ATCC) was grown in EMEM supplemented with 10%
FCS, 1% nonessential amino acids, and penicillin/streptomycin
as above. The T47D human breast cancer cell line (ATCC) was
grown in RPMI 1640 supplemented with 10% FCS, 5 �ig/ml
insulin, L-glutamine, and penicillin/streptomycin as described
above. Confluent cells growing in culture were released from
monolayer by trypsinization, diluted into single-cell suspension,
and then solidified by centrifugation into a pellet, which was
subsequently exposed to 10 pA of fibrinogen (50 mg/mi) and 10
ii.l of thrombin (50 units/mi) for 30 mm at 37#{176}C.Fibrin clots
containing tumor were cut into pieces - 1 .5 mm in diameter.
Each piece of tumor was implanted under the kidney capsule of
an immunodeficient ICR-SCID mouse (Taconic Farms) that
weighed -25 g, as described previously (13, 18). Estrogen
supplementation of mice was required for the growth of MCF-7
and T47 tumors. Supplementation was accomplished by s.c.
implantation of a Silastic tubing capsule containing solid E, (2
mm in length) inserted on the day of tumor implantation. Tumor
growth was then monitored during survival laparotomy at 10-
day intervals by measurement of the diameters of the short and
long axes of each tumor, using a dissecting microscope
equipped with an ocular micrometer ( 13). Tumor volumes were
calculated using the formula ir/6(d)2D, assuming the tumor
shape to be an ellipsoid of revolution around its long axis (D).
Mean tumor volumes ± SE are shown in the figures. However,
for the purposes of group comparisons, the percentage of in-
crease in tumor size in each mouse of a group at each time point
was calculated. The mean increase in tumor size for each group
of 8-10 replicate mice was calculated. The significance of
differences between groups was assessed using the Wilcoxon
rank-sum test.
Quantitation of AFP and Estrogen. The Abbott IMx
automated immunofluorescence system was used to quantitate
AFP. In this system the analyte initially contacts and becomes
bound to microparticles that are coated with monoclonal anti-
bodies to human AFP. Subsequently the particle-bound AFP is
exposed to polyclonal anti-human AlP conjugated to alkaline
phosphatase. After washing to remove excess material, substrate
for the alkaline phosphatase (4-methylumbelliferyl phosphate)
is added, and the fluorescent product is measured in the optical
system of the instrument.
Estradiol was quantitated in a competitive RIA using tubes
coated with rabbit antibody to estradiol (Coat-A-Count, Diag-
nostic Products Corp., Los Angeles, CA). ‘25I-labeled estradiol,
which was provided in the assay kit, was used to compete with
estradiol in the sample for antibody sites on the coated tubes.
Samples (100 �i.l) and ‘25I-E, (1.0 ml) were added to a tube, and
the tubes were incubated for 3 h at room temperature. The tubes
were thoroughly decanted and then counted in a gamma counter.
Final counts in the tubes were inversely related to the amounts
of E2 present in the samples. A predetermined standard curve
generated with standards provided in the assay kit was used to
relate counts to concentration.
Assessment of Estrogen Receptor Antagonism. Com-mercially obtained rabbit uterus (Pel-Freez Biologicals, Rogers,
AR) was used as a source of estrogen receptor. Uteri were
pulverized in a stainless steel impact mortar under liquid nitro-
gen and homogenized (20% w/v) in assay buffer [10 msi Tris
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2880 Inhibition of Breast Cancer Growth by AFP
30
25
5)
E
0>
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2
20
15
10
a
V � � 20 30
Days After Tumor Implantation
30
EE 25
5,
E
0>
I-
0E
I-F...
U-C.)2C
2
20
b
I
15
10
1 10 100 1000
AFP Dose (pg)
Fig. 4 a, effect of HepG2-derived AFP on the growth of estrogen-dependent human MCF-7 breast cancer xenografts. Treatment conditions for thevarious mouse groups were: 0, no E2: #{149}, E,; & E2 and 1 p�g of AFP; A, E2 and 10 p.g of AFP; E, E2 and 100 p.g of AFP; U, E, and 150 p.g of
AFP. SCID mice were estrogenized (E2) at the time of tumor implantation by s.c. implantation of sealed Silastic tubes containing solid estradiol (E,).
AFP-treated mice received daily i.p. injections of the indicated dose of AFP. Tumor volumes in each mouse were measured at the time of tumor
implantation and at 10-day intervals thereafter during survival laparotomies. There were 8-10 replicate mice/treatment group. Tumor volumes are
presented as mean values; bars, SE. b, AFP dose-response curve derived from a, using the tumor volumes obtained 30 days after tumor implantation.
AFP doses of 10, 100, and 150 p.g/mouse/day all resulted in significant suppression of tumor growth compared with the E2-alone group at 30 days
after tumor implantation. P < 0.05: Wilcoxon rank-sum test.
(pH 7.4), 1.5 mM EDTA, 10% glycerol, 10 m�i monothioglyc-
erol, and 10 mM sodium mohybdate] on ice. Centrifugation
(50,000 X g) for 1 h yielded a supernatant containing cytosol,
which was adjusted with assay buffer to 2.5 mg protein/mh. All
incubations were carried out in triplicate, each containing 100 p.1
of cytosol, 20 p.1 of 10 mrsi 6,7-[3H]estradioh (50 Ci/mmol;
DuPont NEN Research Products, Boston, MA), and 80 pA of
putative antagonist in assay buffer. Total count tubes received
20 p.1 of [3H]estradiol and 1 80 p.1 of assay buffer. After incu-
bation overnight at 4#{176}C,all but the total count tubes received
300 p.1 of dextran-coated charcoal suspension; tubes were agi-
tated for 15 mm and then centrifuged (1,000 X g) for 15 mm.
Supernatants were decanted into counting vials, scintillant was
added, and protein-bound tritium was determined by liquid
scintillation counting.
RESULTSHepG2 cells were grown to confluence in 25-cm2 tissue
culture flasks. For bulk production of AFP, multiple 25-cm2
flasks were preferable to fewer flasks with larger surface areas
because confluent monolayers tended to peel away from the
edges in the larger flasks when these cells were being maim-
tamed in serum-free medium. As shown in Fig. 1 , when HepG2
cells were switched to serum-free medium, AFP production
increased by approximately fivefold. Levels plateaued after 7 to
10 days in serum-free medium and were then sustained for as
long as 100 days. Gel electrophoresis indicated that there were
multiple proteins in addition to AFP present in the culture
supernatant (Fig. 2). The dense protein band migrating at a
molecular weight of Mr 69,000 (Fig. 2), consistent with the
molecular weight of AlP, was subsequently confirmed to be
AFP by Western blot (data not shown). AFP in this protein
mixture was purified to a single band, using immunoaffinity
chromatography (Fig. 2); the presence of AFP in that band was
again verified by Western blot (data not shown).
Protein in the culture supernatant was concentrated by
centrifugal force-augmented hydrostatic pressure using Cen-
triprep concentrators with Mr 10,000 cutoff (Amicon, Beverly,
MA). Injection of concentrate containing 100 p.g of AFP into
immature female mice significantly inhibited their uterotrophic
response to E2 (Table 1). This procedure was used as a screening
assay for antiestrotrophic activity in our preparations because of
its rapid turnaround time and cost-effectiveness. Optimal activ-
ity was found in supernatants from cells that had been in
serum-free medium for 7 to 21 days. After immunoaffinity
purification, the chromatographic fraction devoid of AFP had no
antiuterotrophic activity, whereas the immumoaffinity-purified
AFP-containimg fraction accounted for all of the activity in the
crude supernatant (Table 1 ). Dose-response studies indicated
that the antiuterotrophic activity plateaued at 50 jig of AFP and
titered out between 1 and 10 p.g AFP/mouse (Fig. 3).
The human MCF-7 breast cancer was grown as a xenograft
under the kidney capsule of SCID mice. As shown in Fig. 4, the
tumor was dependent for growth on exogenous estrogen sup-
plied via a Silastic capsule, placed s.c., that contained solid E2,
which yielded a steady-state plasma E, level of - 100 pg/mi.
Daily injections of AFP inhibited the growth of this tumor in a
dose-dependent manner (Fig. 4). Over a 30-day period, AFP at
10 p.g/mouse/day retarded the growth of the MC-7 breast cancer
and 100 p.g/mouse/day completely stopped the growth of MC-7
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MCF-7147
Fig. 5 Effect of HepG2-de-
rived AlP on the growth of es-trogen-dependent and estrogen-independent human breast
cancer xenografts. Open sym-
bols represent groups that were
not estrogenized: closed sym-
bols represent groups that were
estrogenized. AFP-treated mice(A, z�) received daily i.p. injec-tions of 100 p.g of AFP. Tamox-
ifen-treated mice (U, LI) re-ceived daily i.p. injections of 50
p.g of Tamoxifen. *, sigmifi-
cantly different from E,-alonegroup: P < 0.05, Wilcoxon
rank-sum test.
*
30
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2CU)
5)2
C.0 10 20 30
Days After Tumor Implantation
MDA-MB-231a-,. 100
E 80
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Days After Tumor Implantation
BT-20
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Days After Tumor Implantation
‘J 0 10 20
Days After Tumor Implantation
Clinical Cancer Research 2881
(Figs. 4 and 5). The growth prevention achieved with AFP at
100 p.g/mouse/day was similar to that obtained with Tamoxifen
at 50 p.g/mouse/day (Fig. 5). Both of these agents produced a
cytostatic effect as evidenced by the resumption of growth after
treatment was stopped and a histological profile showing no
evidence of necrosis, but rather a significant reduction in mitotic
figures and an increase in cells with GO/GI ploidy in growth-
inhibited tumors. This dose and schedule of AlP also inhibited
the estrogen-dependent growth of the T47 human breast cancer
(Fig. 5). Xenografts of two human tumors that grew independent
of estrogen (MDA MB 231 and BT2O) were not inhibited by
daily treatment with 100 p.g of AFP (Fig. 5). Blood samples
taken at the end of 30 days of AFP treatment contained higher
levels of E, than blood samples from animals not given AlP
(Table 2), suggesting that AFP was interfering with another E2
response, the feedback at the hypothalamic-pituitary axis, which
normally would decrease the output of follicle-stimulating hor-
mone in the presence of elevated serum E2 levels and prevent
such an increase in E2. The mechanism by which AlP interferes
with E2 responses is not clear at this time. It is known that
human AlP does not have a high affinity binding site for E2
(19); therefore, sequestering of E2 does not seem to be a likely
mechanism. In our own studies, we examined whether AFP
interferes with the ability of E2 to bind to the estrogen receptor.
As shown in Fig. 6, AlP does not have the characteristics of a
typical estrogen receptor antagonist.
DISCUSSION
The results of this study demonstrate that human AlP can
interfere with estrotrophic responses, including the estrogen-
stimulated growth of human breast cancer xenografts. The es-
trogem receptor-positive MCF-7 and T47 human breast cancers
did not grow in the presence of AFP. In contrast, the estrogen
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0)C
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80
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40
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.� T � Albumin
� -� -�------� AFP
T
--�D�--�D LY156758
‘b.i 1 10 100 1000 10000
2882 Inhibition of Breast Cancer Growth by AFP
Table 2 Eff ect of AFP on serum estradiol levels
Treatment group” Serum E, concentration” (pg/mi)-
Saline 56 ± 7
E,’ 100±5E2,AF�’ 187±8
“ Treatment groups were taken from those shown in Fig. 5.
,, E, concentrations in mouse sera were determined by a competi-tive RIA as described in “Materials and Methods.” Values are the
mean ± SE of five replicate mice per group. The E, concentration in thegroup treated with E, and AFP is significantly greater than that in the
group treated with E2 alone, P < 0.05, Wilcoxon rank-sum test.‘ Mice were estrogenized by s.c. implantation of sealed Silastic
tubes (2 mm in length) containing solid E2.
“ Mice received i.p. injections of AFP at 100 p.g/mouse/day for 30
days, and serum was collected 20 h after the last AFP injection.
receptor-negative MDA-MB-23 1 and BT2O human breast can-
cers grew unabated in the presence of AFP. These results are in
agreement with those of Sonnenschein et a!. (10), who reported
that rat AFP inhibited the growth of an estrogen-dependent
syngeneic rat mammary tumor as well as an estrogen-dependent
rat pituitary tumor. We find that this effect of AFP is not specific
for malignant tissue in that E2-stimulated growth of normal
mouse uterus was also inhibited by AFP. Similarly, Pool et a!.
(20) showed that uterine weight was decreased and that castra-
tion cells were present in the anterior pituitaries of rats bearing
an AFP-secreting rat hepatoma. In addition, Sparks and Grubbs
(21) reported that AFP suppressed the estrogen-induced prolif-
eration of mouse vaginal epithelium. The experiments described
herein showed lack of species specificity in the action of AFP in
that human AFP inhibited the estrogen-stimulated growth of
both normal mouse uterus and of human breast cancer xe-
nografts.
This effect of AFP was dose-dependent and reached its
maximum effect at 100 p.g/mouse/day. That dose resulted in a
blood AFP level which was well below the fetal physiological
range of AFP, since normal fetal blood levels of AlP are -1-3
mg/ml (1). This may very well explain why no evidence of
toxicity, such as weight loss or reduction in activity, was seen in
mice that received this dosing regimen of AFP. It also suggests
that AFP would be well tolerated if it was applied as a thera-
peutic agent in humans. The mechanism of the antiestrotrophic
effect of AFP is not clear at this time. Sequestering of estrogen
by AFP is unlikely because human AFP does not have a high-
affinity binding site for estrogen (19). In fact, the AFP found in
most species other than rodents (22) does not have a high-
affinity binding site for estrogen (23). In our own studies, we
found that human AFP did not compete with estrogen for
binding to the estrogen receptor (Fig. 6). Thus it would seem
that there is a point downstream from the binding of estrogen to
its receptor where AlP, or a signal emanating from AFP asso-
ciating with its receptor, interferes with a signal coming from
the receptor-bound estrogen. Additional study of this area is
ongoing. The fact that AFP does not behave as a classical
estrogen receptor antagonist may offer advantages in that it may
be additive or synergistic in combination with classical estrogen
receptor antagonists, or it may be effective against estrogen
receptor-positive tumors that have become resistant to the clas-
sical estrogen receptor antagonists. However, it does not appear
Inhibitor Concentration (nM)
Fig. 6 Effect of AFP on binding of estradiol to its receptor. Rabbit
uterine cytosol was used as a source of estrogen receptor. All incuba-
tions were performed in triplicate, each containing 100 p.1 ofcytosol, 20
�.tl of 10 nM 6,7-3H estradiol (50 Ci/mmol), and 80 p.1 of test agents atthe final concentrations indicated on the abscissa. E, LY 156758, a pure
estrogen receptor antagonist, was kindly provided by Eli Lilly Co.
(Indianapolis, IN); 0, human albumin was purchased from Sigma
Chemical Co. (St. Louis, MO): �, AlP was purified from HepG2 cellsas described in “Materials and Methods.” Details of the assay are
described in “Materials and Methods.” Concentration of [3HIE,-com-
plex with receptor in the presence of different concentrations of test
agents is expressed as a percentage of the amount of complex formed in
the absence of test agent.
that AFP will impact on the estrogen receptor-negative breast
cancers, which are some of the most difficult breast cancers to
treat with chemotherapy.
Several reports suggest that AFP interferes with estrogen-
induced responses in normal and malignant tissues in humans.
For example, premenopausal women with AFP-secreting hepa-
tomas experience amenorrhea (24, 25) and elevations in serum
E, (26). After surgical removal of the hepatoma, normal
monthly menstruation resumes and serum E, returns to normal
levels. Elevations of E2 in women with AFP-secretimg hepato-
mas are in agreement with our results and can be explained by
AFP-imduced interference with the E, feedback response at the
hypothalamic-pituitary axis, which would normally reduce fol-
licle-stimulating hormone secretion and maintain a steadier
blood level of E2. With regard to the effects of AFP on human
malignancies, the growth of most human breast cancers, at least
in their early stages, is stimulated by estrogen, and interference
with that response has been a therapeutic strategy in the man-
agement of this disease. That AlP may be playing a role in the
control of this disease is supported by several reports in the
literature, which, when taken together, suggest that individuals
who experience elevations in AFP are subsequently at reduced
risk for breast cancer. These reports are summarized in Table 3.
For example, women who have experienced full-term pregnancy
have had elevations of AFP in their serum (27), and subse-
quenthy they have been found to be at reduced risk of breast
cancer when compared with women who have never experi-
enced pregnancy (28). Furthermore, there are factors in preg-
nancy, such as maternal race, weight, hypertension, number of
fetuses in utero, and neural tube defect in the fetus, where
maternal serum (MS) AFP is substantially elevated above nor-
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Clinical Cancer Research 2883
Table 3 Association of pregnancy AFP levels with subseque nt risk of breast cancer
Maternal conditionMaternal serum
AFP concentration
Maternal lifetimebreast cancer risk1 2
Pregnant Nonpregnant
Pregnant, black Pregnant, whitePregnant, lean Pregnant, obesePregnant, hypertensive Pregnant. normotensivePregnant with multiple fetuses Pregnant with a single fetus
Pregnant, fetus with neural tube defect Pregnant, fetus no neural tube defectPregnant, consuming no alcohol Pregnant, consuming alcohol
1 > 2 (27)”
1 > 2 (29)
1 > 2 (29)
1 > 2 (32)
1 > 2 (34)
I > 2 (36)
1 > 2 (38)
1 < 2 (28)”
1 < 2 (30)
1 < 2 (31)
1 < 2 (33)
1 < 2 (35)
1 < 2 (37)
1 < 2 (39)a Numbers in parentheses are the reference sources for the data.
mal pregnancy levels (27, 29, 32, 34). In all of these cases, we
noticed a consistent and striking association: the risk of breast
cancer is lower than when pregnancy produces a normal eleva-
tion in MSAFP (30, 31, 33, 35, 36). In one situation (alcohol
abuse during pregnancy) where MSAFP is lower than in a
normal pregnancy (37), the breast cancer risk is higher than that
found in women who have experienced a normal pregnancy
(38). Recently, Richardson et a!. (39) reported measuring the
association more directly. They found that the concentration of
AlP in cryogenically stored maternal sera was inversely corre-
lated to the risk of breast cancer in these mothers 20-30 years
after their pregnancies. We speculate that the AFP that crosses
the placenta and enters the maternal circulation extinguishes
microscopic premalignant foci that later on in life would be
promoted with the help of estrogen into clinically detectable
breast cancers.
AlP seems to be a protein with an attractive potential for
further development into an agent that could be used in the
prevention and treatment of breast cancer. Recombinant prepa-
rations of this molecule have been made, and these preparations
have retained the biological activities found with natural AlP
(13, 40). Recently, Festin et a!. (41) reported on the production
of the third domain of AFP through recombinant DNA tech-
niques, and Mizejewski et a!. (42) have synthesized a peptide
derived from the third domain of AlP; both of these prepara-
tions possess the biological activities found in the natural, full-
length protein molecule. As these preparations become more
available and as insights into their structure and into the mech-
anism of the antiestrotrophic activity of AFP are developed, the
use of this protein for the treatment and prevention of human
breast cancer becomes more of a possibility.
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xenografts.growth of estrogen-dependent human breast cancer Alpha-fetoprotein derived from a human hepatoma prevents
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