Selective Retention of Estrogen Isomers in Estrogen-Dependent Breast Tumors of Rats Demonstrated by in Vitro Methods

[CANCER RESEARCH 28, 328-337, February Ig68]

Selective Retention of Estrogen Isomers in Estrogen-dependent Breast Tumors of Rats Demonstrated by in Vitro Methods

Lars Terenius Departmen~ of Pharmacology, University of Uppsala, Uppsala, Sweden

SUMMARY trogen-responsive DMBA tumors to be 8-20 times the muscle concentration around 100 minutes after subcutaneous injection Huggins' rat mammary tumors were induced by treatment in saline. She also found a lower accumulation in estrogen-un- with 7,12-dimethylbenz (a) anthracene (DMBA) and the estro- responsive DMBA tumors, indicating that estrogen uptake and gen-dependent ones were selected. The tumors avidly took up retention is characteristic for the estrogen dependence of the potent estrogens 17fl-estradiol and meso-hexestrol in vivo, DMBA-induced tumors. but they did not take up the much less estrogenic racemic This communication describes estrogen uptake and wash-out hexestrol. The potent estrogens in the tumor were not easily experiments with the DMBA-induced tumor. The character- washed out in vitro. Diaphragm neither took up nor retained istics and specificity of these processes is studied. Most ex- potent estrogens significantly. periments have been carried out in vitro, which permits the The selectivity in vitro was most clearly visible as a selective use of slices from the same tumor in experiments under different retention of the potent estrogens in the tumor. When tumor conditions. By this means one can relate all effects to appro- slices and diaphragm strips were incubated with labeled 17fl- priate controls and the problem of variation between tumors estradiol, the tumor/diaphragm concentration ratios became is eliminated. Methods for analysis of estrogen uptake in vitro about 2; but after a subsequent wash-out, in the presence of have been developed for the mouse uterus (22, 25). Similar a large excess unlabeled 17fl-estradiol, the concentration ratios methods have been used for the tumor. Strips of diaphragm increased to about 4. meso-Hexestrol, but not the less estrogenic from the tumor-bearing rat are included in all experiments, racemic hexestrol, was selectively retained by the tumor in the the diaphragm being regarded as a non-target, control tissue. same way. Unlabeled 17a-estradiol, 17fl-estradiol, the synthetic After this work was completed a communication by Jenscn estrogens meso-hexestrol, racemic hexestrol, or the demethylated et ol. (13) appeared, also dealing with the uptake in vitro of analog of the estrogenic acid, methallenestril, suppressed the 17fl-estradiol by the DMBA-induced rat mammary tumor. In retention of labeled 17fl-estradiol by the tumor but not by the many respects, the observations made by these authors agree diaphragm when present during uptake. These estrogenic with the present ones, even if the experimental conditions stereoisomers were inhibitory to an extent which was correlated differ. with their estrogenic potencies. The degree of uptake and retention of 17fl-estradiol in the tumor was diminished by a MATERIALS AND METHODS sulfhydryl inhibitor, N-ethylmaleimide, present during uptake or wash-out. The uptake of 17fl-estradiol by adipose tissue was Radioactive Compounds and Nonradioactive Estrogen high, but showed no specificity. Preparations. Tritium-labeled 17fl-estradiol (labeled in the 6, In all these respects the selectivity of estrogen uptake and 7 positions) was obtained from New England Nuclear Corp. retention in the DMBA tumor is similar to that of the mouse Its specific activity was 132 ~c//~g. Tritium-labeled isomers uterus. This similarity indicates the presence of specific es- of hexestrol were prepared by the author as described else- trogen receptors in the tumor and favors the view that estro- where (21). Their specific activities were 150 ~c/~g. The gens exert a direct influence on the growth of this tumor. radiochemical purity of the radioactive compounds was con- trolled at intervals by thin-layer chromatography on silica gel INTRODUCTION with chloroform:acetic acid (85:15, v/v) or chloroform:di- ethylamine (80:20, v/v) as developer. At times of use the Estrogen-responsive tissues, such as the mammalian uterus radiochemical purity was about 98%. and vagina, take up and retain estrogens (e.g., 8, 14, 20). Most 2 Abbreviations : DMBA, 7,12-dimethylbenz(a) anthracene ; 17fl- rat mammary tumors induced by treatment with DMBA 2 also estradiol, estra-l,3,5(10)triene-3,17fl-diol; hexestrol, 3-4-bis(p-hy- are estrogen-responsive (6, 9). It has been found that this droxyphenyl)-n-hexane; methallenestril, dl-3-(6-methoxy-2-naph- tumor retains systemically administered 17fl-estradiol (15). thyl)-2,2-dimethylpentanoic acid; KRP, Krebs-Ringer phosphate Mobbs (17) found the concentration of 17fl-estradiol in es- buffer; KRPA, Krebs-Ringcr phosphate buffer with 2% albumin; NEM, N-ethylmaleimide; U-11100A, 1-(2-[p-(3,4-dihydro-6-meth- 1 Supported by the Swedish Cancer Society. oxy-2-phenyl- 1,1-naphthyl) phenoxy]-ethyl) pyrrolidine hydro- Received May 25, 1967; accepted October 15, 1967. chloride.

328 CANCER RESEARCH VOL. 28

Nonradioactive 17fi-estradiol and the hexestrol isomers were Table 1

of the same preparation as previously used (21). Methallenes- Content of radioactivity a tril was a commercial specimen, m.p. 225-228~ (Vallestril | Rat Tumor Diaphragm Tumor G. D. Searle & Co., Chicago, Ill.), and the corresponding No. Treatment Medmm Medium I)iaphragra free phenol, dl-3- (6-hydroxy-2-naphthyl)-2,2-dimethylpentanoic A. Uptake a 3.6 -4- 0.13 e 1.85 __+ 0.09 1.95_+ 0.12 acid, m.p. 175-176~ was prepared by the author from 1 B. Wash-out 22~ b 2.2 ___ 0.13 0.42_+ 0.02 5.1 ___ 0.40 methallenestril by demethylation with hydrobromic acid. C. Wash-out 37~ c 1.53 ___ 0.10 0.29_ 0.01 5.5 _ 0.39 Animals and Assay Procedure. Albino rats of the Sprague- A. Uptake 3.8 _ 0.4 2.0 --4-_ 0.11 1.9 ___ 0.23 Dawley strain were supplied through the courtesy of Dr. 2 B. Wash-out 22~ 1.57 ___ 0.05 0.52_ 0.06 3.0 _ 0.35 Charles Huggins from his laboratory. Females were treated C. Wash-out 37~ 1.19 _ 0.06 0.26 _ 0.01 4.6 _ 0.29 with DMBA when they were 50, 53 and 56 days old. On these days 2 mg DMBA in 0.4 ml 15% fat emulsion (obtained from A. Uptake 5.2 __+ 0.4 2.6 ___ 0.11 2.0 -4- 0.18 3 B. Wash-out 22~ 2.5 -*- 0.12 0.94__+ 0.19 2.6 ___ 0.55 Dr. Huggins) was injected intravenously (10). About one C. Wash-out 37~ 1.68 _ 0.10 0.55_+ 0.03 3.1 _+ 0.19 month later the animals were inspected twice weekly and mammary tumors located by palpation. When a rapidly growing Uptake and effect of temperature on retention of 17fl-estradiol tumor appeared, the tumor-bearing animal was ovariectomized by the DMBA tumor and diaphragm in vitro. a Incubation for 1 hour at 37~ in 6 ml of KRPA (Krebs- (in most cases). When regression of the tumor was ascertained, Ringer phosphate buffer with 2% w/v albumin) and 0.003 #g 1.5 t~g of 17fl-estradiol-3-benzoate was injected subcutaneously 17fl-estradiol-3H (1.8 • 10 -9 M solution). in 0.15 ml olive oil every second day. If the regressed tumor b Preincubation as Group A; then incubation 1 hour at 22~ started to grow rapidly on the estrogen treatment, it was con- in other flasks with 6 ml of KRPA and 0.3 #g unlabeled 17fl- sidered to be estrogen-responsive and used for experimentation. estradiol (1.8 • 10 -7 M solution). Only a minor fraction (about 20%) of the animals developed c Preincubation as Group A; then incubation 1 hour at 37~ in tumors which were considered useful by these criteria. The other flasks with 6 ml of KRPA and 0.3 #g unlabeled 17fl- tumor was used for experimentation when its diameter was estradiol. 1-2 cm, 4 days after the last treatment with 17fl-estradiol-3- Concentration ratios tissue/medium calculated as benzoate. In cases where several tumors existed in one rat, only dpm/mg wet tissue "Medium" in this and all subsequent table dpm/#l medium " the most clearly estrogen-responsive one was utilized. headings refer to the medium in which the tissues were loaded After sacrifice of the animal the tumor was dissected free with radioactivity. from surrounding connective tissue. Tumor areas free from e Mean _ S.E. There were 6 tissue slices/group. necrosis and hemorrhage were sliced with a tissue slicer (type Stadie-Riggs). The thickness varied but always was ~1 mm. The first slice was discarded to exclude the outer capsule of Chemical identification of the radioactivity accumulated in the tumor. From the slices, strips weighing approximately 10 the tissues was performed as described earlier (21). mg were cut out. Simultaneously, diaphragm strips of the same weight were prepared. Two tumor strips and two diaphragm RESULTS strips were put into an incubating flask containing 6 ml of KRP pH 7.4, prepared from reagent chemicals and redistilled In the tables each animal has been treated separately since water (5). In most cases the KRP contained 2% (w/v) bovine the differences between tumors from different animals have albumin (Cohn fraction V, Sigma B grade). Aliquots of alco- been considered too great to permit averaging. holic stock solutions of radioactive as well as nonradioactive Ovariectomized Animals in Vitro. From the author's ex- compounds were taken to dryness and redissolved in the buffer. perience of in vitro experiments with the mouse uterus (22-25) The incubation flasks contained air as the gas phase. They were the present experimental conditions were adopted. Slices of shaken in a Warburg apparatus at constant temperature, in tumor tissue and strips from the diaphragm were incubated most cases at 37~ After incubation the tissues were gently for 1 hour in solutions containing radioactive 17fi-estradiol. blotted between two filter papers and then weighed wet on a The diaphragm was included as a control tissue. The concentration of radioactive 17/?-estradiol in the incubation bath was torsion balance. Each tissue slice was put singly into a scintil- the lowest possible. (The limiting factor was the necessity to lation vial and solubilized with 1 ml of Hyamine (obtained recover reliable radioactivity in the tissues. Since the specific from Packard Instrument Co):toluene (1:3, v/v) in a shaking activity of the 17/?-estradiol used was almost the theoretical incubator at 70~ Five ml of scintillation solution (22) was maximum for the compound with two tritium atoms, still lower added, and the samples were measured in a Tri-Carb liquid 17/?-estradiol concentrations cannot easily be attained.) At the scintillation spectrometer model 3324 (Packard Instrument concentration used, the tumor concentration of radioactivity Co.) until at least 10,000 counts were recorded or in samples became about twice that of the diaphragm (Table 1, Treat- with very low activities measured for 50 minutes. Counting ment A). Other slices from the same tumor and diaphragm efficiency was 23-26%, determined either by the addition of were allowed to accumulate radioactive 17fi-estradiol in the internal standard or by external standardization. The recorded same manner, but this treatment was followed by a second counts were then transformed to disintegrations per minute incubation of the tissues at two different temperatures in fresh and mg wet weight (dpm/mg). media containing high concentrations of unlabeled 17fi-estradiol.

FEBRUARY 1968 329

Table 2

Content of radioactivity Rat No. of Tumor Diaphragm Tumor No. slices Treatment Medium Medium Diaphragm 6 A. Uptake a 1.62 • 0.04 ~ 1.21 • 0.04 1.4 ___ 0.06 6 B. Wash-outb 0.70 • 0.02 0.29 • 0.02 2.5 • 0.18 6 C. Saturated uptake, wash-outC 0.33 • 0.01 0.27 ___ 0.01 1.2 ___ 0.06

4 A. Uptake 1.92 • 0.09 1.14 • 0.05 1.7 ___ 0.11 4 B. Wash-out 0.69 • 0.08 0.16 • 0.02 4.0 • 0.72 4 C. Saturated uptake, wash-out 0.33 • 0.02 0.16 ___ 0.01 2.1 • 0.18

4 A. Uptake 2.53 -+- 0.08 1.15 • 0.03 2.2 • 0.09 4 B. Wash-out 0.72 ___ 0.05 0.16 • 0.01 4.3 • 0.41 4 C. Saturated uptake, wash-out 0.19 • 0.01 0.14 ___ 0.01 1.4 ___ 0.12 Uptake and capacity of retention of 17fl-estradiol by the DMBA tumor and diaphragm in vitro. a See Group A, Table 1. b See Group C, Table 1. c Preincubation 1 hour at 37~ in a mixture of 0.003 t~g 17fl-estradiol-3H and 0.3 ttg unlabeled 17fl-estradiol in 6 ml of KRPA (see Table 1) ; then incubation in other flasks for 1 hour at 37~ with 6 ml of KRPA and 0.3 #g unlabeled 17fl-estradiol. a Mean • S.E.

Table 3

Content of radioactivity e Rat Tumor Diaphragm Fat Tumor Fat No. Treatment Medium Medium Medium Diaphragm "Diaphragm A. Uptake (no albumin) a 18.0 • 0.6 11.4 • 0.2 51.1 • 1.6 1.6 • 0.06 4.5 • 0.16 B. Uptake b 5.0 • 0.2 1.6 ___ 0.05 7.8 • 0.7 3.2 • 0.16 4.9 • 0.47 C. Wash-outC 2.1 ___ 0.2 0.26 • 0.03 1.4 • 0.2 7.9 • 1.2 5.1 ___ 0.99 D. Saturated uptake, wash-outS 0.31 • 0.01 0.35 ___ 0.02 1.6 ___ 0.07 0.9 ___ 0.06 4.5 • 0.33

A. Uptake (no albumin) 30.1 • 0.9 22.8 ___ 2.6 101.0 ___ 8 1.3 ___ 0.16 4.4 ___ 0.62 8 B. Uptake 3.0 • 0.09 1.7 -+- 0.1 8.4 • 0.4 1.8 • 0.12 4.9 • 0.37 (Partly C. Wash-out 0.66 • 0.02 0.24 • 0.01 1.3 • 0.2 2.6 -+- 0.14 5.1 • 0.86 necrotic D. Saturated uptake, tumor) wash-out 0.31 • 0.003 0.25 • 0.01 1.3 • 0.06 1.3 ___ 0.05 5.5 • 0.38 Uptake and retention of 17fl-estradiol by the DMBA tumor, diaphragm, and fat under different in vitro conditions. a Incubation in 6 ml of KRP (Krebs-Ringer phosphate; no albumin) with 0.003 #g 17fi-estradiol-~H for 1 hour at 37~ b See Group A, Table 1. c See Group C, Table 1. See Group C, Table 2. e Mean ___ S.E. There were 6 slices/group.

The radioactivity was found to be more easily washed out from estradiol. The following wash-out was performed exactly as the diaphragm than from the tumor (Table 1; Treatments B, described under B. It was found that unlabeled 17fi-estradiol C). The concentration ratios tumor-diaphragm were somewhat in the uptake solution strongly suppressed the content, of radio- higher following washing at 37~ than at 22~ activity left after wash-out in the tumor but not in the dia- Saturation experiments are illustrated in Table 2. The up- phragm (ef. Groups B, C). The above experiments were re- take experiments A and wash-out experiment B were done as peated, but included also fat as a nontarget tissue. Fat tissue described in Table 1 (Groups A and C, respectively). In the is known to concentrate the lipophilic estrogens in vivo (18). experiment denoted C the tissues were preincubated in a mix- In the present in vitro experiments 17fi-estradiol had a high ture of the labeled and a hundred-fold excess of unlabeled 17fl- affinity for fat tissue (Table 3). The table shows (Fat/dia-

330 CANCER RESEARCH VOL. 28

Table 4 than the/?-isomer (22). The estrogenic acid methallenestril and

Content of radioactivity b its demethylated analog were also included. It is known that Rat Radioactive Tumor Diaphragm Tumor methallenestril has to be demethylated in order to suppress the No. estrogen Medium 1V~edium Diaphragm uterine retention of 17/?-estradiol (23). In the following experi- 9 meso- ments, slices from tumor and diaphragm were subjected to tIexestrol a 0.35 __+ 0.02 0.10 _ 0.004 3.6 • 0.24 analogous experiments. They were first preincubated in a mix- 9 racemic ture of the putative nonradioactive inhibitor and radioactive Itexestrol 2.0 • 0.08 1.8 _ 0.08 1.1 • 0.07 17fi-estradiol, and then washed in fresh buffer which contained 10 meso- a large amount of nonradioactive 17fl-estradiol (Table 5). It ttexestrol 0.34 _ 0.01 0.09 + 0.01 4.0 +_ 0.42 was found that 17a-estradiol less effectively suppressed the 10 racemic level of labeled 17/?-estradiol in the tumor than the much more Hexestrol 2.4 _ 0.09 1.7 ___ 0.04 1.5 • 0.06 estrogenic 17/?-isomer (experimental animals 11-13). The tumor Retention of hexestrol isomers by the DMBA tumor and dia- from Animal 13 had a lower concentration of labeled 17/?- phragm in vitro. estradiol than the others; still the difference between the two a All incubations: 1 hour at 37~ in 6 ml of KRPA (see Table estradiol isomers is clearly visible. The concentration of labeled 1) with 0.006 #g of either tritiated hexestrol isomer; then incubation for 1 hour at 37~ in 6 ml of KRPA with 0.6 /~g of the ap- 17fi-estradiol in the tumors from Animals 14 and 15 was dif- propriate unlabeled hexestrol isomer. ferentially reduced by the hexestrol stereoisomers; meso-hexes- b Mean • S.E. There were 6 slices/group. t rol was more active than the much less estrogenic racemic isomer. Animal 16 had a very low concentration of labeled 17/?- estradiol in a partly necrotic tumor which was not affected by phragm column) that the degree of uptake and retention of the inhibitors. Finally, the concentration of labeled 17/?-estra- 17/?-estradiol by the adipose tissue and the diaphragm was diol in the tumor from Animal 17 was not affected by methal- affected similarly by the different experimental conditions. The lenestril, but it was suppressed by the corresponding free tumor/diaphragm concentration ratios, on the other hand, in- phenol. Summing up, tumors from all animals except No. 16 creased on albumin addition or washing and were depressed by took up -rod retained 17/?-estradiol more avidly than the dia- nonradioactive 17fi-estradiol in the uptake solution. Thus, of phragms. The content of 17/?-estradiol in the tumor was re- the tissues investigated, only the tumor appeared to concen- duced by natural as well as by synthetic estrogen isomers. The trate estrogen in a selective manner. most estrogenic isomer was also that which most strongly re- Estrogenic activity is not confined to the natural estrogens. duced the concentration of 17fi-estradiol in the tumor. Methal- A great number of synthetic compounds, often structurally very lenestril had no effect; the corresponding free phenol was highly different from the natural estrogens, are also potent estrogens. active. Provided that the observed accumulation of 17/?-estradiol was Certain sulfhydryl reagents block the uptake by the mouse a consequence of its estrogenicity, synthetic estrogens should uterus of 17/?-estradiol. At least one of the sulfhydryl reagents, behave similarly. The stereoisomers of the synthetic estrogen NEM, also accelerates the wash-out of accumulated 17/?-estra- hexestrol have very different estrogenicities. From experiments diol from the uterine tissue (24). For comparative purposes with immature mice, it is known that the potent meso-hexestrol, similar experiments were carried out on the tumor. It was but not the hundred-fold less active racemic hexestrol, is taken found ~hat NEM strongly reduced the level of 17fi-estradiol in up by the uterus more avidly than by striated muscle tissue the tumor (Table 6), whether present during the uptake or in vivo (21) and in vitro (22). In wash-out experiments meso- wash-out phase of the experiment. The presence of NEM dur- hexestrol is more strongly retained by the uterus than by the ing the uptake phase of the experiment elevated the diaphragm diaphragm. Racemic hexestrol, however, is strongly retained to level above the controls. The selectivity of the NEM blockade a high extent both by the uterus and the diaphragm (25). or' estrogen uptake by the tumor is further stressed by this Analogous experiments with tumor slices were performed and finding. similar observations were made (Table 4). The potent estrogen, Intact and Pregnant Animals in Vitro. Tumors from intact meso-hexestrol, was much more strongly retained by the tmnor (not castrated) animals were used in a few experiments for than by the diaphragm, while the difference is uncertain with comparative purposes. The tumors had developed early after racemic hexestrol. The retention of racemic hexestrol by both the DMBA treatment and were rftpidly growing at the time of tissues, however, was much higher than that of the meso- isomer. use. Such tumors are by experience the best candidates for Some additional experiments were performed in order to estrogen dependence. As can be seen from Table 7, these tumors ascertain the specificity of the accumulation in the tumor. No also preferentially concentrated tritiated 17/?-estradiol, the re- more estrogens labeled to a high specific activity were avail- tention of which was reduced by isotope dilution. able to the author. It was possible, however, to analyze the It is well established that pregnancy strongly accelerates problem indirectly. It has been shown for the mouse uterus that mammary tumor growth in D~IBA-treated rats (11, 16). Ani- unlabeled estrogens inhibit the selective uptake of radioactive mals which did not develop fast-growing mammary tumors for 17fi-estradiol in a very specific manner (22). In order to fur- several months were used for experiments. They were mated ther sharpen the analysis, stereoisomers were used: (a) the and, in most animals, tumors grew out rapidly. Pregnancy is stereoisomers of hexestrol and (b) 17a-estradiol and 17/?- thus the probable reason for the sudden tumor growth. After estradiol, the a-isomer being a hundred times less estrogenic about 15 days of pregnancy rapidly growing tumors were used

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Table 5

Content of radioactivity Rat No. of Addition Tumor Diaphragm Tumor No. slices during uptake ~edium Medium Diaphragm 4 None a 2.21 ~ 0.11 c 0.24 ~ 0.02 9.2 ~ 0.90 11 6 17a-Estradiol, 0.63 __+ 0.02 021 • 0.01 3.0 • 0.17 0.3 #gb 6 17fl-Estradiol, 0.25 -+- 0.01 0.23 + 0.02 1.1 -+- 0.10 0.3 #g

6 None 1.4 + 0.06 0.23 • 0.02 6.0 _ 0.27 12 6 17a-Estradiol, 0.47 __+ 0.03 0.26 ~ 0.01 1.8 ~ 0.14 0.3 #g 6 17fl-Estradiol, 0.19 ~ 0.02 0.24 • 0.01 0.77 + 0.09 0.3 #g

4 None 0.52 ~ 0.02 0.26 -+- 0.03 2.0 _ 0.24 13 6 17~-Estradiol, 0.41 -+- 0.02 0.24 ___ 0.01 1.7 • 0.11 (Slow- 0.3 ~g growing 6 17fl-Estradiol, 0.33 -+- 0.01 0.29 • 0.01 1.1 • 0.05 tumor) 0.3 #g

4 None 0.92 -+- 0.05 0.25 +__ 0.01 3.6 • 0.25 14 6 racemic Hexestrol, 0.63 ~ 0.02 0.23 -+- 0.01 2.7 ~ 0.15 0.9 #g 6 meso-tiexestrol, 0.31 • 0.01 0.23 • 0.01 1.4 • 0.07 0.9 #g

4 None 1.1 • 0.09 0.22 + 0.02 5.0 • 0.61 15 6 racemic ttexestr01, 0.79 + 0.04 0.23 ~ 0.01 3.5 -+- 0.23 0.9 #g 6 meso-ttexestrol, 0.48 ~ 0.03 0.19 +__ 0.01 2.5 -+- 02,1 0.9 #g

4 None 0.34 +__ 0.002 0.25 -+- 0.01 1.4 • 0.06 16 6 racemic Hexestrol, 0.36 • 0.01 0.26 -+- 0.01 1.4 ~ 0.06 (Partly 0.9 #g necrotic 6 meso-Hexestrol, 0.37 -+- 0.01 0.25 ___ 0.01 1.5 • 0.07 tumor) 0.9 #g

4 None 0.84 __+ 0.08 0.30 _ 0.01 2.8 • 028 17 6 Methallencstril, 0.88 • 0.05 0.32 -+- 0.01 2.7 __+ 0.18 10 #g 6 Methallenestril 0.33 -4- 0.01 0.27 -+- 0.01 1.2 • 0.06 (free phenol), 10 #g Effect of natural and synthetic estrogen isomers on the retentiori of 17fl-estradiol in the DMBA tumor and diaphragm in vitro. a See Group C, Table 1. b Preincubation for 1 hour at 37~ in a mixture of indicated amounts of unlabeled inhibitor and 0.003 #g 17fl-estradiol-3H in 6 ml of KRPA (see Table 1); then incubation as controls for 1 hour at 37~ with 0.3 #g unlabeled 17fl-estradiol in 6 ml of KRPA. c Mean -+- S.E. in i~ vitro experiments (Table 7). There was a high preferen- Ovariectomized Animals in Vivo/in Vitro. The in vitro tial concentration of 17fl-estradiol in these tumors. experiments showed that the DMBA tumor retains estrogen In Chart 1 all observed tumor/diaphragm concentration more strongly than the diaphragm (Table 1). In the following ratios of 17fi-cstradiol following uptake and wash-out under experiments, tumors which had been loaded in vivo were sub- "standard conditions" are depicted. There is considerable jected to wash-out in vitro under the "standard conditions." scatter among the values but tumors from ovariectomized ani- One hour after the intravenous injection of 17fl-estradiol the mals have concentration ratios centered around 4; in intact content of radioactivity in tumor tissue was about 10 times animals the ratio is perhaps a little lower and pregnant animals that of the diaphragm and half to a fourth of that of the have high values. uterus (Table 8). Following wash-out in vitro about 50% of

332 CANCER RESEARCH VOL. 28

Table 6

Part of experiment Content of radioactivity Rat No. of with NEM addition Tumor Diaphragm Tumor N'o. slices Uptake Wash-out Medium Medium Diaphragm 8 -- __a 0.53 _ 0.05 d 0.14 ___ 0.01 3.7 +_. 0.45 18 8 -- _fib 0.18 • 0.02 0.17 -+" 0.02 1.1 _ 0.17

6 -- -- 0.73 • 0.05 0.15 + 0.01 5.0 ___ 0.47 19 4 .fi _c 0.28 • 0.03 0.34 _ 0.01 0.83 --4-_ 0.08 6 -- Jr- 0.27 • 0.03 0.19 ___ 0.01 1.4 ~ 0.18

6 -- -- 0.69 ___ 0.04 0.16 + 0.01 4.3 -+- 0.37 20 6 .fi -- 0.20 ___ 0.01 0.41 _ 0.01 0.49 ~ 0.03 6 -- .fi 0.27 __+ 0.01 0.21 __+ 0.01 1.3 -+- 0.08 Effect of NEM (N-ethylmaleimide) on the retention of 17fl-estradiol by the DMBA tumor and diaph,-agm in vitro. a See Group C, Table 1. b Preincubation for 1 hour at 37~ with 0.003 #g 17fl-estradiol-aH; then incubation for 15 rain at 37~ with 6 ml of 10 -3 M NEM in KtlP; finally, incubation for 1 hour at 37~ in 6 ml of 10 -a M NEM in KRPA (see Table l) with 0.3 pg unlabe!ed 17fl-estradiol. CPreincubation for 15 rain at 37~ in 6 ml of KRP with 10 -3 _',~ NEM; |hen incubation for ] hour at 37~ in 6 ml of IKRPA containing 10-:~ _~ NEM and 0.003 #g 17fl-estradiol-3H; finally, incubation for 1 hour at 37~ in 6 ml of KRPA with 0.3 #g unlabeled 17fi-estradiol. Mean _+ S.E.

Table 7

Content of radioactivity ~ Rat Tumor Diaphragm Tumor No. Treatment Mediun~ Medium Diaphragm 21, intact A. Uptake a 2.7 -+- 0.18 2.2 _ 0.18 1.2 --+-_ 0.13 B. Wash-outb 0.82 • 0.02 0.43 _ 0.01 1.9 ~ 0.07 C. Saturated uptake, 0.43 • 0.01 0.39 _ 0.01 1.1 ~ 0.04 wash-outC

22, intact A. Uptake 3.0 • 0.24 2.1 -+- 0.18 1.4 ~ 0.17 B. Wash-out 0.95 • 0.07 0.36 -+- 0.02 2.7 _ 0.24 C. Saturated uptake, 0.34 ___ 0.02 0.36 -+- 0.01 0.9 -+- 0.06 wash-out

23, intact A. Uptake 3.4 ___ 0.21 2.6 _ 0.05 1.3 _ 0.08 B. Wash-out 1.5 _+ 0.09 0.43 _+ 0.02 3.5 -+- 0.27 C. Saturated uptake, 0.38 ~ 0.01 0.45 ~ 0.02 0.8 -+- 0.04 wash-out

24, intact A. Uptake 4.6 -+- 0.39 2.5 ~ 0.03 1.9 _ 0.16 B. Wash-out 1.5 -+- 0.09 0.39 • 0.0i 3.9 +_ 0.25 C. Saturated uptake, 0.34 • 0.02 0.38 • 0.02 0.9 _ 0.07 wash-out

25, pregnant A. Uptake 5.2 _ 0.24 2.1 • 0.10 2.5 _ 0.16 B. Wash-out 2.8 _ 0.19 0.40 ~ 0.03 7.1 _ 0.71

26, pregnant A. Uptake 8.0 __+ 1.0 2.1 +__ 0.07 3.9 ~ 0.49 B. Wash-out 2.5 -+- 0.19 0.35 _+ 0.03 7.1 _ 0.82 Uptake and retention itt vitro of 17fl-estradiol by the DMBA tumor and diaphragm from intact and pregnant rats. a See Group A, Table 1. b See Group C, Table 1. c See Group C, Table 2. Mean -~ S.E. There were 6 slices/group.

FEBRUARY 1968 333

the accumulated activity was retained by the tumor tissue. On hour after the injection. Following wash-out in vitro about 50% treatment with NEM much less activity was retained. remained in the tumor. Exposure to NEM significantly reduced Table 9 depicts a similar experiment with the hexestrol this retention (P ~ 0.01). The content of racemic hexestrol in stere 9 Because the radioactive isomers were in short the tumor, on the other hand, was actually lower than that of supply, only a few experiments were made and low activities the diaphragm at 1 hour after injection. Following wash-out had to be used. The content of meso-hexestrol in tumor was the retention by the tumor was, if anything, less than that of 3-4 times that in diaphragm and a third of that in uterus 1 the diaphragm. NENI caused a slight reduction of the tumor content of radioactivity (P -- 0.02) but it had no effect on the 10 diaphragm. Chemical Identification of Radioactivity Accumulated by the Tumors. Tissue samples from tumors which had accumulated radioactivity in vivo or in vitro were extracted with cold 8 - ethanol and the ethanolic extract subjected to partition. The predominating phenolic fraction was also subjected to thin-layer z 9 9 o 7 chromatography. The partition behavior and chromatographic mobility of the radioactivity corresponded well to authentic -1- O 6 compounds (Table 10). p- a The phenolic fraction was also cocrystallized with a large Ol 9 O o amount of carrier. The specific activities of successive crystal OoOo 9 crops remained essentially constant (not illustrated). E o. o| 9 DISCUSSION

(o) 9 9 The high level of the potent estrogens, 17fi-estradiol or meso- ( 9 9 hexestrol in the DMBA tumor after systemic administration ( 9 in vivo is not easily reduced by a subsequent wash-out in the presence of a more-than-thousand-fold excess of estrogen I SPAYED I INTACT I PREGNANT I (Tables 8, 9). Tumor slices loaded in vitro with these estrogens also retain them during the wash-out much better than the dia- Chart 1. Content of 17fl-estradiol in tumor tissue relative to phragm strips (e.g., Table 1). The mechanism for estrogen diaphragm tissue following incubation in vitro. Experimental con- maintenance in the tumor is, therefore, probably not continuous ditions were identical to those of Group C, Table 1. There were 4-8, in most cases 6, slices of each tissue per point. Points within pumping. A more probable mechanism would be binding. This parentheses refer to partly necrotic or exceptionally slow-growing assumption is supported by the findings by others that protein tumors. fractions in estrogen-responsive tissues such as the calf uterus

Table 8

Amount Tumor Rat injected a Treatment Content of radioactivity (dpm/mg) a content, % No. (tLg) in vitro Uterus Tumor Diaphragm of untreated None 2166 -+- 89 567 __+ 21 57 -+- 2 27 0.5 Wash-outb 310 _ 13 55 NEM, wash-cute 107 -+- 14 19 None 575 -+- 20 309 + 17 28 • 1 28 0.2 Wash-out 154 ___ 17 50 NEM; wash-out 34 _ 5 11 None 111_ 8 9• 29 0.1 Wash-out 61 • 3 55 NEM, wash-out 8 + 2 7 Uptake and effect of N-ethylmaleimide (NEM) on the retention of 17fl-estradiol by the DMBA tumor and diaphragm after systemic administration. a Injected intravenously in 0.3 ml saline into 240- to 260-gm rats, killed 1 hour after injection. b Incubation for 1 hour at 37~ in 6 ml of KRPA (see Table 1) with 0.3 #g unlabeled 17fl- estradiol. r Incubation for 15 min at 37~ in 6 ml of KRP with 10-3 M NEM; incubation for 1 hour at 37~ in 6 ml of KRPA with 10 -3 M NEM and 0.3 #g unlabeled 17fl-estradiol. a Mean _ S.E. There were 6 slices/group. Diaphragm activity after wash-out was too low to be measured with accuracy (~10 dpm/mg).

334 CANCER RESEARCH VOL. 28

Table 9

Content of Radioactive radioactivity, % Rat estrogen Treatment Content of radioactivity (dpm/mg) e of untreated No. (//.g)a in vitro Uterus Tumor Diaphragm Tumor Diaphragm meso- 30 Itexestrol, 0.1 A. None 15 _ 1 4

meso- A. None 187 -+- 4 54 • 5 15 _ 2 31 I-Iexestrol, 0.4 B. Wash-outb 30 ~ 3 3 52 C. NEM, wash-outc 17 _ 2 4 32

racemic A. None 6 4- 0.3 8 • 2 32 ttexestrol, 0.1

racemic A. None 68 • 2 77 • 27 97 • 2 33 Hexestrol, 0.4 B. Wash-out 34 4- 1 53 __+ 4 44 54 C. NEM, wash-out 26 • 3 56 • 1 34 58 Uptake and effect of N-ethyhnaleimide (NEM) on the retention of radioactive hexestrol isomers by the DMBA tumor and diaphragm after systemic ~dministration. a Injected intravenously in 03 ml saline into 240- to 260-gm rats, killed 1 hour after injection. b Incubation for 1 hour at 37~ in 6 ml of KRPA (see Table 1) with 0.6 ttg of the appropriate unlabeled isomer. c In(~ubation for 15 rain at 37~ in 6 ml of KRP with 10-3 ~{ NEM; incubation for 1 hour at 37~ in 6 ml of KRPA with 10 -a M NEM and 0.6 #g unlabeled hexestrol isomer. a Mean _+ S.E. There were 6 slices/group. Radioactivity <10 dpm/mg must be considered uncertain.

Table 10

Chromatography of Fraction from partition a phenolic fraction a Radioactive Water- Estradiol Estrone Estriol estrogen Treatment Protein soluble Lipid Phenolic zone zone zone 17fi-Estradiol In vivoa 2.0 0.8 7.0 90.2 89.0 3.2 1 17fl-Estradiol In vitrob 2.9 1.0 10.6 85.5 97.0 1 <1 17fl-Estradiol In vitro 2.9 6.0 21.6 69.5 93.6 2 15 DMBA tmnors have a lower uptake of 17/?-estradiol than the following wash-out. On the other hand, several nontarget mouse responsive ones. It has also been observed that there is a higher tissues (stomach, spleen, kidney, heart, or thymus) do not re- accumulation of radioactive meso-hexestrol in the human breast tain 17fi-estradiol in vitro; the tissue/diaphragm concentra- tumors which respond to bilateral adrenalectomy and oophorec- tion ratios become less than 2 following wash-out (25). The tomy than in those tumors which show no response (7). There

FEBRUARY 1968 335

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1968 American Association for Cancer Research. Lars Terenius is thus a good deal of evidence that estrogen-responsive breast activity of which could be measured separately from low energy tumors have the property in common with the reproductive /3-emitters such as 14C and all. However, such a method cannot organs to concentrate estrogens, while the unresponsive tumors correct for nonspecific uptake in a lipid-rich tissue. By the use do not. of albumin in the incubation media and a subsequent washing, Only the potent estrogens were found to be retained in the however, one can reduce the interference of nonspecific uptake DMBA tumor. The potent synthetic estrogen meso-hexestrol (Table 3). Jensen et al. (13) have also developed methods for was more strongly retained by the tumor both in vitro (Table the analysis of interactions between estrogens and tissue slices. 4) and in vivo (Table 9) than by the diaphragm. On the other They incubate DMBA tumor slices and diaphragm strips in hand, the much less estrogenic racemic hexestrol was not pref- saline media with 10 -l~ M tritiated 17fl-estradiol. After 1 hour erentially retained by the tumor in vitro or in vivo. Isotope incubation at 38~ the tumor/diaphragm concentration ratio dilution demonstrated that the selective retention of 17fl- was 3.7, after 4 hours, 4.0. The effect of the anti-estrogen estradiol in the tumor was saturable (Table 2). The structure U-11100A on in vitro uptake was also studied. Tumor slices specificity of the estrogen retention was also demonstrated by (or uterus slices) and diaphragm strips were incubated in inhibition analysis. The dilution of radioactive 17fl-estradiol saline media with 10-1~ tritium-labeled 17fl-estradiol with with the much less potent 17a-estradiol, the stereoisomeric and without 10 -5 M U-11100A. The concentration of radio- hexestrols, and another synthetic estrogen (a methallenestril activity in the DMBA tumor (and in the rat uterus) was analog) suppressed the level of radioactivity in the tumor but strongly reduced by U-11100A while the concentration in the not in the diaphragm. It appeared that those substances which diaphragm was slightly elevated. This finding has been con- are potent estrogens suppressed the retention of 17fl-estradiol finned with the present experimental conditions. (U-11100A in the tumor more strongly than the less active isomers (Table was added at 2 • 105 times molar excess over the concentra- 5). Such activity differences have been found in similar ex- tion of radioactive 17fl-estradiol, and the experiments were periments with the mouse uterus (22, 25). Both tissues thus performed as in Table 5. The tumor level of radioactivity was appear to concentrate estrogens in a specific manner. This reduced about 50%.) The inhibition by an anti-estrogen of the finding might have a bearing on the controversy, whether or uptake of estrogen provides additional evidence that the inter- not the effects of estrogen on the DMBA tumor are mediated action between tissue and estrogen is specific. In the present via the pituitary (6). Sterental et al. (19) claim that estrogens author's opinion, however, it seems safer to use estrogens in stimulate DMBA-induced tumor growth only indirectly since the inhibition analysis than an anti-estrogenic compound such they act only in the presence of the pituitary. This may be so, as U-11100A, which radically differs in structure from the po- but the high level of potent estrogens in the tumor makes one tent estrogens and which has to be used at a great concen- suspect that they act there and directly stimulate tumor growth, tration excess. and that absence of pituitary inhibits growth by other mecha- Most radioactivity present in the DMBA tumor after the nisms. The situation would be analogous to that of the mam- injection of, or the incubation with, 17fl-estradiol or meso- mary gland in rats (1). hexestrol was found to behave as the original compounds by Tumors differed considerably in ability to retain 17fi-estra- chromatography (Table 10) and crystallization. King et al. diol in vitro (Chart 1). A similar scatter in uptake of 17fl- (15) made the same observation with regards to 17fl-estradiol estradiol in vivo between different DMBA tumors was found in the DMBA tumor by the use of other methods (column by Mobbs (17). This individuality of the tumors is not un- and paper chromatography as well as crystallization of the expected since they differ macroscopically and have different original compound and acetylated products). Incubation of growth patterns. The in vitro or combined in vivo and in vitro DMBA tumor slices with comparatively large amounts of 17fl- technics used in this paper are thus advantageous because they estradiol result in some metabolism; estrone and estriol appear permit the use of a single tumor in both control and various as metabolites (4). ttowever, these metabolites are evidently experimental groups. Moreover, fundamental mechanisms of not retained by the tumor tissue after wash-out (Table 10). uptake and retention are more easily studied in vitro and such methods may be useful in similar investigations on human ACKNOWLEDGMENTS neoplasms. In vitro methods for the study of sex hormone uptake to I would like to thank Dr. Charles B. Huggins who generously human breast tumors have been reported briefly by other work- donated breeding animals and DMBA in lipid emulsion. Dr. E. V. ers (3, 12). The uptake of radioactive testosterone, proges- Jensen kindly made available unpublished manuscripts. Mrs. terone, or 17fl-estradiol by tumor slices in protein-free saline Carola Engstr6m offered competent technical assistance. media was studied. In such media there is a high degree of nonspecific uptake of lipophilic substances such as the sex REFERENCES steroids (cf. Table 3). James and Braunsberg (12) actually report that the uptake of 17fl-cstradiol as well as of testoster- 1. Ahr6n, K., and Jacobsohn, D. Mammary Gland Growth in Hypophysectomized Rats Injected with Ovarian Hormones one by adipose tissue was 5 to 10 times higher than by breast and Insulin. Acta Physiol. Scand., 87: 190-201, 1956. tumors or "normal" breast tissue. In a recent report they de- 2. Braunsberg, H., and Guyver, A. Automatic Liquid Scintilla- scribe a method which corrects for the amount of medium tion Counting of High Energy fl-Emitters in Tissue Slices and radioactivity left in and on the tissue samples after incubation Aqueous Solutions in the Absence of Organic Scintillator. (2). To the incubation medium 2*NaC1 was added, the radio- Anal. Biochem., 10: 86-95, 1965.

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3. Braunsberg, H., and James, H. T. Studies on the Uptake of nism of Estrogen Action. Recent Progr. Hormone Res., 18: Steroid Hormones by Human Tissues in vitro. Biochem. J., 387-408, 1962. 90: 15P, 1964. 15. King, R. J. B., Cowan, D. M., and Inman, D. R. The Uptake 4. Breuer, H., Breuer, J., and Schm~ihl, D. Stoffwechsel yon of (6,7-3H) Oestradiol by Dimethylbenzanthracene-induced 0stradiol-17fl in dem durch 7,12-Dimethyl-benzanthrazen- Rat Mammary Tumours. J. Endocrinol., 32: 83-90, 1965. induzierten Mammarcarcinom der Ratte. Z. Krebsforsch., 57: 16. McCormick, G. M., and Moon, R. C. Effect of Pregnancy and 247-254, 1965. Lactation on Growth of Mammary Tumours induced by 7,12- 5. Cohen, P. P. Suspending Media for Animal Tissues. In: W. W. Dimethylbenz(a)anthracene (DMBA). Brit. J. Cancer, 19: Umbreit, R. H. Burris and J. F. Stauffer (eds.), Manometric 160-166, 1965. Techniques and Tissue Metabolism, Ed. 4, pp. 131-133. 17. Mobbs, B. G. The Uptake of Tritiated Oestradiol by Dimethyl- Minneapolis: Burgess Publishing Co., 1964. benzanthracene-induced Mammary Tumours of the Rat. J. 6. Dao, T. L. Carcinogenesis of Mammary Gland in Rat. Progr. Endocrinol., 36: 409-414, 1966. Exptl. Tumor Res., 5: 157-216, 1964. 18. Pearlman, W. H., De Hertogh, R., Loumas, K. R., Bruegge- 7. Folca, P. J., Glascock, R. F., and Irvine, W. T. Studies with mann, J. A., and Pearhnan, M. R. J. Metabolism and Tissue Tritium-Labelled Hexoestrol in Advanced Breast Cancer. Uptake of Steroid Sex Hormones in Patients with Advanced Lancet, 2: 796-798, 1961. Carcinoma of the Breast and in Normal Rats. In: G. Pincus, 8. Glascock, R. F., and Hoekstra, W. G. Selective Accumulation T. Nakao and J. F. Tait (eds.), Steroid Dynamics, p. 159-175. of Tritium-Labelled Hexoestrol by the Reproductive Organs New York: Academic Press, 1966. of Immature Female Goats and Sheep. Biochem. J., 72: 673- 19. Sterental, A., Dominguez, J. M., Weissman, C., and Pearson, 682, 1959. O. H. Pituitary Role in the Estrogen Dependency of Experi- 9. Huggins, C., Grand, L. C., and Brillantes, F. P. Mammary mental Mammary Cancer. Cancer Res., 23: 481484, 1963. Cancer Induced by a Single Feeding of Polynuclear Hydro- 20. Stone, G. M., Baggett, B., and Donnelly, R. B. The Uptake of carbons, and its Suppression. Nature, 189: 204-207, 1961. Tritiated Oestrogens by Various Organs of the Ovariectomized 10. Huggins, C., Grand, L., and Fukunishi, R. Aromatic Influ- Mouse following Intravenous Injection. J. Endocrinol., 27: ences on the Yields of Mammary Cancers following Adminis- 271-280, 1963. tration of 7,12-Dimethylbenz(a)anthracene. Proc. Natl. Acad. 21. Terenius, L. The Uptake of Radioactive Isomers of a Syn- Sci. U.S., 51: 737-742, 1964. thcti(', Oestrogen in Various Organs of Immature Mice. Acta 11. Huggins, C., Moon, R. C., and Morii, S. Extinction of Experi- Endocrinol., 53: 84-92, 1966. mental Mammary Cancer I. Estradiol-17fl and Progesterone. 22. Terenius, L. Specific Uptake of Oestrogens by the Mouse Proc. Natl. Acad. Scl. U.S., ~8" 379-386, 1962. Uterus b~ Vit~'o. Acta Endocrinol., 58: 611-618, 1966. 12. James, H. T., and Braunsberg, H. Metabolism of Steroid 23. Terenius, L. Inhibition of 17fl-Oestradiol Uptake in Mouse Hormones. Ann. Rept. Brit. Empire Cancer Campaign, 41 (2): Uterus by Doisynolic and Allenoic Acid Derivatives: An in 326-328, 1963. Vitro Differentiation between True Oestrogens and Pro- 13. Jensen, E. V., DeSombre, E. R., and Jungblut, P. W. Estrogen Oestrogens. Acta Pharnlac'ol. Toxicol., 25: 313-322, 1967. Receptors in Hormone Responsive Tissues and Tumors. In: 24. Terenius, L. SH-Groups Essential for Estrogen Uptake and R. W. Wissler, T. L. Dao and S. Wood, Jr. (eds.), Endogenous Retention in the Mouse Uterus. Mol. Pharmacol., 8: 423-428, Factors Inttuencing Host-Tulnor Balance. Chicago: Chicago 1967. University Press, 1967. 25. Terenius, L. Oestrogen Binding in the Uterus of Immature 14. Jensen, E. V., and Jacobson, H. I. Basic Guides to the Meeha- Mice. Acta Endocrinol., in press.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1968 American Association for Cancer Research. Selective Retention of Estrogen Isomers in Estrogen-dependent Breast Tumors of Rats Demonstrated by in Vitro Methods

Lars Terenius

Cancer Res 1968;28:328-337.

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