Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death.

BACKGROUND: Oestrogen (E2) induces apoptosis in long-term E2-deprived MCF7 cells (MCF7:5C). Taxanes have been used extensively in the treatment of early and advanced breast cancer. We have interrogated the sequence of events that involve the apoptotic signalling pathway induced by E2 in comparison with paclitaxel. METHODS: DNA quantification and cell cycle analysis were used to assess proliferation of cancer cells. Apoptosis was evaluated using annexin V and DNA staining methods. Regulation of apoptotic genes was determined by performing PCR-based arrays and RT-PCR. RESULTS: E2-induced apoptosis is a delayed process, whereas paclitaxel immediately inhibits the growth and induces death of MCF7:5C cells. The cellular commitment for E2-triggered apoptosis occur after 24 h. Activation of the intrinsic pathway was observed by 36 h of E2 treatment with subsequent induction of the extrinsic apoptotic pathway by 48 h. Paclitaxel exclusively activated extramitochondrial apoptotic genes and caused rapid G2/M blockade by 12 h of treatment. By contrast, E2 causes an initial proliferation with elevated S phase of cell cycles followed by apoptosis of the MCF7:5C cells. Most importantly, we are the first to document that E2-induced apoptosis can be reversed after 24 h treatment. CONCLUSIONS: These data indicate that E2-induced apoptosis involves a novel, multidynamic process that is distinctly different from that of a classic cytotoxic chemotherapeutic drug used in breast cancer.

Beyond raloxifene for the prevention of osteoporosis and breast cancer.

Selective oestrogen receptor modulators (SERMs) can build bone in the postmenopausal woman and lower circulating cholesterol. These oestrogen-like properties contrast with the anti-oestrogenic properties observed in the breast where SERMs inhibit the oestrogen-mediated development and growth of ER positive breast cancers. The two clinically useful SERMs, tamoxifen and its chemical cousin raloxifene, are currently used successfully either for the treatment and prevention of breast cancer (tamoxifen) or the treatment and prevention of osteoporosis (raloxifene). However, raloxifene has the beneficial side-effect of breast cancer prevention. These multifunction medicines provide proof of concept that novel molecules can be selectively targeted to diseases mediated by the endocrine system.

Overview from the International Conference on Long-Term Tamoxifen Therapy for Breast Cancer.

The development of tamoxifen therapy to treat selected patients, with all stages of breast cancer, has provided the clinical community with an efficacious and safe drug for long-term therapy. Issues of safety are under constant review, but justified concerns about high doses of tamoxifen acting as a promoter of liver cancer in rats or as a promoter of endometrial cancer in women have not, as yet, proved to be of clinical relevance. The situation will continue to be reviewed during the development of the prevention studies in Europe and the United States because an improvement in women's health is the ultimate goal of these programs. The hallmark for the successful development of tamoxifen has been the close cooperation between the laboratory and the clinic. The clinical strategy of long-term tamoxifen therapy is a direct application of a laboratory concept. Furthermore, potential problems in the clinic have been identified in the laboratory, and the clinical community has responded quickly to evaluate the real risks to the patient population. This close cooperation will continue. Issues of drug resistance, new antiestrogen development, and the application of the knowledge about steroid receptors to develop targeted gene therapies are being addressed so that additional treatment approaches for breast cancer will be in place by the turn of the century.

Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor.

BACKGROUND: The growth of estrogen receptor (ER)-positive breast cancer cells is hormonally regulated, but the majority of breast cancers are ER negative and unresponsive to hormonal therapy. PURPOSE AND METHODS: To test whether hormonal control over replication can be re-established in ER-negative cells, we transfected ER-negative MDA-MB-231 (clone 10A) cells with sense and antisense constitutive ER expression vectors containing the gene for either wild-type or mutant ER linked to the gene for neomycin resistance aminoglycoside phosphotransferase (neo). A Northern blot analysis was done on total RNA from eight of the 10 transfectant clones produced to detect messenger RNA coding for ER and neo, and a Western blot analysis was done on protein extracted from the cells of one mutant and two wild-type ER sense transfectant clones to determine the molecular weight of the ER in transfectants. Levels of ER in transfectants were measured both by enzyme immunoassay and by ligand-binding methods. To ascertain whether the ER in wild-type and mutant sense transfectants was functional, we tested the effects of 17 beta-estradiol (E2) and/or an antiestrogen, ICI 164,384, on 1) ER-activated gene regulation (by transient transfection of these cells a second time with a reporter plasmid containing an estrogen response element linked to the chloramphenicol acetyl transferase [CAT] gene), 2) induction of progesterone receptor, 3) DNA replication, and 4) cell cycle kinetics. RESULTS: Messenger RNA coding for ER and for neo was detectable in both sense and antisense transfectant clones. Sense transfectants (both mutant and wild-type) expressed ER protein with a molecular weight similar to that found in ER-positive control cells. By the ligand-binding method high levels of ER were detected in both wild-type and mutant transfectants, although by the enzyme immunoassay method lower levels were detected in mutant transfectants. ER from both wild-type and mutant sense transfectants appeared functional, since E2 stimulated the expression of reporter-linked CAT and of progesterone receptor in these transfectants. E2 inhibited DNA replication in wild-type sense transfectants at a concentration of 10(-10) M and mutant sense transfectants at a concentration of 10(-8) M, and ICI 164,384 blocked this effect. CONCLUSION: ER-negative breast cancer cells stably transfected with either a mutant or wild-type ER gene regain hormonal responsiveness; however, E2 inhibits rather than stimulates cell growth. IMPLICATION: Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.

Selective estrogen receptor modulation and reduction in risk of breast cancer, osteoporosis, and coronary heart disease.

The recognition of selective estrogen receptor modulation in the laboratory has resulted in the development of two selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, for clinical application in healthy women. SERMs are antiestrogenic in the breast but estrogen-like in the bones and reduce circulating cholesterol levels. SERMs also have different degrees of estrogenicity in the uterus. Tamoxifen is used specifically to reduce the incidence of breast cancer in premenopausal and postmenopausal women at risk for the disease. In contrast, raloxifene is used specifically to reduce the risk of osteoporosis in postmenopausal women at high risk for osteoporosis. The study of tamoxifen and raloxifene (STAR) trial is currently comparing the ability of these SERMs to reduce breast cancer incidence in high-risk postmenopausal women. There is intense interest in understanding the molecular mechanism(s) of action of SERMs at target sites in a woman's body. An understanding of the targeted actions of this novel drug group will potentially result in the introduction of new multifunctional medicines with applications as preventive agents or treatments of breast cancer and endometrial cancer, coronary heart disease, and osteoporosis.

Suppression of mouse mammary tumorigenesis by long-term tamoxifen therapy.

A sustained release of tamoxifen, which produced decreasing serum levels of this drug (24 to 4 ng/mL) over 6 months, suppressed mammary tumorigenesis in virgin or once pregnant C3H/OUJ female mice. Tamoxifen was consistently more effective than early ovariectomy, which only delayed tumorigenesis. Tamoxifen prevented the stimulatory action of cyclical (alternate-month) progesterone administration on mouse mammary tumorigenesis. However, when tamoxifen treatment (12 months) was stopped, progesterone treatment initiated tumorigenesis. In contrast, when long-term tamoxifen treatment was stopped in mice that had not undergone ovariectomy, and estrous cycle returned, the majority of these mice remained tumor free. A comparison of different durations (3, 6, and 12 months) of tamoxifen treatment of virgin mice, starting at approximately 4 months of age, showed an equivalent effect on mammary tumorigenesis. All virgin mice developed tumors by 18 months of age, whereas 80% of the tamoxifen-treated mice were tumor free. Nevertheless, cyclical progesterone administration caused rapid development of tumors after 3 months of tamoxifen treatment; only 15% of these mice were tumor free at 18 months. Cyclical progesterone administration caused an increase in tumorigenesis after 6 months of tamoxifen treatment; 50% of these mice were tumor free at 18 months of age. These data demonstrate the efficacy of tamoxifen to suppress mouse mammary tumorigenesis and demonstrate that continuous tamoxifen therapy is necessary to prevent the development of tumors by progesterone, a stimulatory hormone.

Alteration of endocrine parameters in premenopausal women with breast cancer during long-term adjuvant therapy with tamoxifen as the single agent.

Tamoxifen is used to treat selected patients at each stage of breast cancer. Although most clinical experience has been obtained with postmenopausal women, increasing numbers of premenopausal women will be treated with 5 or more years of adjuvant tamoxifen therapy following surgery. Indeed, the proposed use of tamoxifen to prevent breast cancer in high-risk women could result in its extended use during the childbearing years. We have monitored the changes in circulating hormone levels from the ovary and pituitary gland in premenopausal (41-47 years old) women with stage I or II breast cancer during adjuvant therapy with tamoxifen as the single agent for 4-72 months following a mastectomy. Each patient (total eight) continued to menstruate, and ovulation occurred. Circulating levels of luteinizing hormone and follicle-stimulating hormone (except in one woman) remained in the normal range (determined in 12 regularly menstruating women in a control group). The levels of estradiol, estrone, and progesterone were elevated onefold to threefold. Prolactin levels decreased by 30%-40%, but the levels of sex hormone binding-globulin were unaffected. These data demonstrate that premenopausal women taking tamoxifen are potentially at risk for pregnancy and must be counseled about barrier contraception. Furthermore, the impact of many years of ovarian stimulation by tamoxifen must be evaluated, especially in women with node-negative disease or in healthy women in whom tamoxifen is used to prevent breast cancer.

Investigation of the mechanism of tamoxifen-stimulated breast tumor growth with nonisomerizable analogues of tamoxifen and metabolites.

BACKGROUND: The nonsteroidal anti-estrogen tamoxifen (TAM) is the front-line endocrine treatment for breast cancer, but disease recurrence is common. Treatment failure may occur because tumors become insensitive to TAM. Alternatively, resistance may occur because tumors become stimulated rather than inhibited by TAM. TAM-stimulated growth of MCF-7 human breast tumors has been observed in athymic mice after prolonged treatment with TAM. PURPOSE: Our purpose was to examine the mechanism of treatment failure by determining whether TAM-stimulated tumors acquire the ability to excrete TAM and its anti-estrogenic metabolites or to convert them to estrogenic compounds with weakened antiestrogenic activity. METHODS: We used high-pressure liquid chromatography to quantitate TAM and its metabolites in serum and tumors from ovariectomized athymic mice and in MCF-7 cells grown in vitro. We treated tumor-bearing mice with subcutaneous sustained-release preparations of estradiol, TAM, or a nonisomerizable (fixed-ring) analogue and then assessed the activity of these compounds on TAM-inhibited parental MCF-7 tumors and on TAM-stimulated MCF-7 TAM tumors. RESULTS: We found negligible differences in intratumoral TAM levels between TAM-inhibited parental MCF-7 tumors and TAM-stimulated MCF-7 TAM variants. We did not detect metabolite E (Met E), an estrogenic TAM metabolite, in serum or tumors. Using MCF-7 cells in vitro, we determined that the (Z) isomer of Met E, the form directly produced by TAM metabolism, must be present in the cell at a concentration of over 1000 ng/g to overcome growth inhibition by physiological levels of TAM and antiestrogenic metabolites, but the (E) isomer of Met E was effective at 10 ng/g. We reasoned that conversion of Met E from the (Z) (a weak estrogen) to (E) isomer (a potent estrogen) would be required if formation of Met E were responsible for TAM-stimulated growth. However, fixed-ring TAM, which can only form (Z) Met E, was shown to be as capable as TAM of initiating and maintaining anti-estrogen-stimulated growth of MCF-7 tumors in athymic mice. CONCLUSION: Metabolism and isomerization of TAM to estrogenic compounds is not the mechanism of TAM-stimulated growth in our model. IMPLICATION: Other potential mechanisms for TAM-stimulated growth, such as estrogen receptor mutation, must be investigated so that effective strategies can be devised to control breast cancer once therapy fails.

Effects of tamoxifen therapy on lipid and lipoprotein levels in postmenopausal patients with node-negative breast cancer.

We conducted a 2-year, randomized, double-blind, placebo-controlled toxicity trial of therapy with tamoxifen (10 mg twice a day) in 140 postmenopausal women with a history of breast cancer and histologically negative axillary lymph nodes. These women had been treated with surgery with or without radiotherapy. At a 3-month evaluation, tamoxifen-treated women showed a significant decrease in fasting plasma levels of total cholesterol and low-density lipoprotein (LDL) cholesterol, which persisted at 6- and 12-month evaluations. During the first 12 months, plasma triglyceride levels increased; small but significant decreases in high-density lipoprotein cholesterol (HDL) were observed in tamoxifen-treated women, but ratios of total cholesterol to HDL cholesterol and of LDL to HDL cholesterol changed favorably. While data relating lipid/lipoprotein profiles and cardiovascular disease are limited in women, current evidence suggests that total cholesterol and possibly low-density lipoprotein cholesterol are risk factors. We conclude that during the first 12 months of treatment, tamoxifen exerts a favorable effect on the lipid profile in postmenopausal women with early stage breast cancer.

High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine.

BACKGROUND: P-glycoprotein mediates resistance to natural-product anti-neoplastic agents like vinblastine through an active transport process resulting in reduced intracellular concentration of these agents. The triphenylethylene antiestrogen tamoxifen and its major metabolite N-desmethyltamoxifen at concentrations of 4-6 microM enhance the intracellular concentration of natural-product antineoplastics and augment the cytotoxicity of such drugs three-fold to 10-fold in a variety of human and murine cell lines. PURPOSE: On the basis of these preclinical findings, we conducted a phase I clinical trial of high-dose, oral tamoxifen administered in conjunction with a 5-day continuous infusion of vinblastine. METHODS: We studied 53 patients with advanced epithelial tumors. Tamoxifen was given orally as a loading dose on day 1, followed by two doses a day on days 2-13. Vinblastine was given as a 120-hour continuous infusion (1.5 mg/m2 per day) on days 9-13 of each tamoxifen course. The starting dose of tamoxifen was 40 mg/m2 administered twice a day following a loading dose of 150 mg/m2. The maximum dose was 260 mg/m2 twice a day following a loading dose of 680 mg/m2. Treatment cycles were repeated every 28 days. RESULTS: The dose-limiting toxic effects of tamoxifen were neurologic and began within 3-5 days after the start of treatment. They consisted of tremor, hyperreflexia, dysmetria, unsteady gait, and dizziness. One patient experienced a grand mal seizure 24 hours after the last tamoxifen dose. Toxic effects were rapidly reversible. Asymptomatic prolongation of the QT interval on electrocardiogram occurred at doses of tamoxifen of 80 mg/m2 or higher given twice a day. No coagulation or ophthalmologic abnormalities occurred. Tamoxifen did not enhance the toxicity of vinblastine. Mean plasma concentrations of tamoxifen or N-desmethyltamoxifen at 260 mg/m2 tamoxifen given twice a day for 13 days were 6.04 and 6.56 microM, respectively. There was no relationship between plasma antiestrogen content and the development of neurotoxic effects. CONCLUSIONS: Tamoxifen at 150 mg/m2 given twice a day following a loading dose of 400 mg/m2 results in plasma levels of tamoxifen and N-desmethyltamoxifen of 4 and 6 microM, respectively, without dose-limiting toxicity. We recommend this dose for phase II trials of tamoxifen to modulate P-glycoprotein-mediated drug resistance. IMPLICATIONS: Our study demonstrates that high-dose tamoxifen can be safely administered and that plasma concentrations that may inhibit P-glycoprotein function can be achieved.

Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth.

BACKGROUND: Tamoxifen has been shown to promote the growth of human endometrial tumors implanted in athymic mice, and it has been associated with a twofold to threefold increase in endometrial cancer. Toremifene, a chlorinated derivative of tamoxifen, and ICI 182,780, a pure antiestrogen, are two new antiestrogens being developed for the treatment of breast cancer. The effects of these drugs on endometrial cancer are currently unknown. Our objective was to evaluate the effects of toremifene and ICI 182,780 on the growth of human endometrial cancer in athymic mice. METHODS: Athymic, ovariectomized mice were implanted with human endometrial tumors and treated with estrogen, tamoxifen, or the new antiestrogens. RESULTS: The effects of tamoxifen and toremifene on the growth of either tamoxifen-stimulated or tamoxifen-naive endometrial tumors in athymic mice were not substantially different. ICI 182,780 inhibited the growth of tamoxifen-stimulated endometrial cancer, in both the presence and the absence of estrogen. CONCLUSIONS: Toremifene and tamoxifen produce identical effects in our endometrial cancer models. Therefore, it is possible that toremifene, like tamoxifen, may be associated with an increased incidence of endometrial cancer. In contrast, ICI 182,780 inhibited tamoxifen-stimulated endometrial cancer, both in the presence and in the absence of estrogen, suggesting that this drug may be safe with regard to the endometrium, even if it is used following tamoxifen, and that it may not result in an increased incidence of endometrial cancer. Indeed, it is even possible that ICI 182,780 may prove useful as an adjuvant agent in early stage endometrial cancer.

Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration.

Long-term tamoxifen (TAM) therapy was examined in athymic mice bearing MCF-7 tumors of different sizes. Six months of TAM treatment caused no increase in tumor size (compared to placebo treatment) for animals treated initially following implantation of tumor pieces (approximately 1 mm3) or for animals with 0.2-cm2 tumors (grown with 1 month of estrogen treatment). Tumors could be regrown with estradiol treatment in animals treated with either therapy and these tumors contained both estrogen and progesterone receptors. However, more tumors could be restimulated with estradiol following pretreatment with TAM than with placebo. A third group of animals had larger tumors (grown with 7 weeks of estrogen treatment to a approximately 0.6-cm2 area) before TAM or placebo treatment. These tumors partially regressed after 4 months of TAM or placebo therapy but began to regrow in both groups until the end of the experiment at 8 months. Tumors that grew in both groups were estrogen receptor positive and when retransplanted into athymic animals could grow with estradiol. However, the tumor that grew during TAM therapy, when retransplanted, could grow successfully only with further TAM treatment. Tumors growing with TAM contained double the estrogen receptor content of the estradiol-stimulated MCF-7 tumors that were not exposed to TAM [390 +/- 37 (SE) fmol/mg protein versus 174 +/- 14 fmol/mg protein]. These results may represent a form of TAM resistance, i.e., TAM dependence that may occur before hormone independence is exhibited.

Reversal of the antitumor effects of tamoxifen by progesterone in the 7,12-dimethylbenzanthracene-induced rat mammary carcinoma model.

Coadministration of progesterone (4 mg/day) opposed the antitumor activity of tamoxifen (100 micrograms/day) in rats bearing 7,12-dimethylbenzanthracene-induced tumors and also partially prevented the inhibition by tamoxifen (50 micrograms/day started 30 days after 7,12-dimethylbenzanthracene administration) of tumor occurrence even after tamoxifen therapy had been established for 1 or 2 mo. Although prolonged progesterone treatment raised progesterone levels, serum total estrogen levels were not raised above control. The reversal by progesterone of the inhibition of tumor occurrence produced by tamoxifen was blocked by the antiprogestin RU 486. These results demonstrate that progesterone can reverse the tumoristatic action of tamoxifen in the 7,12-dimethylbenzanthracene-induced tumor model and that this may be via a progesterone receptor-mediated mechanism.

Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines.

The expression and stability of the estrogen receptor (ER) is the result of a complex process that is modulated by estrogens and antiestrogens. Regulation of the steady-state ER mRNA and protein levels in breast cancer cells appears to be the result of either of two distinct regulatory mechanisms. Estrogen exposure causes a rapid down-regulation of the steady-state level of ER mRNA and protein in model I regulation, as exemplified by the MCF-7:WS8 cell line. Conversely, in model II regulation, as observed in the T47D:A18 cell line, estrogen exposure causes an increase in the steady-state ER mRNA level and a maintenance of the ER protein level. In both these cell lines, the nonsteroidal antiestrogen 4-hydroxytamoxifen has little effect on the mRNA level but causes a net accumulation of the ER protein over time. In contrast, the pure antiestrogen ICI 182,780 causes a dramatic reduction of the ER protein in both the MCF-7:WS8 and T47D:A18 cell lines. This loss has little effect upon the ER mRNA level in the MCF-7:WS8 cells but leads to a decline in the ER mRNA in the T47D:Al8 cells. The estrogen-independent MCF-7:2A cell line, which has adapted to growth in estrogen free media, expresses two forms of the ER, a wild-type Mr66,000 ER and a mutant Mr77,000 ER (ER77). ER77 is the product of a genomic rearrangement resulting in a tandem duplication of exons 6 and 7 (J. J. Pink et al, Nucleic Acids Res., 24:962-969,1996). This exon duplication has abolished ligand binding by this protein. Here we demonstrate that the loss of ligand binding has eliminated the effects of 4-OHT and ICI 182,780 on the steady-state ER77 protein level. However, in the MCF-7:2A cells, antiestrogens affect the wild-type ER protein in the same manner as observed in the MCF-7:WS8 and T47D:A18 cells. Estrogen regulates the ER mRNA and wild-type ER and ER77 proteins in the MCF-7:2A cells in the same manner as observed in the MCF-7:WS8 cells. Interestingly, treatment of the MCF-7:2A cells with ICI 182,780 causes a slight increase in ER mRNA, which is reflected in a net increase in the ER77 protein but a dramatic decrease in the wild-type ER. The models presented here describe the response of two human breast cancer cell lines in short-term studies. These distinct regulation pathways are predictive of the response of these cell lines to long-term estrogen deprivation. This study illustrates two alternative regulation pathways that are present in ER-positive, estrogen-dependent breast cancer cells. This variable response highlights the diversity of responses potentially present in the heterogeneous cell populations of clinically observed breast cancer.

Development of antiestrogens and their use in breast cancer: eighth Cain memorial award lecture.

This paper describes the laboratory discovery and clinical testing of the first nonsteroidal antiestrogen, MER-25 (ethamoxytriphetol). The compound blocks estrogen action in all species tested and has only slight but transient estrogenic effects. No other antisteroidal actions are noted. MER-25 is antiestrogenic in primates and was investigated in the clinics in a wide range of gynecological conditions, including breast and endometrial cancer. Unfortunately toxic side effects (hallucinations, etc.) precluded further investigation. A derivative of triphenylethylene, clomiphene, has some partial agonist (estrogen-like) actions in laboratory animals and following clinical evaluation is now an established agent for the induction of ovulation in subfertile women. Although clomiphene is active in advanced breast cancer, it was not developed further. In the late 1960s a related compound, tamoxifen, was evaluated to treat a number of estrogen-responsive disorders but was successfully introduced in the 1970s for the treatment of advanced breast cancer. Although there was only modest initial interest in the palliative use of tamoxifen, an enormous increase in basic and applied studies with antiestrogens resulted in a definition of the target site-specific and tumoristatic actions of tamoxifen. Close cooperation between laboratory and clinical evaluation has guided the subsequent development of tamoxifen which is now available to treat all stages of breast cancer. Long-term adjuvant tamoxifen therapy, a concept developed in the laboratory, is currently the treatment strategy of choice. The considerable success of tamoxifen has focused attention on new antiestrogens with different pharmacological properties for other potential clinical applications.

Antiestrogenic action of toremifene on hormone-dependent, -independent, and heterogeneous breast tumor growth in the athymic mouse.

The antiestrogen toremifene has been used to study the growth control of hormone-dependent (MCF-7), -independent (MDA-MB-231), or mixed tumor cell populations in athymic mice. Maximal MCF-7 tumor growth was produced in ovariectomized athymic mice by circulating estradiol levels of approximately 200 pg/ml (produced by 0.5-cm silastic capsules implanted s.c.). The antiestrogen toremifene (77 +/- 4 micrograms/day from a 2-cm silastic capsule) inhibited estradiol (0.5-cm capsule)-stimulated growth by more than 70%. No tumor growth was observed in mice treated with toremifene alone, although toremifene acted as a weak partial agonist on the mouse uterus. The growth of hormone-independent MDA-MB-231 breast tumors implanted in athymic mice was not influenced by either estradiol (0.5-cm capsule) or toremifene (2-cm capsule) when administered alone or in combination. Furthermore, even very large doses of toremifene (5 mg/day p.o.) did not alter the rate of MDA-MB-231 tumor growth. Mixtures of MCF-7 and MDA-MB-231 cells in 9:1 and 99:1 ratios inoculated into athymic mice produced tumors which grew in the absence of estradiol but responded to estradiol supplementation (0.5-cm capsule) with a more rapid rate of tumor growth. Tumors grown from inoculated MCF-7:MDA-MB-231 cells (99:1 ratio) in the presence of estradiol had estrogen receptor levels of 33.2 +/- 9.2 fmol/mg of protein at Day 44 compared to 84.8 +/- 4.8 fmol/mg of protein in pure MCF-7 tumors. Toremifene (2-cm capsule) treatment inhibited the estrogen stimulation of these mixed tumors (99:1 starting ratio) to that of toremifene alone. However, toremifene-alone treatment produced a more rapid rate of tumor growth than control or tumors grown from irradiated MCF-7 cells mixed with viable MDA-MB-231 cells. Increasing the ratio of MCF-7:MDA-MB-231 cells (999:1) initially inoculated resulted in tumors which developed less rapidly than the lower ratio (99:1). Toremifene (2-cm capsule) again produced partial inhibition of 17 beta-estradiol-stimulated tumor growth while increasing tumor growth above control when the antiestrogen was administered alone. These results demonstrate that toremifene is effective in inhibiting estrogen stimulation of hormone-dependent tumors and partially successful at controlling mixed hormone-dependent/independent tumors; however, the antiestrogen cannot control the growth of a hormone-independent tumor in this model.

The key to the antiestrogenic mechanism of raloxifene is amino acid 351 (aspartate) in the estrogen receptor.

The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17beta-estradiol and raloxifene [A. M. Brzozowski et al., Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a correctly positioned alkylaminoethoxy side chain. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously identified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mutation is at AA351 of LBD, where the aspartate is changed to tyrosine (Asp351Tyr). In this report, we compared and contrasted the pharmacology of raloxifene to block or induce E2-stimulated increase in TGF-alpha mRNA in stable transfectants of ER-negative human breast cancer cells with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to the antiestrogenic activity of raloxifene.

Antitumor actions of keoxifene and tamoxifen in the N-nitrosomethylurea-induced rat mammary carcinoma model.

We have compared the antitumor activities of the antiestrogens, keoxifene (LY 156758) and tamoxifen (TAM), using the N-nitrosomethylurea (NMU) rat mammary carcinoma model. To establish an effective antitumor dose of TAM in this model, rats were treated 2 wk after initiation with NMU for 8 wk with s.c. daily injections of 6.25 micrograms, 25 micrograms, or 100 micrograms of TAM in peanut oil. At the 25-micrograms and 100-micrograms daily doses, TAM completely inhibited tumor appearance during the therapy period. An effective antitumor dose of TAM (100 micrograms daily) was compared to 20-, 100-, or 500-micrograms daily doses of keoxifene 7 wk after NMU initiation. None of the keoxifene-treated groups prevented the appearance of tumors as effectively as TAM during 13 wk of therapy. When keoxifene was compared to TAM at equivalent daily doses of 100 and 500 micrograms daily starting 2 wk after NMU injection, the keoxifene groups again failed to prevent the appearance of all tumors during 10 wk of therapy. TAM, however, completely suppressed any tumor formation. In the same experiment, animals treated with 500 micrograms of TAM had therapy stopped after 10 wk, and tumors started to appear 6 wk later. No tumors appeared when animals (n = 25) were treated continuously for 23 wk with 100 micrograms of TAM. In separate experiments, keoxifene (500 micrograms daily) and TAM (500 micrograms and 100 micrograms daily) administered for 1 wk blocked the binding of [3H]-estradiol in NMU tumors and in uteri. The effect lasted for up to 5 wk after antiestrogen therapy was stopped. These experiments demonstrate that keoxifene is not as effective in its antitumor action as TAM in the NMU model.