1. No category

Reversal of the Antitumor Effects of Tamoxifen by Progesterone
in the 7,12-Dimethylbenzanthracene-induced Rat Mammary
Carcinoma Model
Simon P. Robinson and V. Craig Jordan
Cancer Res 1987;47:5386-5390.
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(CANCER RESEARCH 47, 5386-5390, October 15, 1987]
Reversal of the Antitumor Effects of Tamoxifen by Progesterone in the 7,12Dimethylbenzanthracene-induced Rat Mammary Carcinoma Model1
Simon P. Robinson and V. Craig Jordan2
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792
ABSTRACT
( »administrationof progesterone (4 mg/day) opposed the antitumor
activity of tamoxifen (100 Mg/day) in rats bearing 7,12-dimethylbenzanthracene-induced tumors and also partially prevented the inhibition by
tamoxifen (SOfig/day started 30 days after 7,12-dimethylbenzanthracene
administration) of tumor occurrence even after tamoxifen therapy had
been established for 1 or 2 mo. Although prolonged progesterone treat
ment raised progesterone levels, serum total estrogen levels were not
raised above control. The reversal by progesterone of the inhibition of
tumor occurrence produced by tamoxifen was blocked by the antiprogestin RI 486. These results demonstrate that progesterone can reverse the
tumoristatic action of tamoxifen in the 7,12-dimethylbenzanthraceneinduced tumor model and that this may be via a progesterone receptormediated mechanism.
INTRODUCTION
The antiestrogen tamoxifen has become the antisteroidal
therapy of choice for the treatment of hormone-dependent
breast cancer (1). However, although tamoxifen is as effective
as the previously used ablative and additive therapies (1), failure
ultimately occurs, even in cases where tamoxifen has produced
a long initial period of tumor remission (1). A number of
possible mechanisms resulting in tumor relapse have been pro
posed (2), but at present the main cause remains unclear.
Combinations of tamoxifen with other forms of antihormone
therapy have not been shown to have any clinical advantage
over tamoxifen alone (1). This suggests that, during tamoxifen
therapy, tumors lose the requirement of hormone stimulation
to maintain tumor growth. However, contrary to this are the
reports that more than half the patients who initially respond
to tamoxifen alone and subsequently relapse have objective
remission following hypophysectomy, androgen therapy (3), or
aminoglutethimide treatment (4, 5).
Recently the sequential use of tamoxifen and progestin has
been investigated (6, 7). The rationale for this is that tamoxifen
has been observed to increase the concentration of progesterone
receptors in breast tumors (8,9) and thus may prime or sensitize
the tumor to high-dose progestin therapy.
However, the increase in progesterone receptor produced by
tamoxifen could also be involved in the therapeutic failure of
this antiestrogen. Low doses of progesterone have been dem
onstrated to produce a positive stimulus in rat mammary tu
mors induced by carcinogens (10, 11) or in the transplanted
MTW9 carcinoma (12). Furthermore, in the mouse, progester
one has been implicated as a factor in the development of
spontaneous mammary tumors during pregnancy (13, 14) and
mammary tumors induced in inbred strains by hypophyseal
isografts (15). The combination of estrone and progesterone
Received 5/6/87; revised 7/22/87; accepted 7/24/87.
The costs of publication of this article were defrayed in part by the payment
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.
1This work was supported through the generosity of the Wisconsin Clinical
Cancer Center Anderson Fund.
1To whom requests for reprints should be addressed, at Department of Human
Oncology, Wisconsin Clinical Cancer Center, 600 Highland Ave., Madison, WI
53792.
has been demonstrated to induce tumors in ovariectomized GF
mice, whereas neither is effective alone (16).
The progestational content of the contraceptive pill has been
related to the risk of breast cancer in humans ( 17,18). Although
this finding is controversial (19), the combination with animal
data is suggestive that progesterone may play a part in breast
cancer growth.
The increase in progesterone receptor number produced by
tamoxifen (6, 7) could therefore prime for endogenous progestins and result in tumor stimulation. In support of this hypoth
esis is the report that low doses of progestin therapy in combi
nation with tamoxifen are less effective for breast cancer therapy
than tamoxifen alone (20). Furthermore, consistent with this
hypothesis, tumors failing tamoxifen therapy would still be
hormone dependent, and thus the reports of objective responses
produced by other endocrine therapies (3-5) after cessation of
tamoxifen treatment would be explained.
In order to investigate these observations we examined the
influence of progesterone on tamoxifen therapy of mammary
tumors in rats exposed to DMBA.3
MATERIALS AND METHODS
DMBA and progesterone were obtained from the Sigma Chemical
Company (St. Louis, MO). Tamoxifen was a gift from Stuart Phar
maceuticals (Wilmington, DE), and RU 486 was a gift from Roussel
UCLAF (Romaineville, France).
Drug administration was by s.c. injection into the loose fold of skin
on the back of the neck. Progesterone and tamoxifen solutions were
prepared by adding a small volume of ethanol to crystalline material
and then stirring with peanut oil. The progesterone solution was
wanned to aid solubility. The ethanol in the tamoxifen solution was
evaporated under N2 once in solution; however, the ethanol (3 to 5%,
v/v) was required in the progesterone solution to prevent crystallization.
RU 486 was prepared as a microcrystalline suspension in peanut oil
which was vigorously mixed before injection. DMBA, dissolved in
peanut oil by stirring overnight, was administered (20 mg in 2 ml) by
gavage to 50-day-old female Sprague-Dawley rats (HarÃ-an SpragueDawley, Indianapolis, IN) as previously described (21).
Progesterone Interaction with the Inhibition of Tumor Growth by
Tamoxifen. Animals from a pool of rats treated with DMBA were
randomly entered into 4 groups when tumors reached measurable size
(diameter, >1 cm). Rats were entered into the groups between 80 and
1SOdays after DMBA. The 4 groups were treated daily for the duration
of the experiment with (a) peanut oil, (/>)4 mg progesterone, (c) 100
nK tamoxifen, and (d) 4 mg progesterone and 100 fig tamoxifen. Tumor
sizes were determined for 11 wk by caliper measurements and calculated
using the formula, length/2 x width/2 x tr.
Progesterone Interaction with the Inhibition of Tumor Development
by Tamoxifen. Thirty days after DMBA administration, rats were
randomized into groups of 25 animals. In an initial experiment 4
groups were treated daily with (c) peanut oil alone, (/) 4 mg proges
terone, (g) 50 UKtamoxifen, and (h) 4 mg progesterone and 50 /ig
tamoxifen. Treatment was given for about 12 wk. Animals were pal
pated at weekly intervals. Groups g and h were followed for about 16
wk after treatment stopped.
In a further study, 4 groups were treated daily with peanut oil alone
(i) or containing 50 ¿igtamoxifen (/, k, and /). After 1 mo, Group j
3The abbreviation used is: DMBA, 7,12-dimethylbenzanthracene.
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TAM AND PROG
received additional treatment with 4 mg progesterone. After 2 mo,
Group k received additional treatment with 4 mg progesterone. Animals
were palpated at weekly intervals.
The Influence of an Antiprogestin on Progesterone Interaction with
the Inhibition of Tumor Development by Tamoxifen. Thirty days after
DM HA administration, 100 rats were randomized into 4 groups of
equal size and treated with peanut oil alone (in) or containing SO Mg
tamoxifen (n, o, and p). After 1 mo, Group o received additional
treatment with 4 mg progesterone, and Group p received additional
treatment with 4 mg progesterone and 5 mg RU 486. Animals were
palpated at weekly intervals to detect tumors.
Hormone Levels and Uterine Weights. Circulating hormone levels
and uterine weights were measured in rats chronically treated (approx
imately 12 wk) with peanut oil, tamoxifen (SO ¿ig/day),or tamoxifen
(50 /ig/day) and progesterone (4 mg/day). Blood samples were taken
approximately 4 h after the final injection by ether anesthesia and
bleeding from the abdominal aorta regardless of the stage in the estrus
cycle. Blood was allowed to clot overnight at 4"( ' and then centrifugea
at 2000 x g, and the serum was removed and stored at -20*C until
500r A.
400-
o
1*
analyzed. Progesterone and total estrogen measurements were made
using commercially available radioimmunoassays designed for human
samples. Progesterone was measured on 50-^1 serum samples by the
progesterone coat-a-count (Diagnostic Products, Los Angeles, CA),
ovariectomized rat serum was used as a test control, and spiked samples
were used to validate the assay (data not shown). Total estrogen
measurements were made on 300-^1 serum samples using RSL I2SI-
o i 25456789
10 II
0
I
23456789
10 II
Weeks
Weeks
500
labeled total estrogen radioimmunoassay (Radioassay Systems Labo
ratories, Inc., Carson, CA). This assay had a low standard of 5 pg/ml,
and the intraassay variation at 53 pg/ml was given as a coefficient of
variation of 5.5%.
Statistical Analysis. The rate of change of tumor size between groups
was analyzed using analysis of variance decomposition (22). The Stu
dent / test was used for other comparisons. Significance was recognized
if P < 0.05.
RESULTS
Progesterone Interaction with the Inhibition of Tumor Growth
by Tamoxifen. The administration of tamoxifen (100 jig/day)
to tumor-bearing rats produced a reduction in tumors to <20%
of their initial size in 10 of 15 tumors and stasis of growth in a
further 3 tumors (see Fig. 1C). This contrasts to the peanut oiltreated control animals in which only 5 of 16 tumors noticeably
decreased in size, and the majority of tumors grew (see Fig.
\A). In the progesterone-treated group a similar pattern of
tumor progression to that of the control group was observed
but with fewer regressing tumors (see Fig. IB). The combina
tions of tamoxifen and progesterone produced a tumor response
midway between the tamoxifen alone- and progesterone alonetreated groups. Tumor regression in the combined treatment
group not only occurred in fewer tumors than that of the
tamoxifen alone group (8 of 18), but also their regression was
less rapid. Furthermore, 7 of the 18 tumors grew very rapidly
(see Fig. ID). Analysis of the rate of change of tumor size
(change in size divided by time span from assignment to ne
cropsy) between treatment groups using analysis of variance
decomposition shows a significant interaction between tamox
ifen and progesterone (P < 0.05) with progesterone partially
reversing the tumor regression produced by tamoxifen.
Progesterone Interaction with the Inhibition of Tumor Devel
opment by Tamoxifen. The treatment with progesterone (4 mg/
day) 30 days after DMBA administration resulted in more (see
Fig. 2) and larger tumors (see Fig. 3) developing in the following
8 wk than in the control group. In contrast, tamoxifen (50 fig/
day) treatment over this same period almost totally prevented
tumor occurrence and development (see Figs. 2 and 3). The
tumor burden in the tamoxifen-treated group was significantly
I 23456789
Weeks
10 II
Weeks
Fig. 1. Effect of tamoxifen and progesterone treatment alone or in combina
tion on the growth of individual DMBA-induced rat mammary tumors. Rats
bearing measurable tumors (diameter, >l cm) were randomly entered into 4
groups and treated daily as follows: peanut oil alone (, i ); 4 mg progesterone (B);
100 ¡igtamoxifen «): 100 /¿gtamoxifen and 4 mg progesterone (/>). All drugs
were administered separately by injection (s.c.) in peanut oil (0.1 ml). The limit
of measurable tumor size is indicated by the hatched area. Analyzing the rate of
change of tumor size between treatment groups using analysis of variance decom
position shows a significant interaction between tamoxifen and progesterone (/'
< O.OS)with progesterone partially reversing the tumor regression produced by
tamoxifen.
different after 8 wk of treatment when compared to control (P
< 0.0001). This antitumor action of tamoxifen was reversed by
the simultaneous administration of progesterone which resulted
in significantly (P < 0.01) more and larger tumors per rat (see
Figs. 2 and 3) and more rats developing tumors than the
tamoxifen alone group.
Cessation of tamoxifen treatment alone after 13 wk resulted
in a loss of the protective antitumor action and the development
of tumors over the next 16 wk (see Fig. 2). Similarly, stopping
progesterone combined with tamoxifen resulted in tumor de-
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Research.
TAM AND PROG
15 13
9.421*1^I••1•i1
•
3.0
8765*£
,.!t*•-i4•
4-Ã-¿UJ
3MCO0r-mediumsmallpoint
'.
Weeks from Start of Treatment
Fig. 2. Effect of tamoxifen and progesterone treatment alone or in combina
tion on the occurrence of rat mammary tumor induced by DMBA. Rats were
administered DMBA 30 days prior to drug treatment. Groups of 25 animals were
injected (s.c.) daily with tamoxifen (50 Mg: O), progesterone (4 mg; •).or
tamoxifen (50 Mg)and progesterone (4 mg; •).All drugs were given separately in
peanut oil (0.1 ml). Control (O) animals received peanut oil alone. Tumor burden
was calculated as the number of tumors per group divided by the number of rats.
Treatment period is indicated by the hatched box. Treatment was stopped in the
tamoxifen and tamoxifen plus progesterone groups at the time indicated by the
arrow.
velopment, although a greater mean tumor burden per animal
was observed by wk 28. However, the animal number in the
combined treatment group was less (14 animals at wk 28) than
the tamoxifen alone group (20 animals at wk 28) due to animal
loss resulting from the earlier development of tumors in this
group.
The reversal of the inhibitory action of tamoxifen (50 Mg/
day) on tumor growth by progesterone (4 mg/day) not only
occurred if the drug administration was started concurrently
but also was observed if progesterone (4 mg/day) was admin
istered when tamoxifen (SO ¿ig/day)treatment had been estab
lished for 1 or 2 mo (see Fig. 4). The addition of progesterone
after 1 mo of tamoxifen therapy caused not only a significantly
(/' < 0.001) greater mean tumor burden but also an increase in
the number of rats bearing tumors from <20% of the group to
>80% of the animals over the following 70 days. A similar
response was seen if progesterone treatment was started 2 mo
after tamoxifen therapy had commenced.
Examination of uterine weights in chronically treated rats
showed that progesterone treatment caused significant (/' <
O.OS)inhibition of uterine weight compared to the peanut oiltreated control group. This reduction in uterine weight with
progesterone was to the same extent as in the tamoxifen- and
progesterone-treated group (see Table 1). The circulating levels
of progesterone produced in these intact rats treated with
progesterone (4 mg/day) were about 3 times the levels measured
in control animals (see Table 1). Total estrogen (estrone plus
estradici) levels were similar in control and progesterone groups
and slightly lower in the tamoxifen- and progesterone-treated
group (see Table 1).
of
detection--"
T
CONT PROG. TAM. PROG.
+
TAM.
[423
C57]
C3]
[15]
Fig. 3. Tumor sizes 8 wk after the start of treatment on animals described in
Fig. 2. Tumors that were too small to measure (<1 cm2) were classified descrip
tively. Tumor numbers in each group are shown in brackets. CONT., control;
PROG., progesterone; TAM., tamoxifen.
Influence of an Antiprogestin on the Interaction of Progester
one on the Inhibition of Tumor Development by Tamoxifen. The
administration of tamoxifen for 1 mo established a therapy that
prevented tumor formation. As previously shown the coadministration of progesterone reversed the inhibition. The further
addition of the antiprogestin, RU 486 (5 mg/day), was signifi
cant (P < 0.05) in preventing the reversal by progesterone (4
mg/day) of the antitumor action of tamoxifen (50 fig/day) after
6 wk of treatment (see Fig. 5). This prevention of tumor
formation by RU 486 was to a level slightly but not significantly
below that of tamoxifen alone.
DISCUSSION
The inhibition of DMBA tumor induction and growth by
tamoxifen is well documented (23). Similarly, this model has
been used extensively in studies of progesterone action on
mammary tumors (24, 25). This was, therefore, a useful model
to investigate the hypothesis that progesterone may reverse the
antitumor action of tamoxifen.
The findings, that tumor regression and the protection
against tumor development by tamoxifen treatment were par
tially reversed by the coadministration of progesterone, support
this hypothesis. However, the mechanism behind the reversal
of the antitumor activity of tamoxifen by progesterone is un
clear.
Previously, Jabara (24) demonstrated progesterone treatment
caused the development of DMBA tumors in rats earlier than
5388
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Research.
TAM AND PROG
Weeks of Treatment
2
4
6
8
IO 12 14 16 18 20
22
Weeks of Treatment
Fig. 4. Effect of progesterone treatment on the occurrence of DMBA-induced
mammary tumors in rats treated with tamoxifen. Rats were administered DMBA
30 days prior to drug treatment. Groups of 25 animals were given injections (s.c.)
daily with tamoxifen (SOng; O), tamoxifen (SO*ig)alone for 1 mo then combined
with progesterone (4 mg; •),or tamoxifen (SO^g) alone for 2 mo than combined
with progesterone (4 mg; •).All drugs were given separately in peanut oil (O.I
ml). Control (O) animals received peanut oil alone. Tumor burden was calculated
as the number of tumors per group divided by the number of rats. Arrows indicate
the time at which progesterone treatment was commenced in the combined
treatment groups.
Table 1 Strum hormone levels and uterine weights taken from animals following
daily treatment with agents for 12 wk
Progesterone and tamoxifen plus progesterone treatments have significantly
raised progesterone levels and reduced uterine weights compared to control by
Student's t test I/' < O.OS).Progesterone treatment has not raised estrogen levels
significantly above controls.
Control
7)*Progesterone
(n =
estrogen
(estrone +
estradici)
(pg/ml)44.4
4.4"44.6
±
7)Tamoxifen (n =
2.833.6
±
+progesterone
wt
(ng/ml)27.4
(mg)41
7.080.6
±
±39.8167.0
1.4
6.679.2
±
10.8160.0
±
±6.7Progesterone ±5.8Uterine
±11.0
(n = 5)Total
•
Mean ±SE.
* Numbers in parentheses, number of estimates for each measurement.
in control animals and produced a faster rate of tumor growth.
Similar findings were observed in this study (Fig. 2). We rea
soned that the promotion of tumor growth by progesterone
may result in tumors undergoing rapid development before
tamoxifen treatment has attained sufficient control of tumor
growth. This possibility was addressed and dismissed by the
study of the influence of progesterone on tumor initiation
following established tamoxifen therapy (1 and 2 mo). These
findings clearly demonstrate that progesterone was acting in
the presence of sufficient tamoxifen to prevent tumor develop
ment.
While reversing the tumoristatic action of tamoxifen in the
mammary gland progesterone acted as an estrogen antagonist
in its reduction of uterine wet weight. This type of effect on
Fig. 5. Effect of Hi 486 on the reversal by progesterone of the antitumor
action of tamoxifen. Rats were administered DMBA 30 days prior to drug
treatment. Groups of 25 animals were given injections (s.c.) daily with tamoxifen
(50 ¿ig)
alone for 1 mo. Rats were then continued on either tamoxifen alone (50
#ig;D), tamoxifen (50 pg) and progesterone (4 mg; •),or tamoxifen (50 „n)and
progesterone (4 mg) and RU 486 (5 mg; •).All drugs were administered separately
in peanut oil. Control (O) animals were administered only peanut oil. Tumor
appearance was calculated as the number of tumors occurring during the treatment
period divided by the number of rats.
uterine tissue has previously been noted and related to estrogen
receptor regulation (26). The inhibitory action of progesterone
in the uterus may explain why combination with tamoxifen did
not stimulate growth of this tissue to that of the control group
(see Table 1) or above the uterine wet weight produced by
tamoxifen treatment alone (data not shown).
Ovarian and adrenal enzymes can convert progesterone to
androgens, and either ovarian or peripheral aromatization could
produce estrogens. Raised circulating estrogen levels resulting
from progesterone administration could thereby reverse tamox
ifen action. The measurement of hormone levels in chronically
treated animals argues against this and shows that, while pro
gesterone treatment has raised circulating levels of progesterone
above the range seen in the rat at peak proestrus (27), the levels
of total estrogens were not raised above control. In addition,
the slightly lower total estrogen levels measured in animals
treated with both progesterone and tamoxifen are inconsistent
with estrogen formation being responsible for the reversal of
the antitumor action of tamoxifen.
The original hypothesis of progesterone reversal of the antitumor action of tamoxifen required progesterone to influence
tumor growth by cellular activation via the progesterone recep
tor. Evidence supporting the reversal of the antitumor action
of tamoxifen by progesterone being via progesterone receptor
activation was obtained using the antiprogestin RU 486 to
prevent this effect. However, although this argues in favor of a
progesterone receptor-mediated reversal, RU 486 is not selec
tive and has considerable antiglucocorticoid activity and mod
erate antiandrogen activity, and it produces a variety of endo
crine-related responses (28) which may also play a role. Fur
thermore, this does not exclude the possibility that progesterone
is influencing tumor growth by receptor binding at sites distant
to the tumor. A single injection of progesterone (10 mg) has
been demonstrated to induce pseudopregnancy in female rats
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TAM AND PROG
and results in a period of increased prolactin production (29).
Since DMBA-induced tumors are prolactin dependent (30), the
raised levels of prolactin may result in increased tumor growth.
However, Kelly et al. (31 ) reported that chronic progesterone
(4 mg/day) treatment of rats did not significantly change cir
culating prolactin levels or the number of prolactin receptors
on DMBA-induced tumors. The treatment did, however, cause
an increase in the early incidence of these tumors.
Overall, the findings demonstrate that progesterone can re
verse the tumoristatic actions of tamoxifen in this model and
that this is possibly through progesterone receptor activation.
How this may relate to the failure of tamoxifen in the human
disease is uncertain; however, these findings suggest that the
use of low doses of progestational agents in patients receiving
tamoxifen therapy may be inappropriate.
Indeed, following the submission of this paper for publication
Hissom and Moore (32) demonstrated the activity of the progestin, R5020, to stimulate the growth of T47D human breast
cancer cells in culture. These cells are known to contain a high
level of progesteróne receptor (33).
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