High Inhibitory Activity of a New Antiestrogen, RU 16117 (Lla-Methoxy Ethinyl Estradiol), on the Development of Dimethylbenz(A)Anthracene-Induced Mammary Tumors

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[CANCERRESEARCH37,76-81,January1977] High Inhibitory Activity of a New Antiestrogen, RU 16117 (lla-Methoxy Ethinyl Estradiol), on the Development of Dimethylbenz(a)anthracene-induced Mammary Tumors

Paul A. Kelly, Jacques Asselin, Marc G. Caron, Jean-Pierre Raynaud, and Fernand Labrie1 MedicalResearchCouncilGroupIn MolecularEndocrinology,LeCentreHospitalierdeI'UniversitdLaval,QuÃ©becGiV4G2,Canada(P.A. K.,J. A., M. G.C., F. L.J,and Centrede RecherchesRoussel-UCLAF,Romainville93230,France(J-P.R.J

SUMMARY have been shown to reduce the number and size of these tumors (4, 5, 9, 21, 23, 25, 26, 30). Moreover, tumor growth From the first day of dimethylbenzanthracene administra can be reinitiated in ovariectomized animals by treatment tion, daily treatment with 8 or 24 @gofthe new antiestrogen with estrogens or PAL (17, 21, 29). lla-methoxy ethinyl estradiol (RU 16117) completely pre Since we have recently found that RU 16117 has more vented the appearance of mammary tumors in all animals potent antiestrogenic properties than did the compounds up to the last time interval studied (130 days after dimethyl previously available (8, 27), it seemed of interest to study its benzanthracene administration). At daily doses of 0.5 and effect on the development of DMBA-induced mammary tu 2.0 @gRU16117, the tumor incidence was reduced to 78.6 mors in the rat. In an attempt to correlate the response to and 40.0%, respectively. Not only was the number of ani antiestrogen treatment with hormone receptor levels, the mals developing tumors reduced after injection of low concentration of receptors for 17f3-estradiol, progesterone, doses of RU 16117, but the number of tumors per rat and PRL, GH, and insulin was also determined in individual the size of tumors were also markedly reduced. The levels of tumors. receptors for estradiol, progesterone, and prolactin in tu mon tissue were reduced after treatment with 2.0 j.tg RU 16117, while the binding of growth hormone and insulin MATERIALS AND METHODS was not affected. Whereas plasma luteinizing hormone 1ev els decreased after treatment with 8 or 24 p@gRU 16117, Treatments.FemaleSprague-Dawleyrats(obtainedfrom plasma prolactin levels slightly increased in animals receiv Canadian Breeding Farms, St. Constant, QuÃ©bec,Canada) ing the highest dose of the antiestrogen. It is thus likely that were administered 20 mg DMBA in 1 ml corn oil by gastric the potent inhibitory effect of RU 16117 on the development gavage at 50 to 55 days of age. Animals were housed 2/cage of dimethylbenzanthnacene-induced mammary tumors re with a lighting regimen of 14 hr light-lO hr darkness (lights suIts from actions at both the hypothalamic-pituitary and on between 5:00 a.m. and 7:00 p.m.), and they received the tumor (mammary gland) levels, the action at the peniph Purina Rat Chow and water ad libitum. eral level possibly being secondary to a reduced sensitivity From the first day that DMBA (Sigma Chemical Co. , St. of the tissue to circulating hormones through lowering of Louis, Mo.) was administered, the animals received daily hormone receptor concentrations. s.c. injections of RU 16117 (Roussel-UCLAF, Romainville, France) at doses of 0.5, 2, 8, or 24 @gorthe vehicle alone (0.1 ml 1% gelatin-0.9% NaCI solution). RU 16117 was first INTRODUCTION dissolved in a small volume of ethanol before further dilu tion with the vehicle. A group of animals was ovaniecto Approximately 35% of patients with breast cancer expeni mized the day after DMBA treatment. At the outset of the ence remission of their disease after endocrine ablative experiment, there were 18 to 20 animals/group. However, surgery or steroid therapy (13, 19). It thus appears important within the 1st 10 days after DMBA administration, 1 to 4 to gain as much knowledge as possible about the mecha animals from each group died, resulting in groups of 15, 14, nisms controlling the hormonal dependency of these can 15,15,19,and 14 ratsinthecontrol,RU16117 (0.2,2,8, cers. The mammary carcinoma induced in the rat by DMBA2 and 24 l@Lg),andovariectomized groups, respectively. After (12) has been the most widely accepted model of hormone the initial period, no further loss of animals was observed dependent breast cancer. Estrogens and PAL have been until very late in the study when 2 animals died as a result of shown to be important for the development and growth of large ulcerated tumors. these mammary tumors. In fact, procedures that reduce Animals were examined for the appearance of mammary circulating levels of PRL (hypophysectomy, ergot drugs) tumors by palpation twice weekly, beginning 45 days after

I Associate of the Medical Research Council of Canada. DMBA administration. The number of animals with 1 or 2 The abbreviations used are; DMBA, 7,12-dimethylbenz(a)anthracene; more tumors divided by the total number of animals in the PRL, prolactin; RU 16117, 11a-methoxy-19-nor-17a-1 ,3,5(10)-pregnatrien 20-yne-3,17-dlol; GH, growth hormone; LH, luteinizing hormone. group at the indicated time points gave the tumor incidence Received February 3, 1976; accepted October 4, 1976. expressed as percentage. The average tumor number per

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tumor-bearing rat was also recorded. The product of the 2 volume of 0.5 ml in 25 mM Tnis-HCI-10 mM MgCl2 (pH 7.4) longest perpendicular diameters of the tumor measured containing 0.1% bovine serum albumin. The incubation was with calipers was used as a standard estimate of the size of stopped by the addition of 3 ml of the incubation buffer. a lesion. These values were expressed as average tumor Bound and free hormone were separated by low-speed cen area (sq cm) and average tumor area (sq cm) per rat. This tnifugation (2000 rpm) at 4Â°inan IEC PR 6000 centrifuge for value is obtained by dividing the sum of tumor areas in a 25 mm. After decantation, the pellets were counted in a LKB group by the total number of animals in that group. Tumor gamma spectrometer. Specific binding was obtained by development was followed in these rats up to 130 days after subtracting nonspecific from total bound radioactivity. This DMBA treatment. Animals were sacrificed by decapitation value was generally expressed as a percentage of the total

. between 8:00 a.m. and 9:00 a.m., and trunk blood was radioactivity added to the tube. Binding values of 125I-la collected in hepaninized beakers. Following separation by beled PAL and GH in membrane fractions of DMBA-induced centnifugation, plasma was stored at â€”20Â°untilhormone mammary tumors remain constant for periods up to at least assays were performed. 3 months, whereas 1251-labeledinsulin binding values begin Preparation of Cytosol and Membrane Fractions. After to decline after 1 month of membrane storage at â€”20Â°(P.A. decapitation of the animals, the mammary tumors were Kelly, unpublished observations). removed, freed of connective and adipose tissue, and Radioimmunoassays. Plasma LH and PAL were mea rinsed in ice-cold Buffer B (10 mM Tris-HCI, pH 7.4, 1.5 mM sured by double-antibody nadioimmunoassays (2, 24) using EDTA, and 10 mM thioglycerol). The tumors were then rat hormones (LH-l-3, LH-RP-1 , PAL-I-i , and PRL-RP-i ) and weighed and homogenized in 3 volumes (w/v) of Buffer A rabbit antisera (anti-rat LH serum I and anti-rat PRL-S-2) (25 mM Tnis-HCI, pH 7.4, 1.5 mM EDTA, 10 m@ thioglycerol, kindly provided by Dr. A. F. Parlow for the National Institute and 10% glycerol) using a Polytron PT-10 homogenizer at a of Arthritis and Metabolic Diseases, Rat Pituitary Hormone setting of 5 for 2 periods of 10 sec with an interval of 10 sec Program. Radioimmunoassay data were analyzed with a for cooling. The homogenate was centrifuged at 18,000 x g desk-top Hewlett-Packard calculator using a program writ for 15 mm, and the resulting supernatant was centrifuged at ten in this laboratory and based on Model II of Rodbard and 105,000 x g for 90 mm to obtain the cytosol (supernatant) Lewald (28). All data are expressed as mean Â±S.E. Statisti fraction. All steroid binding assays were performed with cal significance was calculated according to the multiple fresh cytosol from tumors removed the day the animals range test of Duncan-Kramer (16). were sacrificed. The 105,000 x g pellet was resuspended in Hormones.17f3-[2,4,6,7-3HlEstradiol(105 Ci/mmole)was 25 mM Tris-HCI-10 mM MgCI2 (pH 7.4) with a Teflon-glass obtained from New England Nuclear, Boston, Mass., while homogenizer and stored at â€”20Â°untilinitiation of peptide [1,2-3HJR5020 and the corresponding unlabeled steroids binding assays. Protein concentration was measured (18) were synthesized at the Roussel Research Center. Ovine using bovine serum albumin as standard. PRL(26 units/mg, NIH-P-S11) and bovine GH (1.9 units/mg, [3H]R5020 and 17/3-(3H]EstradiolBindingAssays. The NIH-B 1003A) were generously supplied by the National potent synthetic progestin R 5020 (17,21-dimethyl-19-nor Institute of Arthritis, Metabolism and Digestive Diseases. pregna-4,9-diene-3,20-dione) was used for measurements lodination with 1251was performed using the lactopenoxi of progesterone receptor levels (1). The use of this com dase procedure as described (33). Specific activities of the pound makes it possible to avoid the difficulties met with iodinated preparations ranged from 40 to 80 @Ci/@gforGH [3H]progesterone, which binds to corticosteroid-binding and PAL and 120 to 150 j.@Ci/@gforinsulin. globulin and has a relatively fast dissociation rate from its receptor. [3H]R 5020 and 17f3-[3H]estradiol binding were measured RESULTS in an assay system using dextran-coated charcoal to sepa rate bound and free hormone. Three-tenths-mI aliquots of As illustrated in Chart 1A, tumors first appeared in control adequately diluted cytosol were incubated with 0.1 ml of 4 x rats 53 days after DMBA administration, and the incidence 10-8 M [3H]R 5020 or 17f3-[3H]estradiol in the presence or of tumors increased gradually (with several periods of no absence of a 100 molar excess of the unlabeled steroid for change) to a maximum of 94.1% recorded at the end of the 16 to 20 hr at 0-4Â°.Unbound steroid was then removed by experiment (130 days). All treatments resulted in a delayed incubation for 10 mm at 0-4Â°with 0.4 ml of 0.5% Nonit onset of tumor appearance. After a delay of 10 days, RU A-0.05% Dextran-T-70 in Buffer C and centnifugation at 16117 at a dose of 0.5 pg/day resulted in a curve similar to 2000 x g for 10 mm at the same temperature. Four-tenths that of controls, although the incidence reached only ml aliquots of the supernatant were then removed, and, 78.6%. When RU 16117 was injected at a dose of 2 pg/day, after addition of 10 ml of Aquasol, the radioactivity was themaximal incidencewas60% between Days 99 and 106, measured in a Beckman liquid scintillation spectrometer at after which the incidence fell to a value of 40% at Day 130, a counting efficiency of 45%. due to the regression and disappearance of lesions in some Binding assays of 1251-IabeledOvine PRL, Bovine GH, animals. The important finding is that RU 16117, at doses of and Porcine Insulin. Specific bindingwas determinedin either 8 or 24 pg/day, completely inhibited tumor develop tumor membrane fractions that had been stored at â€”20Â°for ment in all animals. Ovaniectomy completely inhibited tu less than 1 week as previously described (14). Briefly, ap mor appearance until Day 95 when 2 of 14 animals devel proximately 100,000 cpm of 125I-labeledhormone were incu oped palpable tumors (14.2%). bated with 300 @gofmembrane fraction protein in a final The average number of tumors per tumor-bearing animal

JANUARY1977 77

A o.â€”o CONTROL B â€¢â€”... 00-16117 (O.Spgl @ o-..o â€˜(2op@) @ h a â€˜ (8$)4pg)

C D

Chart 1. Effect of treatment with increasing doses of RU 16117 or ovariectomy (OVX) on the development of DMBA-induced mammary tumors. Injections began the day DMBA was administered and continued for the next 130 days. Ovariectomy was performed the day after DMBA administration. Animals were examined twice weekly for the presence of tumors, and, when present, tumor area (length x width) was measured. A, tumor incidence as a function of time after DMBA; B, average tumor number; C, average tumor area (sq cm); D, average tumor area (sq cm) per animal. This value is obtained by dividing the sum of tumor areas in a group by the total number of animals in that group.

is shown in Chart lB. Once again, control animals showed a Chart 1D, which shows a steady increase in the average gradual increment, reaching a maximum of 3.3 tumors/rat tumor size per rat to 4.4 sq cm/rat on Day 130. In animals 130 days after DMBA administration. At doses of 0.5 and 2 treated with 0.5 or 2 @gRU16117, the tumor size was 3.1 mg/day, RU 16117 resulted in a reduction to 2.5 and 1.5 and 1.5 sq cm/rat, respectively, and the tumor averaged 0.8 tumors/animal, respectively. As mentioned earlier, only 1 sq cm/rat in ovaniectomized animals. The certainty of this tumor developed in each of the 2 ovaniectomized rats. reduction in tumor size in animals receiving RU 16117 re These 2 tumors, however, became quite large (Chart 1C). mains to be verified in view of the fact that there is a large Therefore, at the end of the experiment in the control, AU inherent variability in the rate at which DMBA tumors grow, 16117 (0.5, 2, 8, and 24 mg), and ovaniectomized groups, even within a single group of 19 animals. therewas a totalof52,27,9,0,0,and 2 tumors,respec The average latency period for the development of DMBA tively. In control rats, the largest average tumor size was 2.6 induced mammary tumors in control rats was 96.0 Â±3.2 sq cm on Day 71, after which the values declined to approxi days. It is interesting to note that, although the appearance mately 1.2 sq cm for the remainder of the study, reflecting of the 1st tumor was delayed in groups treated with 0.5 or the development of smaller, new tumors late in the study. 2.0 j@gRU16117, the average latency period was not signifi Treatment with 0.5 @gAU16117 had no significant effect on cantly different from that of controls. In other words, in all tumor size, whereas rats receiving 2 @gAU16117 showed a groups, tumors that appeared did so at about the same time more complex pattern of tumor growth. Initially, tumor size after DMBA administration. increased to approximately 1.2 sq cm, after which there was Specific binding of [3H]estradiol, [3H]R 5020, and 1251.. a diminution in size to an average tumor area of only 0.4 sq labeled ovine PAL to DMBA-induced mammary tumors is cm on Day 130. It should be mentioned, however, that this shown in Chart 2, A, B, and C, respectively. Binding of 1713- increase in average tumor area between Days 102 and 130 in [3H]estradiol was 5.1 Â±1.0 pmoles/g tissue in tumors from the group treated with 2.0 @gAU16117 is due to only 1 large control animals, whereas, after daily treatment with 0.5 and tumor that accounted for 80% of the total tumor area of this 2.0 @gRU16117, reductions of binding to 2.7 Â±0.3 and 1.9 group at the end of the experiment. In the absence of this Â± 0.6 pmoles/g tissue were observed. Binding of [3H]R 5020 tumor, average tumor area would have continued to decline was 8.9 Â±1.5 pmoles/g tissue in the control group. At the in this group to reach a value of 0.4 sq cm on Day 130. The dose of 0.5 @tg,RU 16117 was without significant effect, overall effect of the treatments is most clearly evident in whereas daily injection of 2 j@gof the steroid resulted in a

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pg/DAY Chart 2. Specific binding of 17/3-[3H]estradiol (A), [3H]R5020 (B), and â€˜251-labeledovinePRL (C) to DMBA-induced rat mammary tumors from rats treated with increasing doses of RU 16117 or ovariectomized (OVX) rats. Rats were given a single injection of DMBA, and treatment with RU 16117 commenced the same day. All tumors with an area larger than 1 sq cm on the day animals were sacrificed (Day 130) were removed. Cytosol and particulate membrane fractions were prepared, and specific hormone binding was determined as described under â€˜Materialsand Methods.â€•Thenumber of tumors assayed per group is indicated in the bars in B. reduction to 4.5 Â±0.5 pmoles/g tissue. In 1 tumor that 0 CONTROL developed in the ovaniectomized group, the level of proges terone receptors was very low at 0.6 pmole/g tissue. RU 0 OVX 16117 treatment caused a reduction of â€˜25l-labeledPALto tumor plasma membranes from 6.3 Â±1.5% in control rats to E 2.9 Â±0.6% in tumors from rats receiving RU 16117 at 2 j.@g/ day (Chart 2C). In the 1 tumor from an ovaniectomized rat, there was very low binding of â€˜25l-labeledovinePAL (1.2%). Specific binding of both â€˜25l-labeledbovineGH and insulin was constantat0.2to0.4% inalltreatmentgroups(datanot shown). I- Since DMBA-induced mammary tumors are PAL depend .@ ent (4, 17, 21, 23, 25, 28), it was of interest to examine the N- effect of RU 16117 treatment on plasma PAL levels. As illustrated in Chart 3, the doses of 2.0 and 8.0 j.@gofsteroids ,@ caused a nonsignificant decrease in plasma PAL levels, 1. while the highest dose (24 @g)increasedthe levels from 21 Â± 7 to 41 Â± 10 ng/ml (p < 0.05). Ovaniectomy decreased the plasma PRL concentration to 2.0 Â±0.3 ng/mI (p < 0.05). As illustrated in Chart 4, treatment with AU 16117 led to a progressive decrease in plasma LH levels, the effects be coming significant at the 8- (p < 0.05) and 24-pg (p < 0.01) Chart 3. Plasma PRL levels in DMBA rats treated with increasing concen doses. Plasma follicle-stimulating hormone concentrations trations of RU 16117 or ovariectomized (OVX) rats. (not shown) were not affected by the antiestrogen treat ment, whereas ovaniectomy led, as expected, to a marked pound has weak estrogenic activity in the mouse (Aubin test increase in both LH and follicle-stimulating hormone = 1/100 of 17f3-estradiol) and castrated rat (Allen Doisey plasma levels. test = 1/20 of 17f3-estradiol) and competes for uterine estra diol cytosol receptor in mouse (1/20 of 17f3-estradiol) and rat (1/10 of 17f3-estradiol) (27). When injected at low doses DISCUSSION (1 or 2 lLg) during estrus, RU 16117 has been shown to (a) inhibit the spontaneous LH peak on the afternoon of proes The present data show that the new antiestrogenic com trus, (b) inhibit the 7- to 10-fold increase of pituitary sensi pound, RU 16117, at the relatively low doses of8 and 24 p.g/ tivity to LH-RH on the afternoon of proestrus, (c) block ovu day, is capable of completely preventing the appearance of lation on the expected morning of estrus, and (d) delay the mammary tumors after DMBA administration. This com appearance of vaginal cornification (Ref. 8; L. Fenland and

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0 CONTROL and F. Labnie, unpublished observations). Moreover, in this same system , while high doses of 17f3-estradiol adminis tered from the time of DMBA administration resulted in a delayed appearance of tumors, RU 16117 led to a complete inhibition up to at least 130 days after DMBA administration a (Chart 1). It now appears quite clear that high levels of estradiol receptors in human breast cancers are associated with a positive response to endocrine therapy in 60 to 75% of 0@ cases (13, 19). It has been suggested that the level of cer = tainty of the prediction is increased when both estradiol and progesterone receptors are present in sufficiently high con

C..) N- centrations in the tumor tissue (10). Hormone-dependent rat mammary tumors were found to specifically bind .@ estradiol both in vivo and in vitro (22, 30). In DMBA-induced = mammary tumors, a correlation has in fact been found between the level of estradiol receptors and the hormonal dependency of the tumor (20). Recent observations indicate that, while the absence of a detectable level of estrogen receptors indicates hormone independence of the tumor, 0.5 2.0 8.0 24.0 the presence of estrogen receptor may not necessarily mdi Chart 4. Plasma LH levels in DMBA rats treated with increasing concen cate hormone dependency (3, 7). These findings indicate trations of RU 16117 or in ovariectomized (OVX) rats. the importance of studying multiple receptor levels in the tumor tissue. Moreover, a positive correlation has been F. Labnie, unpublished observations). observed between the binding of 125l-Iabeled PAL to a mi Whilethe 2 highestdosesof AU 16117 ledto complete crosomal fraction of DMBA-induced mammary tumors and inhibition of tumor development, the 2 lower doses also had the response of these tumors to stimulation of growth by significant effects. The net effect is best illustrated in Chart injected PAL (14). 1D where it can be seen that treatment with 0.5 or 2.0 @gAU The present data clearly show that treatment with 2 @tgRU 16117 led to inhibition of average tumor size at Day 130 to 161 17 led to a 40 to 60% reduction in the levels of receptors 75 and 37% of the control values, respectively. This impor for estradiol, progesterone, and PAL in the mammary tu tant effect resulted from a lower incidence and smaller mors. Levels of progesterone and PAL receptors were re tumors after treatment with low doses of the antiestrogen. duced to 10 to 15% of control after ovariectomy, a finding Although requiring much higher doses, other antiestro confirmed on a larger scale in subsequent experiments (1). gens have been found to inhibit DMBA-induced tumor de These observations may suggest that tumors unresponsive velopment and growth. For example, i-[2-p-[a(p-methoxy (or less responsive) to hormonal treatment (RU 16117 or phenyl)-f3-nitrostyryl]phenoxyethyl pynrolidine (Cl-628), at a ovaniectomy) have low spontaneous levels of hormone re dosage of 1 mg/kg, was found to be somewhat less efficient ceptors. It is possible, however, that the low levels of recep than ovaniectomy in inhibiting DMBA-induced mammary tons are secondary to the hormonal treatment itself. It may tumor growth (6). At the daily dosage of 5 mg for 40 days be that part of the mechanism of action of AU 16117 in starting 20 days before DMBA administration, i-[p-(2-di volves a lowering of hormone receptor levels, thus render ethylaminoethoxyphenyl)]-2-(p-methoxyphenyl)-i -phenyleth ing tumor tissue less responsive to its hormonal environ anol (MER-25) reduced tumor incidence to only 40% of con ment. trol (15). When given every other day at a dosage of 1 mg/kg While the inhibitory effect of daily doses of 2 or 8 @gAU for 30 days in rats that had developed tumors, nafoxidine 16117 may be secondary to inhibition of both LH and PAL (U-iii00 A) led to an important inhibition of tumor growth secretion, the complete absence of tumor development in (31 , 32), while, in another study, the same drug at the daily the presence of elevated levels of plasma PAL indicates that dosage of 1 mg/kg begun 2 weeks after DMBA adminis the 24-pg dose of the steroid is exerting at least part of its tration almost completely prevented tumor development inhibitory action directly at the tumor level. It thus appears measured after 6 on 12 weeks (9). likely that the potent inhibitory activity of AU 16117 on the The fact that estrogens can have a dual effect on mam development of DMBA-induced mammary carcinoma re mary carcinoma induced by DMBA is well known. Adminis suIts from actions at both the hypothalamic-pituitary and tration of estrogens stimulates tumor development in ovani tumor or mammary gland levels, the action at the mammary ectomized animals (17), while in animals with developed tissue level possibly being exerted through modification of tumors, large doses of estrogens can lead to tumor negres steroid and PAL receptor levels. sion (ii) or inhibition of tumor development (15). That the effect of AU 16117 is not due to its low estrogenic activity is indicated by the findings that doses of 17/3-estradiol equiva REFERENCES lent to the doses of RU 16117 used have no inhibitory effect 1. Asselin, J., Labrie, F., Kelly, P. A., Philibert, D., and Raynaud, J. P. on the development of DMBA-induced tumors (P. A. Kelly Specific Progesterone Receptors in Dimethylbenzanthracene (DMBA)

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Paul A. Kelly, Jacques Asselin, Marc G. Caron, et al.

Cancer Res 1977;37:76-81.

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