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Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo
J. steroid
Biuchem.
0022-4731:8453.00+ 0.00
Vol. 20, No. 48. pp. 1033-1037.1984
Copyright @, 1984 Pergamon Press Ltd
Printed in Great Britain. All righu nserwd
BIOLOGY
AND RECEPTOR INTERACTIONS OF ESTRIOL AND ESTRIOL DERIVATIVES IN VI7’Ml AND IN VIVO
BENTA S. KATZEN~LL~~~~N Department of Physiology and Biophysics, University of Illinois and University of Illinois College of Medicine, Urbana, IL 61801, U.S.A. Snmmary-The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in uiuo and in oifro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely. l7z-ethynyl estriol, estriol-3-cyclopentyl ether, and 17z-ethynyl estriol-3cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in ho. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endomettial cells in primary tissue culture, E, has 12% the affinity of estradiol (EJ for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentmtions of El (IO-“’ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E, to be only slightly less effective than E,. In these endomettial and breast cancer cell systems in z:ifm, there is no metabolism of E, while E, is metabolized to estrone. Hence, estriol is an effective estrogen in rim. In viuo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.
INTRODUCtION It has long been known that the biological potency of a compound, which depends on its persistence in the tissue, is a function both of receptor affinity and clearance from the circulation. In z&o, estriol shows an activity that is of short duration and it has frequently been referred to as a weak or impeded estrogen [I -3). In this paper, we compare the biopotency of estriol (E,) in three systems-rat uterus, human endometrial cells and MCF-7 human breast cancer cells-and we compare the potency and effects of E, in vitro and in vivo. In these tissues and cells, E, has modest affinity for receptor, approx 20-30x that of estradiol (E, ) and, at least based on the end points we have monitored, it evokes a similar spectrum of effects as does E,, if the effective concentration of E, is kept equivalent to that of E,. In vitro, where clearance is not a factor, E, appears to be especially effective as an estrogen, being more effective than is seen for E, in viva
EXPERIMENTAL
Studies in rats utilized immature female Holtzman rats (from Holtzman Company, Madison, WisconAddress for correspondence: Department of Physiology and Biophysics, 524 Burt-ill Hall, University of Illinois, 407 South Goodwin Avenue, Urbana, IL 61801, U.S.A.
sin) 19-23 days of age. Estriol and related compounds were administered sc in saline once daily [4]. Induced protein synthesis and receptor translocation was determined in whole immature rat uteri incubated in vim as described ($61. Human endometrial primary cell cultures were prepared and maintained exactly as described previously and estrogen receptor and progesterone receptor levels were determined in cultured cells utilizing [3H]estradiol and the synthetic progestin [‘H]R5020 [7,8]. MCF-7 human breast cancer cells (from Dr M. Lippman, National Institutes of Health, Bethesda, Maryland) were grown in plastic T- 150 flasks in Improved Minimal Essential Medium (IMEM, Gibco) containing IOmM HEPES buffer, gentamicin (50 fig/ml), penicillin (100 U/ml), streptomycin (0.1 mgjml), hydrocortisone (3.75 ngjml), insulin (Lilly, 0.02 U/ml) and So/, fetal calf serum. To determine the effects of E, and E, on ceil growth, MCF-7 cells were grown in IMEM medium described above but containing 5% charcoal dextrantreated calf serum for 2 weeks prior to being seeded into T-25 flasks for growth experiments. At 2 days after seeding (ca 1.5 x lO’cells/flask), various concentrations of E,, E,, or vehicle (0.1% ethanol) were added and cells were grown in this IMEM medium with all ingredients except insulin and containing 5”/, charcoal treated calf serum for 12 days. Media were changed every 2 days, and triplicate flasks of cells were counted at several time points during the growth period.
1033
1034
Fig. I. Concentrations of estradioi, estriol and estrone required for 50% saturation for cytosot (C) and nuclear(N) binding sites and 50y0 of maximal induction of estrogeninduced protein (IP) synthesis. From Ref. 6.
RESULTS
AND DISCUSSION
in the rat uterus, a carefully programm~ time sequence of events OCCWS after estrogen administration [I, 9, IO]. One of the earliest responses to estrogen involves synthesis of a protein, termed induced protein by Notides and Gorski[ll], now identified as the BB isozyme of creatine kinase [ 121. The synthesis of this protein can be elicited by estrogens in tritro [S, S] and there is a good correlation between the magnitude of nuclear estrogen receptor levels at 1 h after estrogen and the magnitude of induced protein stimulation. In studies with rat uteri iz vitro, a comparison of the naaomolar concentrations of estradioi. estriol and estrone required to elicit 50% occupancy of cytosol estrogen receptors, 50% translocation of receptor to the nucleus and 50% of maximal stimulation of induced protein synthesis (Fig. 1) reveals that El is quite an effective estrogen. Only two times as much E!, as Ez is needed to give 50% of maximal receptor localization in the nucleus and 50y0 of maximal induced protein stimulation.
Fig. 2. Dose-response curves showing the ut~rotrophic activities of estdol, estradiol and estriol derivatives. Rats (21-days old) were injected subcutaneously with the indicated daily dose of compound in 0.5 ml of saline once daily at 24-h intervals on three successive days, and uterine wet weights were determined at 24 h after the last injection. Control animals received saline alone. Each value is the mean of determinations from at least five individual animals f SEM. E,, estradiol; E,, estriol; EE,, 17a-ethynyl estriol; E, CPE, estriol-3-cyclopentyl ether; EE,CPE, 17x-ethynyl estriol-3-cyclopentyl ether. From Ref. 4.
Under it? t&o conditions, however. E, appears a much weaker estrogen (1. 21,and high concentr~~ti~~~s of E, are required to evoke substantial increases in uterine weight. We have studied the basis of the weak uterotrophic activity of E, and the effects of chemical mod~ficati~?n of E, on the biopotency of E;. The modifications we have examined introduce an ethynyl group at the 17%position to reduce metabolic inactivation of the compound and to increase the binding affinity for the estrogen receptor and a cyclopentyl ether group at the 3 position, to provide a prohormonal form of the estrogen. These modi~~d~ions~ it was believed, should increase the biological persistence of the estriol molecule. The compounds we have studied are estriol (EJ and several E,-derivatives prepared by the Eli Lilly Company, namely, 17x-ethynyl estriol (EE,), estriol-3-cyc~openty~ ether (E,CPE), and 17~-ethynyl estriol-3-cyclopen~yl ether (EE,CPE). Dose--response curves indicating the effectiveness of these compounds in increasing uterine weight are plotted in Fig. 2. It is clear that E, is the least elective of the compounds tested. Addition of a cyclopentyl ether group at the Sposition of estriol (E,CPE) shifts the dose-response curve slightly to the left, but E,CPE appears to be a significantly better estrogen than estriol only when given at a high dose (10 [lg;rat). If an ethynyl group is introduced at the 17~ position of estrioi (EE,), the estrogenic activity is increased dramatically. And the very high estrogenic potency of EE,CPE is apparent at dose Ievels above 0.3 /ig/day. After a single injection of 5 pg doses of these compounds, the prolonged and marked estrogenic effect of EEJIPE on uterine weight can be clearly seen (Fig. 3). All compounds elicit an early (3-6 h) increase in uterine weight. While the onset of the increase is delayed in the case of EE,CPE (no increase at 1 h), presumably owing to the req~iremeut for metabolic activation in the animal, most striking is the very prolonged and marked elevation of uterine
036
I.2
24
36
HOURS
48
60
Fig 3. Uterine wet weight as a functiot~ of time after a singie injection of estriol, estradiol or estriol derivatives. Rats (21-days old) were injected with 5 /ig of compound or saline (controls) at the time indicated prior to killing and excision of uteri. Each weight value is the mean of determinations from 5 animals + SEM. From Ref. 4.
Estriol biology and receptor interaction
I 2-
-60
E2
IO
0
-70
11 3 6
g 12
24
36 HOURS
/ 48
60
7;’
Fig. 4. Time course of nuclear estrogen receptor levels and uterine wet weight after a single injection of 5 pg of EE,CPE, EE,, E, or E,. From Ref. 4.
weight seen after EECPE. Following the initial ca 6 h peak in uterine weight, there is a very prolonged and even more pronounced elevation of uterine weight at 36-48 h, with weight still remaining over 2-fold above the control after 72 h. FE, and E,, which are considerably more potent than E; in the 3-day uterotrophic assay, give a more prolonged weight elevation than does E,; yet neither of these compounds results in a uterine weight that is much above the control at 72 h. Hence, of the four compounds, EE,CPE clearly evokes the most pronounced uterine growth. Figure 4 compares the time course of nuclear receptor levels and uterine weight after a single subcutaneous injection of 5 pg of estriol, estradiol, or estriol derivatives. EE,, E, and E2 all elicit a rapid uptake of receptor into the nucleus and show an equivalent wet weight response at 3 h. However, there is a rapid decline in nuclear receptor levels after
1035
E, injection (at control by 24 h), and this is paralleled by a corresponding inability of E, to maintain uterine wet weight; nuclear receptor levels decline less rapidly after EE, or E, and uterine weight also remains elevated for a longer time. Of the four compounds, EE,CPE shows the most dramatic and prolonged maintenance of high nuclear receptor levels and elevated uterine weight. Thus, chemical modifications of the estriol molecule which result in a prolonged stimulation of uterine growth also result in a prolonged maintenance of hormone-receptor complexes in the nucleus. These studies indicate that E, is a weak estrogen because of its brief duration of interaction with receptor and gives strong support to the concept [9, 10, 13-151 that true uterine growth requires the direct and prolonged influence of the estrogen-receptor complex in the nucleus, A marked increase in plasminogen activator activity accompanies estradiol action in the uterus f16- 1S] and this increase in plasminogen activator activity is estrogen-sp~ific, being stimulated only by compounds that interact with the estrogen receptor. Figure 5 compares the efficacies of E3 and several other estrogens in causing elevation of uterine plasminogen activator activity (left panel) and in stimulating uterine weight gain (right panel). Although E, evokes only a slight increase in uterine weight over the doses examined, E, does evoke increases in plasminogen activator activity at the 1 and 10 pg doses. The more potent estrogens E, and diethylstilbestrol (DES) evoke increases in plasminogen activator activity at much lower hormone concentrations (0.03-0.1 pg}; and the decrease in uterine plasminogen activator activity at the higher concentrations of E, and DES is most likely due to a secretion of plasminogen activator from the uterine tissue into the luminal fluid in these highly stimulated uteri [18, 191. In primary cell cultures prepared from human endometrium, estradiol in vitro is able to increase the cellular levels of progesterone receptor in a concentration and time-dependent manner. In human endometrium, we find that E, has an affinity for estrogen receptor 12% that of E, and there is no metabolism of E, by the endometrial cells [8]. In these cell cultures, we have evaluated the effectiveness of E, and other compounds in increasing cellular progesterone receptor levels (Fig. 6). Values are expressed relative to maximal progesterone receptor stimulation by E,, which is arbitrarily set at lOO’%.These dose-response curves indicate the effectiveness in stimulating progesterone receptor levels to be: P1496 > 17/I-E, 2 17u-E, > E3 > estrone (E,) $ C1628. Estriol is weaker than Ez in stimulating progesterone receptor levels but E, is more effective than perhaps expected. This reflects the absence of E, metabolism, while E2 is substantially converted to E,
1036
BENITAS.
c
1
KAT~E~~LLENB~~~~
Estrone ./ ._4
1
5
600-
I
./
DAILY
DOSE
OF
ESTROGEN
(pgl
Fig. 5. Dose-response curves comparing the efficacies of estriol and several estrogenic compounds in causing elevation of uterine plasminogen activator activity (left panel) and uterine weight gain (right panel). Nineteen-day old animals (3 per group) received se. injections for three days of the indicated daily dose of 17/?-estradiol, 17x-estradiol, estrone, estriol, diethylstilbesterol (DES), or the fungal estrogenic compound zearalanol (P-1496). Values are the mean & SEM. Significant differences from controls at P < 0.01 (*) are represented only for 17/3-E,, from which comparisons can be made for the other estrogenic compounds. From Ref. 18.
by the endometrial
cells and the fact that a smaller of E, is bound by serum in the media (30% of added E, is serum bound while 70-80x of proportion
E, is serum bound) so that at each concentration, more E, is available for interaction with the cells. MCF-7 human breast cancer cells contain high levels of estrogen receptors and their growth is stimulated by E, in vitro. Figure 7 compares the dose-response curves for E, and E, stimulation of cell proliferation. It is clear that E, is nearly as effective N 160 w
I
,
1
,
T
, ?
?ii 140 c
I
0. I
I
I
I
IO
CONCENTRATION
as E, in stimulation of cell proliferation. Marked stimulation is achieved with lo-” M concentrations of both estrogens with IO-‘” M concentrations giving maximal stimulation. In these cells, El, has an affinity for receptor only 25% that of estradiol (20; A. Nardulli and B. S. Katzenellenbogen, unpublished). As in human endometrial cells, there is no metabolism of E, in the MCF-7 cells and, again, a larger proportion of the E, is free in the serum-media so that the effective, available E, concentration is higher at each added dose compared with E,.
I
1496
-i
I
I
loo
IO00
I
lO,cao
(nM)
I I I L Fig, 6. Ability of E, and other estrogens and antiestrogen I I , ,o-"i lo-~~ ,QIO lO-9 10-9 lo-7 10-9 C1628 to increase cytosol progesterone receptor in human endometrial cells. Cells were incubated with hormoneMOLAR CONCENTRATION containing media (changed daily) for 3 days. The cells were Fig. 7. Dose-response curves for the effects of E, and E, on then harvested and homogenized, and the progesterone receptor content was determined by hydroxylapatite assay the growth of MCF-7 breast cancer cells. Cells were grown using j3H]R5020. Results are expressed as the percent of in T-25 Aasks in medium lacking insulin but containing SO,:, maximal progesterone receptor increase caused by E,, with charcoal-dextran treated calf serum in the presence of the maxima1 increase defined as the response obtained after a indicated concentrations of E, or E, and media and hor3-day incubation with 1 x 10m8M E,. Each point represents mones were changed every 48 h. After 12 days of growth, the mean of 2-3 separate samples. Data obtained from 15 triplicate flasks of cells were counted. Data are expressed as separate uterine samples are included in this figure. From the percent of the control cell number measured after 12 days of growth and represent the mean & SEM. Ref. 8.
Estriol biology and receptor interaction These studies in rat uterus in vivo and in rat uterus in vitro
and in human endometrial and MCF-7 human breast cancer cells in vitro provide strong evidence that El is a very effective estrogen in vitro. Most target tissues do not metabolize E, and it has a moderate affinity (10-25x that of E,) for estrogen receptor. In vivo, E, is cleared quite rapidly and hence it appears to be an estrogen of low potency. However, if it is provided at effective levels over a period of time, it can be a very potent estrogen. Hence, when given either in a prohormonal form that is metabolized relatively little (such as EE,CPE) or is given by
repeated injection or by implant [21,22], E3 can be a quite potent estrogen, evoking a spectrum of biological responses similar to that elicited by estradiol. Acknowledgements-The
studies reported here were supported in part by NIH grants USPHS HD06726 and CA181 19. I am grateful to the collaborators from my laboratory in these studies, in particular, Richard Eckert, Mark Kneifel, Nancy Lan and Ann Nardulli. REFERENCES 1. Szego C. M. and Roberts S.: Steroid action and interaction in uterine metabolism. Recent Prog. Horm. Res. 8 (1953) 419-469. 2. Hisaw F. L.: Comparative effectiveness of estrogens on fluid inhibition and growth of the rat’s uterus. Endocrinology 64 (1959) 276-289. 3. Terenius L.: Structure-activity relationships of anti-
1037
estrogens in vitro. J. clin. Endocr. Metab. 52 (1981) 699-708. 9. Katzenellenbogen B. S. and Gorski J.: Estrogen actions on synthesis of macromolecules in target cells. In Eiochemical Actions of Hormones (Edited by G. Litwack). Academic Press. New York. Vol. 3 (1975) DD. 187-243. 10. Katzenellenbogen B. S.: Dynamics of steroid hormone 11 receptor action. A. Rev. Physiol. 42 (1980) 17-35. Notides A. C. and Gorski J.: Estrogen-induced syn’ thesis of a specific uterine protein. Proc. natn. Acad. Sci., U.S.A. 56 (1966) 230-235.
12. Reiss N. A. and Kaye A. M.: Identification of the major component of the estrogen-induced protein of rat uterus as the BB isozyme of creatine kinase. J. biol. Chem. 256 (1981) 5741-5749. 13. Anderson J. N., Peck E. J. Jr. and Clark J. H.: Nuclear receptor-estrogen complex: Relationship between concentration and early uterotrophic responses. Endocrinology 92 (1973) 1488-1495.
14. Anderson J. N., Peck E. J. Jr and Clark J. H.: Estrogeninduced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology % (1975) 160-164.
15. Gorski J. and Raker B.: Estrogen action in the uterus: The requisite for sustained estrogen binding in the nucleus. Gynec. Oncol. 2 (1974) 249-256. 16. Albrechtson 0. K.: Effect of estradiol on fibrinolytic activity of rat uterus. Proc. Sot. exp. Biol. Med. 94 (1957) 700-705.
17. Katz J., Troll W., Levy M., Filkens K., Russo J. and Levitz M.: Estrogen-dependent trypsin-like activity in the rat uterus. Archs Biochem. Biophys. 173 (1976) 347-356.
endocr., Copenh. 66 (1971) 431-438. 4. Lan N. C. and Katzenellenbogen
18. Kneifel M. A., Leytus S. P., Fletcher E., Weber T., Mange1 W. F. and Katzenellenbogen B. S.: Uterine plasminogen activator activity: Modulation by steroid hormones. Endocrinology 111 (1982) 493-499. 19. Peltz S. W., Katzenellenbogen B. S., Kneifel M. A. and Mange1 W. F.: Plasminogen activators in tissues of the immature and estrogen-stimulated rat uterus and in uterine luminal fluid: Characterization and properties.
(1976) 220-227. 5. Katzenellenbogen B. S. and Gorski J.: Estrogen action in vitro: Induction of the synthesis of a specific uterine protein. J. biol. Chem. 247 (1972) 1299-1305. 6. Ruh, T. S., Katzenellenbogen B. S., Katzenellenbogen
20. Lippman M., Monaco M. E. and Bolan G.: Effects of estrone, estradiol and estriol on hormone-responsive human breast cancer in long-term tissue culture. Cancer
with with oestrogens regard to interaction 17/I-oestradiol in the mouse uterus and vagina. Acta B. S.: Temporal relationships between hormone receptor binding and biological responses in the uterus: Studies with shortand long-acting derivatives of estriol. Endocrinology 98
J. A. and Gorski J.: Estrone interaction with the rat uterus: In vitro response and nuclear uptake. Endocrinology 92 (1973) 125-134. 7. Pavlik E. J. and Katzenellenbogen
B. S.: Human endometrial cells in primary tissue culture: Estrogen interactions and modulation of cell proliferation. J. clin.
Endocr. Metab. 47 (1978) 333-344. 8. Eckert R. L. and Katzenellenbogen
B. S.: Human endometrial cells in primary tissue culture: Modulation of progesterone receptor level by natural and synthetic
Endocrinology 112 (1983) 890-897.
Res. 37 (1977) 1901-1907.
21. Clark J. H., Paszko Z. and Peck E. J. Jr: Nuclear binding and retention of the receptor estrogen complex: Relation to the agonistic and antagonistic properties of estriol. Endocrinology 100 (1977) 91-96. 22. Clark J. H., Peck E. J. Jr, Hardin J. W. and Eriksson H.: The biology and pharmacology of estrogen receptor binding: Relationship to uterine growth. In Receptors and Hormone Action (Edited by B. W. O’Malley and L. Birnbaumer). Academic Press, New York, Vol. 2 (1978) pp. l-32.
Biuchem.
0022-4731:8453.00+ 0.00
Vol. 20, No. 48. pp. 1033-1037.1984
Copyright @, 1984 Pergamon Press Ltd
Printed in Great Britain. All righu nserwd
BIOLOGY
AND RECEPTOR INTERACTIONS OF ESTRIOL AND ESTRIOL DERIVATIVES IN VI7’Ml AND IN VIVO
BENTA S. KATZEN~LL~~~~N Department of Physiology and Biophysics, University of Illinois and University of Illinois College of Medicine, Urbana, IL 61801, U.S.A. Snmmary-The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in uiuo and in oifro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely. l7z-ethynyl estriol, estriol-3-cyclopentyl ether, and 17z-ethynyl estriol-3cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in ho. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endomettial cells in primary tissue culture, E, has 12% the affinity of estradiol (EJ for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentmtions of El (IO-“’ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E, to be only slightly less effective than E,. In these endomettial and breast cancer cell systems in z:ifm, there is no metabolism of E, while E, is metabolized to estrone. Hence, estriol is an effective estrogen in rim. In viuo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.
INTRODUCtION It has long been known that the biological potency of a compound, which depends on its persistence in the tissue, is a function both of receptor affinity and clearance from the circulation. In z&o, estriol shows an activity that is of short duration and it has frequently been referred to as a weak or impeded estrogen [I -3). In this paper, we compare the biopotency of estriol (E,) in three systems-rat uterus, human endometrial cells and MCF-7 human breast cancer cells-and we compare the potency and effects of E, in vitro and in vivo. In these tissues and cells, E, has modest affinity for receptor, approx 20-30x that of estradiol (E, ) and, at least based on the end points we have monitored, it evokes a similar spectrum of effects as does E,, if the effective concentration of E, is kept equivalent to that of E,. In vitro, where clearance is not a factor, E, appears to be especially effective as an estrogen, being more effective than is seen for E, in viva
EXPERIMENTAL
Studies in rats utilized immature female Holtzman rats (from Holtzman Company, Madison, WisconAddress for correspondence: Department of Physiology and Biophysics, 524 Burt-ill Hall, University of Illinois, 407 South Goodwin Avenue, Urbana, IL 61801, U.S.A.
sin) 19-23 days of age. Estriol and related compounds were administered sc in saline once daily [4]. Induced protein synthesis and receptor translocation was determined in whole immature rat uteri incubated in vim as described ($61. Human endometrial primary cell cultures were prepared and maintained exactly as described previously and estrogen receptor and progesterone receptor levels were determined in cultured cells utilizing [3H]estradiol and the synthetic progestin [‘H]R5020 [7,8]. MCF-7 human breast cancer cells (from Dr M. Lippman, National Institutes of Health, Bethesda, Maryland) were grown in plastic T- 150 flasks in Improved Minimal Essential Medium (IMEM, Gibco) containing IOmM HEPES buffer, gentamicin (50 fig/ml), penicillin (100 U/ml), streptomycin (0.1 mgjml), hydrocortisone (3.75 ngjml), insulin (Lilly, 0.02 U/ml) and So/, fetal calf serum. To determine the effects of E, and E, on ceil growth, MCF-7 cells were grown in IMEM medium described above but containing 5% charcoal dextrantreated calf serum for 2 weeks prior to being seeded into T-25 flasks for growth experiments. At 2 days after seeding (ca 1.5 x lO’cells/flask), various concentrations of E,, E,, or vehicle (0.1% ethanol) were added and cells were grown in this IMEM medium with all ingredients except insulin and containing 5”/, charcoal treated calf serum for 12 days. Media were changed every 2 days, and triplicate flasks of cells were counted at several time points during the growth period.
1033
1034
Fig. I. Concentrations of estradioi, estriol and estrone required for 50% saturation for cytosot (C) and nuclear(N) binding sites and 50y0 of maximal induction of estrogeninduced protein (IP) synthesis. From Ref. 6.
RESULTS
AND DISCUSSION
in the rat uterus, a carefully programm~ time sequence of events OCCWS after estrogen administration [I, 9, IO]. One of the earliest responses to estrogen involves synthesis of a protein, termed induced protein by Notides and Gorski[ll], now identified as the BB isozyme of creatine kinase [ 121. The synthesis of this protein can be elicited by estrogens in tritro [S, S] and there is a good correlation between the magnitude of nuclear estrogen receptor levels at 1 h after estrogen and the magnitude of induced protein stimulation. In studies with rat uteri iz vitro, a comparison of the naaomolar concentrations of estradioi. estriol and estrone required to elicit 50% occupancy of cytosol estrogen receptors, 50% translocation of receptor to the nucleus and 50% of maximal stimulation of induced protein synthesis (Fig. 1) reveals that El is quite an effective estrogen. Only two times as much E!, as Ez is needed to give 50% of maximal receptor localization in the nucleus and 50y0 of maximal induced protein stimulation.
Fig. 2. Dose-response curves showing the ut~rotrophic activities of estdol, estradiol and estriol derivatives. Rats (21-days old) were injected subcutaneously with the indicated daily dose of compound in 0.5 ml of saline once daily at 24-h intervals on three successive days, and uterine wet weights were determined at 24 h after the last injection. Control animals received saline alone. Each value is the mean of determinations from at least five individual animals f SEM. E,, estradiol; E,, estriol; EE,, 17a-ethynyl estriol; E, CPE, estriol-3-cyclopentyl ether; EE,CPE, 17x-ethynyl estriol-3-cyclopentyl ether. From Ref. 4.
Under it? t&o conditions, however. E, appears a much weaker estrogen (1. 21,and high concentr~~ti~~~s of E, are required to evoke substantial increases in uterine weight. We have studied the basis of the weak uterotrophic activity of E, and the effects of chemical mod~ficati~?n of E, on the biopotency of E;. The modifications we have examined introduce an ethynyl group at the 17%position to reduce metabolic inactivation of the compound and to increase the binding affinity for the estrogen receptor and a cyclopentyl ether group at the 3 position, to provide a prohormonal form of the estrogen. These modi~~d~ions~ it was believed, should increase the biological persistence of the estriol molecule. The compounds we have studied are estriol (EJ and several E,-derivatives prepared by the Eli Lilly Company, namely, 17x-ethynyl estriol (EE,), estriol-3-cyc~openty~ ether (E,CPE), and 17~-ethynyl estriol-3-cyclopen~yl ether (EE,CPE). Dose--response curves indicating the effectiveness of these compounds in increasing uterine weight are plotted in Fig. 2. It is clear that E, is the least elective of the compounds tested. Addition of a cyclopentyl ether group at the Sposition of estriol (E,CPE) shifts the dose-response curve slightly to the left, but E,CPE appears to be a significantly better estrogen than estriol only when given at a high dose (10 [lg;rat). If an ethynyl group is introduced at the 17~ position of estrioi (EE,), the estrogenic activity is increased dramatically. And the very high estrogenic potency of EE,CPE is apparent at dose Ievels above 0.3 /ig/day. After a single injection of 5 pg doses of these compounds, the prolonged and marked estrogenic effect of EEJIPE on uterine weight can be clearly seen (Fig. 3). All compounds elicit an early (3-6 h) increase in uterine weight. While the onset of the increase is delayed in the case of EE,CPE (no increase at 1 h), presumably owing to the req~iremeut for metabolic activation in the animal, most striking is the very prolonged and marked elevation of uterine
036
I.2
24
36
HOURS
48
60
Fig 3. Uterine wet weight as a functiot~ of time after a singie injection of estriol, estradiol or estriol derivatives. Rats (21-days old) were injected with 5 /ig of compound or saline (controls) at the time indicated prior to killing and excision of uteri. Each weight value is the mean of determinations from 5 animals + SEM. From Ref. 4.
Estriol biology and receptor interaction
I 2-
-60
E2
IO
0
-70
11 3 6
g 12
24
36 HOURS
/ 48
60
7;’
Fig. 4. Time course of nuclear estrogen receptor levels and uterine wet weight after a single injection of 5 pg of EE,CPE, EE,, E, or E,. From Ref. 4.
weight seen after EECPE. Following the initial ca 6 h peak in uterine weight, there is a very prolonged and even more pronounced elevation of uterine weight at 36-48 h, with weight still remaining over 2-fold above the control after 72 h. FE, and E,, which are considerably more potent than E; in the 3-day uterotrophic assay, give a more prolonged weight elevation than does E,; yet neither of these compounds results in a uterine weight that is much above the control at 72 h. Hence, of the four compounds, EE,CPE clearly evokes the most pronounced uterine growth. Figure 4 compares the time course of nuclear receptor levels and uterine weight after a single subcutaneous injection of 5 pg of estriol, estradiol, or estriol derivatives. EE,, E, and E2 all elicit a rapid uptake of receptor into the nucleus and show an equivalent wet weight response at 3 h. However, there is a rapid decline in nuclear receptor levels after
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E, injection (at control by 24 h), and this is paralleled by a corresponding inability of E, to maintain uterine wet weight; nuclear receptor levels decline less rapidly after EE, or E, and uterine weight also remains elevated for a longer time. Of the four compounds, EE,CPE shows the most dramatic and prolonged maintenance of high nuclear receptor levels and elevated uterine weight. Thus, chemical modifications of the estriol molecule which result in a prolonged stimulation of uterine growth also result in a prolonged maintenance of hormone-receptor complexes in the nucleus. These studies indicate that E, is a weak estrogen because of its brief duration of interaction with receptor and gives strong support to the concept [9, 10, 13-151 that true uterine growth requires the direct and prolonged influence of the estrogen-receptor complex in the nucleus, A marked increase in plasminogen activator activity accompanies estradiol action in the uterus f16- 1S] and this increase in plasminogen activator activity is estrogen-sp~ific, being stimulated only by compounds that interact with the estrogen receptor. Figure 5 compares the efficacies of E3 and several other estrogens in causing elevation of uterine plasminogen activator activity (left panel) and in stimulating uterine weight gain (right panel). Although E, evokes only a slight increase in uterine weight over the doses examined, E, does evoke increases in plasminogen activator activity at the 1 and 10 pg doses. The more potent estrogens E, and diethylstilbestrol (DES) evoke increases in plasminogen activator activity at much lower hormone concentrations (0.03-0.1 pg}; and the decrease in uterine plasminogen activator activity at the higher concentrations of E, and DES is most likely due to a secretion of plasminogen activator from the uterine tissue into the luminal fluid in these highly stimulated uteri [18, 191. In primary cell cultures prepared from human endometrium, estradiol in vitro is able to increase the cellular levels of progesterone receptor in a concentration and time-dependent manner. In human endometrium, we find that E, has an affinity for estrogen receptor 12% that of E, and there is no metabolism of E, by the endometrial cells [8]. In these cell cultures, we have evaluated the effectiveness of E, and other compounds in increasing cellular progesterone receptor levels (Fig. 6). Values are expressed relative to maximal progesterone receptor stimulation by E,, which is arbitrarily set at lOO’%.These dose-response curves indicate the effectiveness in stimulating progesterone receptor levels to be: P1496 > 17/I-E, 2 17u-E, > E3 > estrone (E,) $ C1628. Estriol is weaker than Ez in stimulating progesterone receptor levels but E, is more effective than perhaps expected. This reflects the absence of E, metabolism, while E2 is substantially converted to E,
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BENITAS.
c
1
KAT~E~~LLENB~~~~
Estrone ./ ._4
1
5
600-
I
./
DAILY
DOSE
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ESTROGEN
(pgl
Fig. 5. Dose-response curves comparing the efficacies of estriol and several estrogenic compounds in causing elevation of uterine plasminogen activator activity (left panel) and uterine weight gain (right panel). Nineteen-day old animals (3 per group) received se. injections for three days of the indicated daily dose of 17/?-estradiol, 17x-estradiol, estrone, estriol, diethylstilbesterol (DES), or the fungal estrogenic compound zearalanol (P-1496). Values are the mean & SEM. Significant differences from controls at P < 0.01 (*) are represented only for 17/3-E,, from which comparisons can be made for the other estrogenic compounds. From Ref. 18.
by the endometrial
cells and the fact that a smaller of E, is bound by serum in the media (30% of added E, is serum bound while 70-80x of proportion
E, is serum bound) so that at each concentration, more E, is available for interaction with the cells. MCF-7 human breast cancer cells contain high levels of estrogen receptors and their growth is stimulated by E, in vitro. Figure 7 compares the dose-response curves for E, and E, stimulation of cell proliferation. It is clear that E, is nearly as effective N 160 w
I
,
1
,
T
, ?
?ii 140 c
I
0. I
I
I
I
IO
CONCENTRATION
as E, in stimulation of cell proliferation. Marked stimulation is achieved with lo-” M concentrations of both estrogens with IO-‘” M concentrations giving maximal stimulation. In these cells, El, has an affinity for receptor only 25% that of estradiol (20; A. Nardulli and B. S. Katzenellenbogen, unpublished). As in human endometrial cells, there is no metabolism of E, in the MCF-7 cells and, again, a larger proportion of the E, is free in the serum-media so that the effective, available E, concentration is higher at each added dose compared with E,.
I
1496
-i
I
I
loo
IO00
I
lO,cao
(nM)
I I I L Fig, 6. Ability of E, and other estrogens and antiestrogen I I , ,o-"i lo-~~ ,QIO lO-9 10-9 lo-7 10-9 C1628 to increase cytosol progesterone receptor in human endometrial cells. Cells were incubated with hormoneMOLAR CONCENTRATION containing media (changed daily) for 3 days. The cells were Fig. 7. Dose-response curves for the effects of E, and E, on then harvested and homogenized, and the progesterone receptor content was determined by hydroxylapatite assay the growth of MCF-7 breast cancer cells. Cells were grown using j3H]R5020. Results are expressed as the percent of in T-25 Aasks in medium lacking insulin but containing SO,:, maximal progesterone receptor increase caused by E,, with charcoal-dextran treated calf serum in the presence of the maxima1 increase defined as the response obtained after a indicated concentrations of E, or E, and media and hor3-day incubation with 1 x 10m8M E,. Each point represents mones were changed every 48 h. After 12 days of growth, the mean of 2-3 separate samples. Data obtained from 15 triplicate flasks of cells were counted. Data are expressed as separate uterine samples are included in this figure. From the percent of the control cell number measured after 12 days of growth and represent the mean & SEM. Ref. 8.
Estriol biology and receptor interaction These studies in rat uterus in vivo and in rat uterus in vitro
and in human endometrial and MCF-7 human breast cancer cells in vitro provide strong evidence that El is a very effective estrogen in vitro. Most target tissues do not metabolize E, and it has a moderate affinity (10-25x that of E,) for estrogen receptor. In vivo, E, is cleared quite rapidly and hence it appears to be an estrogen of low potency. However, if it is provided at effective levels over a period of time, it can be a very potent estrogen. Hence, when given either in a prohormonal form that is metabolized relatively little (such as EE,CPE) or is given by
repeated injection or by implant [21,22], E3 can be a quite potent estrogen, evoking a spectrum of biological responses similar to that elicited by estradiol. Acknowledgements-The
studies reported here were supported in part by NIH grants USPHS HD06726 and CA181 19. I am grateful to the collaborators from my laboratory in these studies, in particular, Richard Eckert, Mark Kneifel, Nancy Lan and Ann Nardulli. REFERENCES 1. Szego C. M. and Roberts S.: Steroid action and interaction in uterine metabolism. Recent Prog. Horm. Res. 8 (1953) 419-469. 2. Hisaw F. L.: Comparative effectiveness of estrogens on fluid inhibition and growth of the rat’s uterus. Endocrinology 64 (1959) 276-289. 3. Terenius L.: Structure-activity relationships of anti-
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estrogens in vitro. J. clin. Endocr. Metab. 52 (1981) 699-708. 9. Katzenellenbogen B. S. and Gorski J.: Estrogen actions on synthesis of macromolecules in target cells. In Eiochemical Actions of Hormones (Edited by G. Litwack). Academic Press. New York. Vol. 3 (1975) DD. 187-243. 10. Katzenellenbogen B. S.: Dynamics of steroid hormone 11 receptor action. A. Rev. Physiol. 42 (1980) 17-35. Notides A. C. and Gorski J.: Estrogen-induced syn’ thesis of a specific uterine protein. Proc. natn. Acad. Sci., U.S.A. 56 (1966) 230-235.
12. Reiss N. A. and Kaye A. M.: Identification of the major component of the estrogen-induced protein of rat uterus as the BB isozyme of creatine kinase. J. biol. Chem. 256 (1981) 5741-5749. 13. Anderson J. N., Peck E. J. Jr. and Clark J. H.: Nuclear receptor-estrogen complex: Relationship between concentration and early uterotrophic responses. Endocrinology 92 (1973) 1488-1495.
14. Anderson J. N., Peck E. J. Jr and Clark J. H.: Estrogeninduced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology % (1975) 160-164.
15. Gorski J. and Raker B.: Estrogen action in the uterus: The requisite for sustained estrogen binding in the nucleus. Gynec. Oncol. 2 (1974) 249-256. 16. Albrechtson 0. K.: Effect of estradiol on fibrinolytic activity of rat uterus. Proc. Sot. exp. Biol. Med. 94 (1957) 700-705.
17. Katz J., Troll W., Levy M., Filkens K., Russo J. and Levitz M.: Estrogen-dependent trypsin-like activity in the rat uterus. Archs Biochem. Biophys. 173 (1976) 347-356.
endocr., Copenh. 66 (1971) 431-438. 4. Lan N. C. and Katzenellenbogen
18. Kneifel M. A., Leytus S. P., Fletcher E., Weber T., Mange1 W. F. and Katzenellenbogen B. S.: Uterine plasminogen activator activity: Modulation by steroid hormones. Endocrinology 111 (1982) 493-499. 19. Peltz S. W., Katzenellenbogen B. S., Kneifel M. A. and Mange1 W. F.: Plasminogen activators in tissues of the immature and estrogen-stimulated rat uterus and in uterine luminal fluid: Characterization and properties.
(1976) 220-227. 5. Katzenellenbogen B. S. and Gorski J.: Estrogen action in vitro: Induction of the synthesis of a specific uterine protein. J. biol. Chem. 247 (1972) 1299-1305. 6. Ruh, T. S., Katzenellenbogen B. S., Katzenellenbogen
20. Lippman M., Monaco M. E. and Bolan G.: Effects of estrone, estradiol and estriol on hormone-responsive human breast cancer in long-term tissue culture. Cancer
with with oestrogens regard to interaction 17/I-oestradiol in the mouse uterus and vagina. Acta B. S.: Temporal relationships between hormone receptor binding and biological responses in the uterus: Studies with shortand long-acting derivatives of estriol. Endocrinology 98
J. A. and Gorski J.: Estrone interaction with the rat uterus: In vitro response and nuclear uptake. Endocrinology 92 (1973) 125-134. 7. Pavlik E. J. and Katzenellenbogen
B. S.: Human endometrial cells in primary tissue culture: Estrogen interactions and modulation of cell proliferation. J. clin.
Endocr. Metab. 47 (1978) 333-344. 8. Eckert R. L. and Katzenellenbogen
B. S.: Human endometrial cells in primary tissue culture: Modulation of progesterone receptor level by natural and synthetic
Endocrinology 112 (1983) 890-897.
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21. Clark J. H., Paszko Z. and Peck E. J. Jr: Nuclear binding and retention of the receptor estrogen complex: Relation to the agonistic and antagonistic properties of estriol. Endocrinology 100 (1977) 91-96. 22. Clark J. H., Peck E. J. Jr, Hardin J. W. and Eriksson H.: The biology and pharmacology of estrogen receptor binding: Relationship to uterine growth. In Receptors and Hormone Action (Edited by B. W. O’Malley and L. Birnbaumer). Academic Press, New York, Vol. 2 (1978) pp. l-32.