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.

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.

Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse.

The effects of the antiestrogen tamoxifen (TAM) on the growth of two hormone-sensitive human tumors have been examined in athymic mice. The endometrial tumor, EnCa101, was stimulated to grow by TAM either alone or when combined with estradiol. This contrasted with the nonstimulation of the breast tumor, MCF-7, by TAM alone and the antagonist action of TAM on estradiol-stimulated growth of MCF-7 tumors. The individual tumor responses were observed even when the two tumor types were implanted on opposite sides of the same animal. This suggests that host metabolism of TAM does not dictate tissue response. The conclusion is supported by the finding of very similar patterns of metabolites in the two tumors after administration of [ring-3H]TAM. Tissue metabolism therefore is unlikely to be involved. Progesterone receptor levels were higher in estradiol (376 +/- 35 fmol/mg cytosol protein)- or TAM (317 +/- 37 fmol/mg cytosol protein)-stimulated EnCa101 tumors than control (42 +/- 5 fmol/mg cytosol protein) and increased further with combined treatment (485 +/- 75 fmol/mg cytosol protein). Estrogen receptor levels, however, were lower in estradiol (45 +/- 11 fmol/mg cytosol protein)-treated tumors than control (92 +/- 13 fmol/mg cytosol protein) but higher than control in TAM (200 +/- 15 fmol/mg cytosol protein)-treated tumors. Tumors grown with estradiol and TAM had lower estrogen receptor levels (130 +/- 7 fmol/mg cytosol protein) than tumors grown with TAM alone. Estrogen receptor levels indicate that TAM may not be acting exactly as estradiol in the EnCa101 tumor. Overall, these findings suggest that the disparate pharmacology of TAM is a tissue-specific phenomenon.

Inhibition of tamoxifen-stimulated growth of an MCF-7 tumor variant in athymic mice by novel steroidal antiestrogens.

This investigation examines the tamoxifen (TAM)-dependent growth in vivo of an MCF-7 tumor variant, MCF-7TAM, previously reported in this journal (M. M. Gottardis and V. C. Jordan, Cancer Res., 48: 5183-5187, 1988). Ovariectomized athymic mice were implanted with 1-mm3 pieces of MCF-7TAM and were treated with Silastic capsules of varying sizes containing TAM to demonstrate dose-dependent growth over a 10-wk experiment. TAM was necessary to maintain tumor growth. Animals whose capsules were removed at 6 wk showed complete tumor stasis after 20 wk of observation. Removal of TAM after 11 wk caused the rate of tumor growth to decrease compared with TAM-treated animals. Tumor areas were significantly different (P less than 0.03) at Wk 20. The growth of TAM-stimulated tumor, MCF-7TAM, was inhibited by the novel steroidal antiestrogens, ICI 164,384 and RU 39,411. TAM-stimulated growth (0.5-cm Silastic capsule) was maintained at control levels by 8 wk of treatment with ICI 164,384 (1 mg s.c. every other day). ICI 164,384 alone had no stimulatory activity. At the same dose, RU 39,411 inhibited TAM-stimulated growth of MCF-7TAM, although not to control levels. RU 39,411 was slightly stimulatory when administered alone. The growth of MCF-7TAM was stimulated by either TAM or 17 beta-estradiol. The antiestrogen, RU 39,411, effectively inhibited estradiol-stimulated tumor growth. Overall, these studies confirm and extend the previous observation on TAM-stimulated growth of breast cancer cells in vivo and demonstrate the possibility of developing novel antiestrogens to prevent this form of drug resistance should it occur in the clinic.

Effect of steroidal and nonsteroidal antiestrogens on the growth of a tamoxifen-stimulated human endometrial carcinoma (EnCa101) in athymic mice.

Tamoxifen (TAM), a nonsteroidal antiestrogen, is used in the adjuvant treatment of breast cancer. Previous studies, however, have indicated that some human breast and endometrial tumors are stimulated to grow with TAM in the athymic mouse. One such TAM-stimulated tumor is the EnCa101 human endometrial adenocarcinoma. Our aim was to evaluate the ability of different doses of TAM or other nonsteroidal antiestrogens to stimulate the growth of EnCa101 tumors in athymic mice. Additionally we have evaluated less estrogenic antiestrogens (two steroidal antiestrogens, RU 39,411 and ICI 164,384, and two nonsteroidal antiestrogens, keoxifene and MER-25) for their ability to inhibit TAM-stimulated growth. All experiments were done in ovariectomized athymic mice transplanted in the axillary mammary fat with 1-mm3 pieces of EnCa101 tumor. Sustained release preparations (0.5-2.0-cm Silastic capsule or 5-mg TAM cholesterol pellet) of TAM caused similar tumor growth. The growth rate was not altered by an additional daily i.p. injection of 1 mg TAM in 0.1 ml peanut oil. A 3-mg TAM daily dose was toxic. Four weeks of treatment (100-micrograms s.c. injections, every other day) with nonsteroidal antiestrogens, trioxifene mesylate, enclomiphene, or nafoxidine stimulated tumor growth. However, keoxifene stimulated this tumor to a lesser degree than TAM and partially inhibited TAM-stimulated growth. ICI 164,384 showed no stimulatory activity (1-mg s.c. injections every other day) alone compared to controls but inhibited TAM-stimulated (0.25-cm Silastic capsule) growth. In a parallel experiment, RU 39,411 (1-mg s.c. injections every other day) stimulated EnCa101 to grow. In contrast when RU 39,411 was administered in a sustained release preparation (2.0-cm Silastic capsule) there was no stimulatory growth compared to controls. Additionally RU 39,411 inhibited TAM-stimulated growth, but the low-potency antiestrogen, MER-25, was less effective in this regard. These data suggest that less "estrogenic" antiestrogens can inhibit TAM-stimulated tumor growth in vivo. Thus these compounds or derivatives may prove useful as a second-line endocrine therapy should TAM-stimulated tumor growth occur in the clinic.

Differential ability of antiestrogens to stimulate breast cancer cell (MCF-7) growth in vivo and in vitro.

We have previously described an MCF-7 breast cancer cell variant, MCF-7TAM, which is stimulated to grow in athymic mice by tamoxifen (TAM) (M. M. Gottardis and V. C. Jordan, Cancer Res., 48:5183-5187, 1988). Earlier experiments have shown that TAM exhibits some profound estrogen-like effects in mice whereas TAM is less estrogenic in the rat. The aim in these studies was to compare the ability of MCF-7TAM to grow in different host environments and to determine whether the TAM-stimulated phenotype could be maintained in vitro. Ovariectomized athymic mice and rats were implanted with 1-mm3 pieces of MCF-7TAM tumor and treated with estradiol, TAM, or control silastic capsules. After 9 weeks of growth in either species, TAM or estradiol-treated groups both had sustained growth of MCF-7TAM compared with the control groups. To determine the effects of estradiol and TAM on immune function in athymic mice, splenocytes from treated or control athymic mice, challenged with poly(I:C), were assayed for natural killer (NK) cell activity against 51Cr-labeled YAC1 target cells. Both estradiol and TAM abolished lytic activity by 12 weeks of treatment. To evaluate the role of a decrease in NK-cell activity in the host on growth of MCF-7TAM xenografts we compared the growth effects in athymic and NK-cell deficient, ovariectomized beige mice. TAM stimulated MCF-7TAM in both beige and athymic mice; however, the tumor grew more rapidly in control beige mice than in control athymic mice. This study demonstrated that TAM-stimulated growth could occur in vivo. However, TAM or 4-hydroxytamoxifen did not cause a stimulation of MCF-7TAM compared with wild-type MCF-7 cells when experiments were conducted in vitro. These studies demonstrate that a suppression immune function can facilitate the growth of MCF-7TAM in athymic animals. However, additional components of the host environment contribute to TAM-stimulated growth in vivo.

Beyond tamoxifen: the retinoid X receptor-selective ligand LGD1069 (TARGRETIN) causes complete regression of mammary carcinoma.

Recently, we reported that LGD1069, a high-affinity ligand for the retinoid X receptors (RXRs), was shown to have an efficacy equivalent to that of tamoxifen (TAM) as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. Furthermore, LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. Due to the high efficacy and benign profile of this RXR agonist as a suppressor of carcinogenesis, we examined its role as a therapeutic agent on established mammary carcinomas. In the rat mammary carcinoma model, N-nitroso-N-methylurea was used to induce tumors, and the tumors were allowed to grow to an established size prior to initiation of treatment. LGD1069-treated animals showed complete regression in 72% of treated tumors and had a reduced tumor load compared to control. In addition, the combination of LGD1069 and TAM showed increased efficacy over either agent alone. Histopathological analysis showed a reduction of LGD1069-treated tumor malignancy, an increase in differentiation, and a sharp decrease in cellular proliferation compared to vehicle-treated control tumors. These data demonstrate that the RXR-selective ligand LGD1069 is a highly efficacious therapeutic agent for mammary carcinoma and enhances the activity of TAM.

Enhanced growth of an estrogen receptor-negative endometrial adenocarcinoma by estradiol in athymic mice.

The aim of this study was to investigate the effects of estradiol and tamoxifen (TAM) on the growth of human endometrial carcinomas in athymic mice. Tissues from primary tumors were implanted into estradiol-treated mice. In passage 2, animals were treated with (a) placebo, (b) estradiol, (c) estradiol plus TAM, and (d) TAM alone. The size of the tumors was measured weekly. Estrogen receptors (ER) were determined with the dextran-coated charcoal method and/or ER enzyme-linked immunoassay. Progesterone receptors were measured with the dextran-coated charcoal technique. Of 16 primary tumors, 2 grew in the athymic mice and were studied further. Tumor EL was positive for ER (145 fmol/mg protein) and progesterone receptors (993 fmol/mg protein). Tumor EL in passage 2 was not significantly stimulated by estradiol, but was stimulated by a combination of estradiol and TAM. Treatments (estradiol, estradiol plus TAM, or TAM) all increased tumor growth in passage 3. Tumor BR and a metastasis BR-MET were ER and progesterone receptor negative, applying dextran-coated charcoal, ER enzyme-linked immunoassay, and immunocytochemistry. The BR and BR-MET cells contain the complete ER gene but do not express any measurable amounts of ER mRNA as quantitated by Northern blot analysis, using a complete ER complementary DNA probe. In all animal passages the growth rate was significantly higher in estradiol-treated mice compared with the control. TAM alone had some growth stimulatory effect, but much smaller than observed in the estradiol group. TAM inhibited estradiol-stimulated growth. These results suggest that estradiol and possibly TAM are capable of stimulating tumor growth in the athymic mice independently from ER, potentially through a host-mediated mechanism.

Chemoprevention of mammary carcinoma by LGD1069 (Targretin): an RXR-selective ligand.

Recently, 9-cis retinoic acid, a high affinity ligand for retinoic acid receptors and retinoid X-receptors (RXRs), was shown to have efficacy superior to all-trans retinoic acid as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To further explore the specific contribution RXR activation may play in suppression of carcinogenesis, the efficacy of LGD1069 (Targretin), an RXR-selective ligand, in the N-nitroso-N-methylurea-induced rat mammary tumor model was studied. LGD1069-treated animals showed a 90% reduction in tumor burden and tumor incidence compared with vehicle-treated rats with an efficacy similar to that achieved with tamoxifen. LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. These data demonstrate that LGD1069, an RXR-selective ligand, can act as a highly effective and benign chemopreventive agent for mammary carcinoma.

Estrogen receptor activation via activation function 2 predicts agonism of xenoestrogens in normal and neoplastic cells of the uterine myometrium.

The possible contribution of endocrine disrupters to human disease, particularly those compounds that modulate the estrogen receptor (ER), has recently drawn considerable attention. The tissue specificity of effects mediated by the ER is well recognized, although the mechanism of this specificity is not understood sufficiently to predict the effects of a particular ligand in different target tissues. Although the divergence of ER-mediated effects in the breast, bone, and uterine endometrium has been described, a frequently overlooked site of estrogen action is the smooth muscle of the uterus. The uterine myometrium is the tissue of origin of an extremely common hormone-responsive tumor, uterine leiomyoma, a tumor with a significant impact on women's health and a possible environmental influence. This report describes an in vitro/in vivo system for identifying the effects of ER ligands in the myometrium and elucidating their mechanism of action. Several natural and synthetic xenoestrogens were evaluated at the cellular and molecular level for their ability to mimic estrogen action in uterine myometrial tissues. Diethylstilbestrol, coumestrol, genistein, naringenin, and endosulfan were able to activate the AF2 function of the ER in vitro and demonstrated agonist activity in estrogen-responsive myometrial cells, as determined by induction of proliferation and increased message levels of progesterone receptor. Compounds that could not activate AF2 function (4-hydroxy-tamoxifen, LY117018, and LY317783) did not act as estrogen agonists. For agonists, rank order of potency was predicted by receptor affinity; however, endosulfan displayed a surprising degree of activity, despite negligible receptor binding. Additionally, diethylstilbestrol and tamoxifen demonstrated prototypical agonist and antagonist effects, respectively, in the intact myometrium of sexually mature rats. The results presented here suggest that some exogenous ER ligands may mimic the effects of endogenous estrogens on uterine leiomyoma and may contribute to a complex hormonal milieu that impacts both normal and neoplastic myometrium.

Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor.

Tamoxifen inhibits estrogen receptor (ER) transcriptional activity by competitively inhibiting estradiol binding and inducing conformational changes in the receptor that may prevent its interaction with coactivators. In bone, the cardiovascular system, and some breast tumors, however, tamoxifen exhibits agonist activity, suggesting that the tamoxifen-ER complex is not recognized identically in all cells. We used phage display to demonstrate that the antiestrogen GW5638 induces a unique structural change in the ER. The biological significance of this conformational change was revealed in studies that demonstrated that tamoxifen-resistant breast tumor explants are not cross-resistant to GW5638. Because of these properties, this drug is currently being developed as a potential therapeutic for tamoxifen-resistant breast cancers.

Synergistic cytotoxicity exhibited by combination treatment of selective retinoid ligands with taxol (Paclitaxel).

The focus of this study was to develop retinoic acid receptor (RAR) RAR alpha/beta selective agonists with anticancer efficacy and reduced toxicity associated with RAR gamma activity. In these studies, we report the identification and characterization of high-affinity RAR alpha/beta selective agonists with limited RAR gamma activity. These compounds inhibited human tumor cell line proliferation with similar efficacy to that observed for a pan-RAR agonist. However, for most tumor cell lines, the efficacy of these compounds was restricted to the micromolar range. To determine whether the RAR alpha/beta selective agonists could be additive or synergistic with existing agents, we investigated the effects of combining RAR alpha/beta selective agonists with various cytotoxic agents. Our results showed that the alpha/beta selective retinoids dramatically lowered the effective dose of Taxol needed to induce cytotoxicity of a wide range of tumor cell lines. This synergy was specific to tubulin-modifying agents and could not be observed with a variety of other cytotoxic agents of diverse function. Examination of pathways common to Taxol and retinoid signaling revealed that this synergy was related in part to effects on Bcl-2 expression/phosphorylation as well as the activity of the c-Jun NH(2)-terminal kinase and activator protein-1. In contrast, the tubulin polymerization induced by Taxol was not further affected by cotreatment with a variety of retinoid receptor ligands. These observations indicate that potent RAR alpha/beta selective agonists may be of therapeutic benefit in combination with Taxol therapy.

Retinoid-induced suppression of squamous cell differentiation in human oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice.

Retinoids are promising agents for therapy of squamous cancers. In vitro, retinoids decrease expression of differentiation markers in head and neck squamous carcinoma cells. Little information is available on effects of retinoids on head and neck squamous carcinoma cell xenograft growth in vivo. To address this issue, head and neck squamous carcinoma cells (line 1483) were established as xenografts in nude mice. Control tumors grew rapidly with doubling times of 4-6 days to mean volumes of 1696 mm3 after 24 days. Histological analyses indicated the formation of well-differentiated squamous carcinoma cells exhibiting pronounced stratification (basal and suprabasal cells) and keratinization (keratin pearls) with abundant stroma. Cytokeratin 19 expression was restricted to the basal cell layers, and cytokeratin 4 expression was abundant in suprabasal cells. Mice were treated daily with 30 mg/kg 9-cis retinoic acid, 20 mg/kg all-trans-retinoic acid, or 60 mg/kg 13-cis retinoic acid by p.o. gavage on a schedule of 5 days/week over 4 weeks. Low micromolar (1.48-3.67 microM) and nanomolar (200-490 nM) concentrations of 9-cis retinoic acid and all-trans-retinoic acid were measured in plasmas and xenografts, respectively, 30 min after dosing. Retinoid treatment produced a marked suppression of the squamous cell differentiation of tumor cells manifest by decreased keratinization, loss of stratification, and accumulation of basal cells. This was accompanied by large decreases in the number of CK4-positive cells and concomitant increases of CK19-positive cells. REtinoic acid receptor-beta expression was also increased by 2.9-9.7-fold after chronic retinoid treatment. 9-cis retinoic acid and all-trans-retinoic acid decreased tumor volumes by 23 +/- 5 (SE) and 19 +/- 3%, respectively (P < or = 0.05); 13-cis retinoic acid was inactive. These retinoids did not decrease the rate of exponential tumor growth but increased the latent period until exponential growth began. These studies demonstrate that retinoids do not universally decrease tumor growth but profoundly suppress squamous cell differentiation in vivo in this xenograft model.

9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice.

Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.

Sensitive and specific detection of retinoid receptor subtype proteins in cultured cell and tumor extracts.

Subtype-specific antipeptide antibodies have been developed against each of the retinoic acid receptors (RARs alpha, beta, and gamma) and each of the retinoid X receptors (RXRs alpha, beta, and gamma). Each antibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by both immunoblot and immunoprecipitation technology. We describe a sensitive and specific assay that combines the binding of cultured cell and tumor extracts to [3H]all-trans-retinoic acid or [3H]9-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by the subtype-specific antibodies to determine the levels of functional retinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compounds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six retinoid receptor proteins ranging in concentration from 9 fmol/mg total protein for RAR beta and RXR gamma to 50 fmol/mg for RXR alpha. Hep G2 and HL60 cells express RAR alpha and RXR alpha proteins at approximately 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (approximately 5 fmol/mg) in Hep G2 cells. MCF-7 cells in culture express RAR alpha (approximately 32 fmol/mg), RAR gamma (approximately 35 fmol/mg), and RXR alpha (approximately 60 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen stimulation and tamoxifen inhibition of leiomyoma cell growth in vitro and in vivo.

Uterine leiomyomas (fibroids) are the most common gynecological neoplasms and may be associated with significant morbidity. Recently, we described a rat model (Eker rat) of fibroid development in which reproductive tract leiomyomas develop spontaneously with high frequency. The present studies describe the estrogen and antiestrogen responsiveness of an Eker rat leiomyoma-derived cell line in vitro and a nude mouse xenograft system in vivo. In this cell line, estradiol stimulated growth in estrogen-depleted medium, whereas the nonsteroidal antiestrogen tamoxifen maximally inhibited cell proliferation in medium containing 10% charcoal-stripped serum. Proliferation was also decreased by the biologically active tamoxifen metabolite 4-hydroxytamoxifen; the metabolite was more effective than the parent compound in exerting this growth inhibition. Compared to placebo-treated controls, estradiol increased the size of tumors that developed in a nude mouse xenograft system, whereas tamoxifen increased tumor latency and decreased tumor size. This study of leiomyoma cells in a well defined system suggests that antiestrogens may prove efficacious in the treatment of this clinically important neoplasm.

Regulation of retinoblastoma gene expression in hormone-dependent breast cancer.

Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.

5-Benzylidene 1,2-dihydrochromeno[3,4-f]quinolines, a novel class of nonsteroidal human progesterone receptor agonists.

A novel series of nonsteroidal progestins, 5-benzylidene-1, 2-dihydrochromeno[3,4-f]quinolines (2), was discovered, and a preliminary structure-activity relationship study around the 5-benzylidene ring generated several potent human progesterone receptor agonists (compounds 8, 16). These new progestins showed biological activities (EC50 = 5.7 and 7.6 nM) similar to progesterone (EC50 = 2.9 nM) in the cotransfection assay with high efficacy (132% and 166%) and binding affinity (Ki = 0.66 and 0.83 nM) similar to medroxyprogesterone acetate (MPA) (Ki = 0.34 nM). A representative analogue, 8, demonstrated similar oral potency to MPA in the uterine wet weight/mammary gland morphology assay in ovariectomized rats.

5-Aryl-1,2-dihydrochromeno[3,4-f]quinolines: a novel class of nonsteroidal human progesterone receptor agonists.

The development of a novel class of nonsteroidal human progesterone receptor (hPR) agonists, 5-aryl-1,2-dihydro-5H-chromeno[3,4-f]quinolines 2, is described. The introduction of a 5-aryl group into the 1,2-dihydrocoumarino[3,4-f]quinoline core 1 is the key for progestational activities. The structure-activity relationship (SAR) studies of the 5-aryl substituents generated a series of potent hPR agonists, which exhibited similar biological activity (EC50 = 8-30 nM) to the natural hormone progesterone (EC50 = 2.9 nM) in cell-based assays with efficacies ranging from 28% to 96%. Most of the analogues displayed similar or greater binding affinity (Ki = 0.41-3.6 nM) than progesterone (Ki = 3.5 nM). Three representative analogues (13, 15, and 24) demonstrated in vivo activities in mammary gland morphology/uterine wet weight assay in ovariectomized rats.

5-Aryl-1,2-dihydro-5H-chromeno[3,4-f]quinolines as potent, orally active, nonsteroidal progesterone receptor agonists: the effect of D-ring substituents.

Several 5-(4-chlorophenyl)-1,2-dihydro-5H-chromeno[3,4-f]quinolines were prepared to determine the effects of substitution at C(8) and C(9) on the progestational activity of this pharmacophore. In combination with a halogen (F or Cl) at C(9), replacement of the C(5) aryl group with variously substituted aryl groups resulted in optimization of the progestational activity, affording compounds with in vitro activity greater than that of progesterone as measured by a cotransfection assay using human progesterone receptor subtype-B (hPR-B). Binding affinities (Ki) to hPR-A were subnanomolar in many cases. These in vitro effects were verified in vivo using a rodent model. Compound 10 (LG120794, 9-chloro-5-(4-chlorophenyl)-1,2-dihydro-2,2,4-trimethyl-5H-chromeno++ +[3,4-f] quinoline) was more potent than medroxyprogesterone acetate at counterpoising the effects of estradiol benzoate in the uterine wet weight assay using immature rats.