(CANCER RESEARCH 40, 3095-3098. September 19801
0008-5472/80/0040-0000$02.00
SynergisticEffect of ChronicProlactinSuppressionand Retinoid
Treatment in the Prophylaxisof N-Methyl-N-nitrosourea.induced
MammaryTumorigenesisin Female Sprague-DawleyRats
Clifford W. Welsch,' Carolyn K. Brown, Margaret Goodrich-Smith,
Jane Chiusano, and Richard C. Moon2
Department of Anatomy, Michigan State University, East Lansing, Michigan 48824 (C. W. W., C. K. B.. M. G-S., J. C.] and lIT Research Institute, Chicago,
Illinois 60616(R. C. M.j
ABSTRACT
Two hundred forty Sprague-Dawley rats were treated i.v.
with 2.5 or 1.25 mg of N-methyl-N-nitrosourea (MNU) per 100
g body weight at 50 and 57 days of age. At 60 days of age,
rats given either dose were divided into 4 groups (30 rats/
group) and treated as follows: Group 1, controls; Group 2, 0.4
mg 2-bromo-a-ergocryptine (CB-154) per 100 g body weight
injected s.c. once daily; Group 3, retinyl acetate (328 mg/kg
diet) fed daily; and Group 4, CB-154 and retinyl acetate treat
ments combined. Rats that received the 2.5-mg dose of MNU
were treated for 129 days; those that received the 1.25-mg
dose of MNU were treated for 175 days. The rats that were
treated with the high dose of MNU were maintained without any
treatment for an additional 13 weeks, after which they were
sacrificed. The rats that were treated with the low dose of the
carcinogen were sacrificed immediately after treatment. All rats
were palpated once weekly for palpable mammary tumors. The
number of rats with mammary tumors and the total number of
mammary tumors at cessation of treatments were, respectively,
as follows. MNU (2.5 mg): Group 1, 22 of 30 (73%), 82; Group
2, 11 of3O(37%), 17;Group3, 11 of3O(37%), 19;Group4,
2 of 30 (7%), 2. MNU (1 .25 mg): Group 1, 8 of 30 (27%), 14;
Group 2, 4 of 30 (13%), 5; Group 3, 3 of 30 (10%), 4; Group
4, 0 of 30, (0%), 0. Thus, chronic CB-154 treatment or retinyl
acetate feeding markedly reduced the percentage of rats bear
ing mammary tumors and the total number of mammary tumors.
The combined treatments were superior to either treatment
alone, inasmuch as mammary tumorigenesis was nearly com
pletely blocked in the rats of Group 4 that received the 2.5-mg
dose of MNU and was totally blocked in the rats of Group 4
that received the 1.25-mg dose of MNU. Retinyl acetate feeding
or CB-154-induced prolactin suppression appear to be equally
effective treatments in the prophylaxis of MNU-induced mam
are most efficacious when used shortly after carcinogen treat
ment; thus, they appear to act primarily at the level of the
incipient neoplasm, and they are not particularly effective when
administered to animals in advanced stages of the disease.
Prolactin is an important hormone for the development of
polycyclic hydrocarbon-induced mammary carcinomas in rats,
inasmuch as drug-induced suppression of this hormone shortly
after carcinogen treatment results in a significant reduction in
tumor incidence (9, 12). The effect of this pituitary peptide on
the genesis of MNU-induced rat mammary carcinomas, a re
cently developed model which reportedly is more closely akin
to human breast cancer than is the polycyclic hydrocarbon rat
model (1), has not been reported. Thus, the purpose of this
study is to ascertain whether or not prolactin is important in the
genesis of MNU-induced rat mammary carcinomas and if so,
whether chronic prolactin suppression in conjunction with ret
inoid treatment will be more effective than either treatment
alone. An interaction of retinoid treatment and endocrine ther
apy has not heretofore been studied, although an inverse
relationship between steroid and retinoid receptors in ovarian
dependent and -independent tumors has been reported (4).
MATERIALS AND METHODS
Two hundred forty female Sprague-Dawley rats (Spartan
Research Animals, Inc., Haslett, Mich.) were divided into 8
groups, housed in a temperature-controlled (24 ± 1O) and
light-controlled (14 hr/day) room, and given a diet of Wayne
laboratory meal (Allied Mills, Inc., Chicago, Ill.) and water ad
!ibitum. The vitamin A content of the Wayne laboratory meal is
15 IU of the vitamin per g of ration. All rats were treated i.v.
with either 1.25 or 2.5 mg MNU per 100 g body weight at 50
and 57 days of age. MNU (Ash Stevens, Inc., Detroit, Mich.)
mary tumorigenesisin rats; the combinedmodality,however, was dissolved in 0.9% NaCl solution (10 mg/mI) acidified (pH
4 to 5) with acetic acid. Pituitary prolactin secretion was
appears to be far superior than either treatment alone.
suppressed by daily s.c. injections of CB-154 (0.4 mg/i 00 g
body weight) beginning at 60 days of age. CB-154 solution
INTRODUCTION
was prepared by dissolving the ergot alkaloid in a minimal
In recent years, increased attention has been turned toward amount of 100% ethanol and diluting with 0.9% NaCI solution
the experimental use of retinoids for the chemoprevention of so that the final concentration was 2.0 mg CB-i 54 per ml.
carcinogenesis. The vitamin A analogs have been very effective Control rats and rats fed the retinoid diet alone were given daily
in suppressing the incidence of a number of experimental injections of the diluent only. Retinoid treatment was begun at
epithelial neoplasms (7), including mammary carcinomas in
60 days of age and was accomplished by blending retinyl
duced by MNU3 and DMBA in rodents (2, 3, 8). The retinoids acetate into the stock diet in the form of stable gelatinized
beadlets at a concentration of 328 mg retinyl acetate per kg
I Recipient
of NIH Contract
NOl -CB-74090.
To whom requests
for reprints
diet. Control rats and rats treated with CB-i 54 alone were fed
should be addressed.
2 RecipIent
of NIH C,ontract
NOl -CB-74207.
gelatinized beadlets without retinyl acetate. The rats that re
3 The
abbreviations
used
are:
MNU,
N-methyl-N-nitrosourea;
DMBA,
7,12
ceived the 1.25-mg dose of MNU (Groups 5 to 8) were treated
dimethylbenzanthracene; CB-154. 2-bromo-a-ergocryptine.
Received January 28, 1980; accepted May 15, 1980.
for 175 days and then were sacrificed. The rats that received
SEPTEMBER1980
3095
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C. W. We!sch et a!.
the 2.5-mg dose of MNU (Groups 1 to 4) were treated for 129
days; treatment was then discontinued, and the rats were
maintained on a standard rat chow (Wayne Lab-Blox) for a
period of 13 weeks.
Beginning 1 month after carcinogen treatment, all rats were
weighed and palpated once weekly for mammary tumors. Blood
was obtained from each rat in Groups 5 to 8 by decapitation
and analyzed by radioimmunoassay for prolactin (National In
stitute of Arthritis, Metabolism, and Digestive Diseases rat
prolactin radioimmunoassay kit). Ovaries, uteri, adrenals, and
pituitaries were removed and weighed from rats of Groups 5 to
8. Inguinal mammary glands were excised from rats of Groups
5 to 8 and prepared for wholemount evaluation. Mammary
tumors were removed from rats of Groups 1 to 8, stained with
hematoxylin and eosin, and examined histologically. Mean
differences between blood prolactin levels, mean latency pe
nod of mammary tumor appearance, and organ weights were
evaluated statistically by Student's t test. Mammary tumor
incidence was analyzed by x2 analysis. For an outline of the
experimental design, see Table 1.
imately two-thirds the total number of palpable mammary tu
mors (Table 1). The percentage of rats with mammary tumors
was also substantially reduced by these treatments. Combined
prolactin suppression and retinoid treatment completely
blocked the development of mammary tumors; no tumors were
observed during the 175-day treatment period.
Blood prolactin levels were sharply reduced in the rats
treated with CB-i 54 [21 .6 ±1.2 (S.E.) ng/mlj when compared
with those of rats treated with retinoid (54.1 ±18.4) and of
controls (69.0 ±14.9). No significant difference was observed
in blood prolactin levels between control and retinoid-treated
rats. Mean ovarian weight (mg/i 00 gm body weight) was
increased (83.6 ±0.3) and pituitary weight was decreased
(3.4 ±0. i ) in the CB-i 54-treated rats when compared with
1Sf
l4(
I3(
l2(
RESULTS
ll(
Rats Treated with 2.5 mg MNU (Groups I to 4). Chronic
suppression of prolactin secretion or retinoid feeding reduced
by nearly four-fifths the total number of palpable mammary
tumors and reduced by one-half the number of rats bearing
mammary tumors (Table 1). Combined prolactin suppression
and retinoid feeding nearly completely blocked the develop
ment of mammary tumors; only 2 of 30 rats developed a total
of 2 mammary tumors. The mean latency period of mammary
tumor appearance (days after carcinogen treatment) was not
significantly affected by CB-i 54 or retinoid treatments. Histo
logically, nearly all the palpable mammary tumors were ade
nocarcinomas or papillary carcinomas. The histological types
of tumors which developed during treatments were similar to
those which developed after treatment. Upon cessation of
retinoid plus CB-i 54 treatment, mammary tumors developed
at a rate comparable to tumor development in the control group
(Chart 1). The same phenomenon occurred upon cessation of
treatments in the groups treated with CB-i 54 (Chart 2) and
retinoid (Chart 3).
Rats Treated with I .25 mg MNU (Groups 5 to 8). Chronic
prolactin suppression or retinoid treatment reduced by approx
lOC
90
coNT@
@30—
+-A@k@W64
I@ oays
I
.
80
@imber
of
M@
Tumors
70
6C
Sc
4c
3C
(3°—
2C
.
IC
7
9
II
3 15 l7 l92l
2325272931
@eks
after MNU
Chart 1. Effect of CB-i 54 treatment plus retinyl acetate on MNU-induced
mammary tumorigenesis in female Sprague-Dawley rats. p < 0.005 for the
number of mammary tumors for controls versus CB-i 54 plus retinyl acetate-fed
rats.
Table 1
Effect of CB-154 and/or retinyl acetate treatments on MNU-induced mammary tumorigenesis in female
Sprague-Dawley rats
8were
Beginning 3 days after carcinogen treatment, Groups 1 to 4 were treated for 129 days, Groups 5 to
p< treated for 175 days. Tumor incidence at cessation of retinyl acetate and/or CB-154 treatments:
versusGroup
0.005 for Group 1 versus Groups 2, 3, and 4; p < 0.05 for Group 2 versus Group 4, Group 3
4, and Group 5 versus Group 7; p < 0.01 for
8.Treatment
Group 5 versus Group
ofGroup
tumors1
No. of rats
MNU
(mg)
30
30
30
30
30
30
30
30
2.50
2.50
2.50
2.50
1.25
1.25
1.25
1.25
822
(controls)
173
194
25
146
(controls)
57
48
0a
Numbers
3096
in parentheses,
Retinyl
acetate CB-154
—
—
+
+
—
—
+
+
—
+
—
+
—
+
—
+
No. of rats with
tumors
Total no.
22 (73)a
11 (37)
11 (37)
2(7)
8 (27)
4(13)
3(10)
0(0)
percentage.
CANCERRESEARCHVOL. 40
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MNU-induced Mammary Tumorigenesis
14C
.
131
12C
CONTROL
t30 rats/gro,.@l
I II
nocebo
or
c8-i54
a
lOC
I@
days
I
91
,
29@
S
SI
S
Numberof
Mammary
Tumors
7C
6C
5C
4C
C8-54
(30rots/@oup@
3C
a
or adrenal gland weights. Mammary gland development in the
control rats was characterized by numerous ducts with exten
sive branching, many end buds, and a small amount of alveoli.
Slightly less duct, end bud, and alveoli development was ob
served in the mammary glands of the retinoid- and/or CB-i 54treated rats. Fewer mammary gland hyperplastic nodules were
observed in the retinoid- and/or CB-i 54-treated rats when
compared to controls. CB-i 54 treatment did not significantly
affect body weight gains. Retinoid treatment reduced body
weights, when compared with control and CB-i 54-treated rats
attermination ofthe study, by i 3% (p < 0.05). Retinoid-treated
rats consumed approximately 18 g of ration per day, in contrast
to the control rats who consumed approximately 20 g of ration
per day. The retinoid-fed rats consumed approximately 6 mg
of retinyl acetate each day. The only apparent undesirable
anomalies in the treated rats were in the retinoid-fed animals.
Many
of these
animals
had scaly
tails,
roughened
coats,
and
pale livers.
DISCUSSION
S
IC
‘79
II
13 5
7
9 21 23 25 27 29 31
Weeksafter MNU
Chart 2. Effect of CB-1 54 treatment on MNU-induced mammary tumorigene
sis in female Sprague-Dawley rats. p < 0.005 for the number of mammary tumors
for controls versus CB-154-treated rats.
ISO
S
I4C
S
l3(
12(
I IC
or
lOC
9C
4—Relinyi-ocetate
l3do@s
I
//
—p
i29doys
S
SC
S
t@kirnberof 7C
Mam@y
Tumors
S
60
Retinyl-ocetaie
(3Ori3t$/@
50
4'
30
20
S
10
@7
It
3 5 i7
9 21 23 25 27 29 3i
Weeks after MNU
Chart 3. Effect of retinyl acetate feeding on MNU-induced mammary tumori
genesis in female Sprague-Dawley rats. p < 0.005 for the number of mammary
tumors for controls versus retinyl acetate-fed rats.
those in control rats (ovarian weight, 22.7 ±0.2; pituitary
weight, 7.7 ±0.2; p < 0.OOi ). Uterine and adrenal gland
weights were not affected by CB-i 54 treatment. Retinoid treat
ment did not significantly affect mean ovarian, uterine, pituitary,
SEPTEMBER
It has been previously reported that CB-i 54-induced inhibi
tion of prolactin secretion has a marked inhibitory effect on the
development and growth of polycyclic hydrocarbon-induced
mammary tumorigenesis in female rats (9, i 2). It appears that
this pituitary peptide is also important in the developmental
phases of MNU-induced mammary tumorigenesis as well. The
number of rats bearing mammary tumors was reduced by 50%
and total number of tumors was reduced by two-thirds to four
fifths in rats treated with CB-i 54 shortly after carcinogen
treatment. An important role for prolactin in the developmental
phases of this experimental mammary tumor model has not
heretofore been reported. It is important to note, however, that
the antitumorigenic effect of CB-i 54 treatment is apparent only
during drug treatment. Approximately 2 weeks prior to cessa
tion of treatment and when the treatment was discontinued,
mammary tumor incidence began to increase (Chart 2). This
slight increase in tumor incidence during the last 2 weeks of
treatment suggests a selection process for prolactin-insensitive
mammary tumors. A previous report has shown that ovariec
tomy of rats either before or shortly after MNU treatment also
sharply reduced the number of mammary tumors (1). It ap
pears therefore, that the MNU-induced rat mammary tumor
model
is a hormonally
responsive
neoplasm
qualitatively
similar
to the polycyclic hydrocarbon rat models.
Retinyl acetate feeding has previously been reported to
suppress the development of MNU-induced mammary cancers
in rats (3). Our results confirm this study. In addition, we have
shown that upon cessation of retinyl acetate feeding, mammary
tumor incidence rate is comparable to that observed in controls
(Chart 3). Thus, the antitumorigenic activities of retinyl acetate
were apparent only when the animals were fed the retinoid. No
enduring antitumor effects after cessation of retinoid treatment
were apparent. It should be pointed out that during the last 2
weeks of retinoid treatment, a substantial increase in tumor
incidence was also noted. Once again, as in the CB-i 54treated rats, a selection for insensitive tumors appears to be
occurring.
Although chronic CB-i 54 treatment or retinoid feeding alone
was effective in markedly reducing mammary tumor incidence,
the combined modality was superior to either treatment alone.
1980
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1980 American Association for Cancer Research.
3097
C. W. We!sch et a!.
The combined treatment completely blocked mammary tumor
igenesis in rats treated with the low dose of MNU (i .25 mg)
and only 2 mammary tumors developed in 30 rats treated with
the higher dose (2.5 mg) of the carcinogen. To our knowledge,
no one has previously attempted to enhance the antitumori
genic efficacy of retinoid feeding by concurrent endocrine
therapy. It is clear from the results of our study that a synergism
between these 2 distinctly different types of therapeutic mo
dalities does occur in the MNU-induced rat mammary tumor
model.It is also apparentthat the antitumorigeniceffect of the
combined modality is effective only during treatments; upon
cessation of treatments, the rate of mammary tumor develop
ment is comparable to placebo-fed control rats (Chart i).
When one compares retinyl acetate feeding with chronic CB
i 54-induced prolactin suppression on the general well-being
of the animal, it is clear that the ergot treatment is considerably
superior to retinoid treatment. CB-i 54 is an effective specific
inhibitor of prolactin secretion; it does not significantly interfere
with other endocrine processes at the dose levels used in this
study(6, 10). Theergot-treatedrats resembledthe controlrats
in every respect; the only major disparity was observed in the
mammary gland, i.e. , there was a conspicuous reduction or
absence of neoplasia in the prolactin-suppressed animals.
[Prolactin-suppressed rats have enlarged ovaries but normal
estrous cycles (1 1) because of decreased prolactin luteolysis.]
In contrast, the retinyl acetate-fed animals had a slight but
significant reduction in body weight gains and a general insa
lubrious condition characterized by mildly roughened coat,
scaly tail, and pale liver. The reduced body weight gains
observed in the retinoid-treated rats (i 3%) are believed to be
of insufficientmagnitudeto significantlyimpedemammarycar
cinogenesis. Since the onset of retinoid treatment occurred 3
days after carcinogen treatment, it is very unlikely that the
retinoids influenced hepatic catabolism of the carcinogen.
Nevertheless, the degree to which the retinoid-induced mild
insalubrity influenced the carcinogenic process in this study
cannot
be resolved
at this time.
Increased
success
in devel
oping synthetic retinoid analogs that are less noxious than
retinyl acetate yet possess comparable antitumorigenic quali
ties is essential if the vitamin A analogs are to have any
3098
significant practical application. Once these analogs are de
veloped, and noted progress is being made in this area (5),
combined endocrine and retinoid therapies in the prophylaxis
of breast carcinogenesis in humans may be a reality.
ACKNOWLEDGMENTS
We thank Dr. Richard L. Elton, Sandoz Pharmaceuticals, East Hanover, N. J.,
for a generous supply of CB-154 and Hoflmann-La Roche Inc., Nutley, N. J., for
a generous supply of retinyl acetate and placebo beadlets.
REFERENCES
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1 1 . Weisch,
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CANCERRESEARCHVOL. 40
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1980 American Association for Cancer Research.
Synergistic Effect of Chronic Prolactin Suppression and
Retinoid Treatment in the Prophylaxis of N-Methyl-N
-nitrosourea-induced Mammary Tumorigenesis in Female
Sprague-Dawley Rats
Clifford W. Welsch, Carolyn K. Brown, Margaret Goodrich-Smith, et al.
Cancer Res 1980;40:3095-3098.
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