The influence of a novel cyclopropyl antiestrogen (compound 7a) on human breast cancer cells in culture

Compound 7a ([Z]-1,1,-dichloro-2,3-diphenyl-2-(4-(2-dimethylamino)ethoxy)phenyl) cyclopropane, dihydrogen citrate salt) is a novel cyclopropyl antiestrogen which was shown to be an estrogen antagonist without estrogen agonist activity. The antiproliferative activity of 7a was examined on estrogen receptor (ER)positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells. Compound 7a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10^–9 to 10^–5M, but did not alter the growth of MDA-MB-231 or A-549 cells. The antiproliferative activity of 7a (10^–7M) on MCF-7 cells was reversed by co-administration of estradiol (10^–8M). An ER-dependent mechanism of action is also supported by the specific ER binding of 7a in MCF-7 cells observed in this study. A study of cell surface morphology using scanning electron microscopy (SEM) revealed that compound 7a at 10^–6M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10^–8M). Compound 7a did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7a inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study support the antiestrogenic action of 7a previously observedin vivo and suggest that 7a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Antiestrogen action in breast cancer cells: Modulation of proliferation and protein synthesis,and interaction with estrogen receptors and additional antiestrogen binding sites

Summary Antiestrogens have proven to be effective in controlling the growth of hormone-responsive breast cancers. At the concentrations of antiestrogens achieved in the blood of breast cancer patients taking antiestrogens (up to 2 × 10^–6 M), antiestrogens selectively inhibit the proliferation of estrogen receptor-containing breast cancer cells, and this inhibition is reversible by estradiol. Antiestrogens also inhibit estrogen-stimulation of several specific protein synthetic activities in breast cancer cells, including increases in plasminogen activator activity, progesterone receptor levels and production of several secreted glycoproteins and intracellular proteins.Antiestrogens bind with high affinity to the estrogen receptor and to additional microsomal binding sites to which estrogens do not bind. These latter sites, called antiestrogen binding sites (AEBS), are present in equal concentrations in estrogen receptor-positive and -negative breast cancer cells and are present in a wide variety of tissues, with highest concentrations being found in the liver. The antiestrogenic and growth suppressive potencies of a variety of antiestrogens correlate best with their affinity for estrogen receptor and not with affinity for AEBS.Antiestrogens undergo bioactivation and metabolismin vivo and hydroxylated forms of the antiestrogen have markedly enhanced affinities for the estrogen receptor. Detailed studies with high affinity radiolabelled antiestrogens indicate that antiestrogens induce important conformational changes in receptor that are reflected in the enhanced maintenance of a 5 S form of the estrogen receptor complex; reduced interaction with DNA; and altered activation and dissociation kinetics of the antiestrogen-estrogen receptor complex. These conformational changes effected by antiestrogens likely result in different interactions with chromatin, causing altered cell proliferation and protein synthesis.Analyses of the rates of synthesis and turnover of the estrogen receptor through pulse-chase experiments utilizing the covalently attaching antiestrogen, tamoxifen aziridine, and studies employing dense amino acid labeling of estrogen receptor reveal that the antiestrogen-occupied receptor is degraded at a rate (t 1/2 = 4 h) similar to that of the control unoccupied receptor. Hence, antiestrogens do not prevent estrogen receptor synthesis and they do not either accelerate or block estrogen receptor degradation.Our findings raise serious doubts about the role of the AEBS in mediating directly the growth suppressive actions of antiestrogens, and suggest that interaction with the estrogen receptor is most likely the mechanism underlying the growth-inhibitory effects of antiestrogens. At present, the role of the AEBS in the actions of antiestrogens or in possible antiestrogen metabolism remains unclear.     

Antitumor effect of triphenylethylene derivative (TAT-59) against human breast carcinoma xenografts in nude mice.

The antitumor activity of a newly synthesized triphenylethylene derivative [(E)-4-[1-[4-[2-(dimethylamino)ethoxy-phenyl]-2-(4-isopropyl)phenyl-1- butenyl] phenyl monophosphate] (TAT-59) was investigated against human breast carcinoma xenografts in nude mice with reference to the changes of hormone receptors. Five strains (MCF-7, Br-10, R-27, ZR-75-1, and T-61) used for the experiments possessed cytosol estrogen receptor (ER), and their growth was estradiol dependent. Five mg of TAT-59 and tamoxifen citrate (TAM) per kg were administered p.o. daily except Sunday. TAT-59 showed a positive antitumor effect against MCF-7 and R-27, whereas TAM was effective on MCF-7, and their adverse effects detected by mortality rate, body weight loss, and spleen weight loss were similar to each other. The reduction of ER and production of progesterone receptor (PgR) after the treatment with TAT-59 were more potent than after TAM, suggesting that TAT-59 exerts its antitumor effect through binding to ER. These findings suggest that TAT-59 might merit use in clinical trials with breast cancers.     

Molecular mechanisms of antiestrogen action in breast cancer

The success of antiestrogen therapy to treat all stages of breast cancer, and the evaluation of tamoxifen as a preventive for breast cancer in normal women, have focused attention on the molecular mechanisms of antiestrogen action and mechanisms of drug resistance. The overall goal of research is to enhance current therapies and to develop new approaches for breast cancer treatment and prevention. Recent studies show that tamoxifen and the new pure antiestrogens appear to have different mechanisms of action: tamoxifen and related compounds cause a change in the folding of the steroid binding domain that prevents gene activation whereas the pure antiestrogens cause a reduced interaction at response elements and cause a rapid loss of receptor complexes. Tamoxifen treatment produces changes in the cellular and circulating levels of growth factors that could influence both receptor negative or receptor positive tumor growth and the metastatic potential of a tumor. These events may explain the survival advantage observed with tamoxifen therapy. However, the current therapeutic challenge is to avoid drug resistance during long-term tamoxifen therapy. Numerous explanations for drug resistance to tamoxifen have been suggested, including elevated estrogen levels, increased tumor antiestrogen binding sites, receptor mutations, and impaired signal transduction. However, it is probable that multiple mechanisms evolve to facilitate tumor survival. Most importantly, current research is examining mechanisms responsible for the beneficial actions of tamoxifen on bones and lipids as well as the potentially deleterious effects of tamoxifen on liver and endometrial carcinogenesis and retinopathy. The urgent need to understand antiestrogenic drug mechanisms and toxicity is being facilitated by the application of the technology developed for basic molecular biology.     

Anti‐breast cancer potential of SS1020, a novel antiestrogen lacking estrogenic and genotoxic actions

Long‐term treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. Several antiestrogens developed in last decades have been discontinued from clinical testing because of their undesirable effects on the uterus. To avoid such serious side‐effect while increasing the drug's anti‐breast cancer potential, new triphenylethylene antiestrogens, 2E‐3‐{4‐[(E)‐4‐chloro‐1‐(4‐hydroxyphenyl)‐2‐phenylbut‐1‐enyl]‐phenyl} acrylic acid (SS1020) and 2E‐3‐{4‐[(Z)‐4‐chloro‐1,2‐diphenylbut‐1‐enyl]phenyl}acrylic acid (SS1010), were designed as safer alternatives. Unlike TAM, SS1020 does not present significant uterotrophic potential in rats; in contrast, SS1010, a compound removing a 4‐OH moiety from SS1020, represented weak uterotrophic activity. The structurally related compounds 4‐hydroxytamoxifen, toremifene, ospemifene, raloxifene (RAL) and GW5638 all have uterotrophic activity. In addition, SS1020 and SS1010 exhibit no genotoxic activity to damage hepatic DNA in rats. Therefore, SS1020 was selected as a safer antiestrogen candidate and used for evaluating the antitumor potential in animals. At the human equivalent doses of TAM, SS1020 had antitumor potential much higher than that of TAM, RAL and GW5638 against 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinoma in rats. The growth of human MCF‐7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS1020. SS1020, lacking estrogenic and genotoxic actions and having strong antitumor potency superior to that of TAM and RAL, could be a safer alternative for breast cancer therapy and prevention.     

Phase II study of second-line treatment with high-dose cyclophosphamide in recurrent metastatic breast cancer

 A total of 78 patients with second recurrence or progression of histologically verified breast cancer were treated with single-agent cyclophosphamide given at 2.5 g/m² by i.v. infusion every 3 weeks along with mesna support. All had previously been treated with epirubicin and cisplatin or epirubicin alone. Toxicity was predominantly hematologic: WHO grade III+IV toxicity was found in 95% of cases. The overall response rate was 26.7% (95% confidence limits, 15.8–41.4%), with 7% of patients achieving a complete response (CR) and 19.7%, a partial response (PR). The median duration of CRs and PRs was 11 and 5 months, respectively. The response rate observed for patients previously treated with epirubicin alone was 30.5% in contrast to the 8.3% recorded for patients previously treated with cisplatin plus epirubicin. Thus, an indication of cross-resistance was absent between cyclophosphamide and epirubicin but possible between cyclophosphamide and cisplatin. Received: 10 January 1995/Accepted: 6 June 1995     

Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells

Cytotoxic activity of drug conjugates of human chorionic gonadotropin (hCG) and doxorubicin alone was investigated compared to doxorubicin in breast cancer cells with and without expression of hCG receptors. Expression of hCG receptor was determined in MCF-7 and MB231 breast cancer cell line using a multiplex nested rt-PCR approach. The entire sequence of mRNA encoding for hCG receptor was detected in MCF-7 but not in MB231 breast cancer cell line. Cytostatic effect of doxorubicin–hCG conjugates was investigated in these cell lines in comparison to unconjugated doxorubicin. The number of viable cells was determined after 24, 48, 72, 96, and 120 h. To exclude non-specific uptake of the carrier hCG from the culture media, a similar experiment was performed with albumin–doxorubicin conjugates. The number of viable cells decreased in a concentration depending manner after doxorubicin and hCG–doxorubicin conjugate treatment. However, the cytotoxic effect of hCG–doxorubicin conjugate was 10-fold increased compared to unconjugated doxorubin in hCG-receptor positive MCF-7 but not in hCG-receptor negative MB231 cells. Albumin–doxorubicin conjugates showed no increased toxicity compared to doxorubicin. We conclude that the cytotoxic effect of hCG–doxorubicin conjugates is mediated specifically via the hCG receptor. By using hCG conjugates, the development of more selective cytostatics can be achieved.     

Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency

Complex DNA damage such as double strand breaks (DSBs) and non-DSB bistranded oxidative clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10bp on opposing DNA strands) are considered the hallmark of ionizing radiation. Clustered DNA lesions are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role during the processing of DSBs. To evaluate the role of DNA-PKcs in the processing of complex DNA damage in human MCF-7 breast cancer cells we used small interfering RNAs (siRNAs) to target the silencing of the gene Prkdc coding for DNA-PKcs. MCF-7 cells with knockdown DNA-PKcs expression showed a marked decrease in their efficiency to process DSBs and OCDL after exposure to radiotherapy-relevant gamma ray doses. For the detection and measurement of complex DSBs and OCDL, we used the gamma-H2AX assay and an adaptation of pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. An accumulation of all types of DNA damage was detected for the siRNA-treated MCF-7 cells compared to controls. These findings point to the important role of DNA-PKcs in the processing of complex DNA damage and its potential association with breast cancer development.     

Experience of intravenous digital subtraction angiography (IV-DSA) in evaluation of neoadjuvant chemotherapy for advanced breast cancer

 Intravenous digital subtraction angiography (IV-DSA) was performed before and after neoadjuvant chemotherapy (NACT) in five patients with locally advanced breast cancer, and the efficacy of NACT was evaluated on the basis of the results of IV-DSA and histopathological examination. Following NACT, the maximum density of tumor enhancement (MAX) in the IV-DSA image decreased by 61.6% in case 1, 50% in case 2, 58.1% in case 3, 90.8% in case 4, and 97.2% in case 5. In all five patients, the efficacy of chemotherapy was rated as a partial response in terms of tumor size, while histological efficacy was rated as slightly effective in cases 1–4 and moderately effective in case 5. The pathological efficacy of NACT was highest in case 5, which showed the greatest decrease in MAX. These results indicate that variations in MAX reflect clinical efficacy, and, to some extent, also permit prediction of pathological efficacy. Received: March 4, 2002 / Accepted: June 10, 2002 Correspondence to:O. Watanabe     

Antiestrogenic activities of alternate-substituted polychlorinated dibenzofurans in MCF-7 human breast cancer cells

Purpose: 1,3,6,8-Substituted alkyl polychlorinated dibenzofurans (PCDFs), typified by 6-methyl-1,3,8-triCDF (MCDF), inhibit 17β-estradiol (E2)-induced responses in the rodent uterus and human breast cancer cells. The major purpose of the experiments reported here was to determine the structure-dependent antiestrogenic activities of several alternate-substituted (1,3,6,8- and 2,4,6,8-) PCDFs. Methods: The antiestrogenic activities were determined in MCF-7 human breast cancer cells using two assays, that is E2-induced cell proliferation and induction of chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with the E2-responsive Vit-CAT plasmid. Results: MCDF (10⁻⁵ M ), 6-isopropyl-1,3,8-triCDF, 6-ethyl-1,3,8-triCDF, 3-isopropyl-6-methyl-1,8-diCDF, and 6-methyl-2,4,8-triCDF, inhibited both E2-induced cell proliferation and CAT activity in MCF-7 cells. All of the remaining ten congeners inhibited either E2-induced cell proliferation or CAT activity, but not both responses. Conclusions: The antiestrogenic activity of the alternate-substituted PCDFs involves interactions between the aryl hydrocarbon and estrogen receptor signaling pathways. Although these compounds exhibited antiestrogenic activity in MCF-7 cells, the effects of individual congeners were response-specific, and there were no apparent structure-activity relationships. Received: 26 July 1996 / Accepted: 16 November 1996     

Biological and clinical evaluation of Lanreotide (BIM 23014), a somatostatin analogue,in the treatment of advanced breast cancer

Summary Recently, compounds having pure antiestrogenic activity have become available. In this study, we examined the activity of the new steroidal antiestrogen EM-170 (N-n-butyl, N-methyl-11-(16-chloro-3,17-dihydroxy-estra-1,3,5-(10)-trien-7-yl) undecanamide) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma stimulated by treatment with estrone (E₁), a steroid known to play an important role as precursor of 17-estradiol (E₂), especially in postmenopausal women. Twenty-five days after ovariectomy (OVX), tumor volume in control OVX animals decreased to 51.4 ± 11% of the initial volume; treatment with E₁, administered by Silastic implants, stimulated tumor growth to 179 ± 21%. Treatment with the antiestrogen EM-170 at a dose of 200 µg (twice daily) not only completely reversed the stimulatory effect of E₁, but also inhibited tumor growth to 30.5 ± 9.6%, an effect that is 41% (P < 0.01 vs OVX control) greater than that of ovariectomy alone. At a relatively low dose of 40 µg (twice daily), 20 days of treatment with EM-170 reversed by 55% the stimulatory effect of E₁ (1.0 µg, subcutaneously, twice daily) on tumor growth in OVX animals. On the other hand, the antiestrogen also induced a significant inhibitory effect on 17-hydroxysteroid dehydrogenase (17-HSD) activity in the DMBA-induced mammary tumors, an effect that is in agreement with the marked reduction caused by the same treatment on tumor estradiol (E₂) levels in E₁-treated OVX animals. The present data show that the new steroidal antiestrogen EM-170 exerts a potent inhibitory effectin vivo on E₁-stimulated growth of DMBA-induced mammary tumors, an effect that is probably mediated by both its antiestrogenic activity at the receptor level and its inhibitory effect on 17-HSD, thus inhibiting local E₂ formation and facilitating the action of the antiestrogen at the receptor level.     

An N-myristoylated protein kinase C-α pseudosubstrate peptide that functions as a multidrug resistance reversal agent in human breast cancer cells is not a P-glycoprotein substrate

 Protein kinase C-α (PKC-α) activation is an important contributing factor in human breast cancer MCF-7 MDR cell drug resistance. We recently reported the use of N-myristoylated PKC-α pseudosubstrate peptides with potent PKC-α inhibitory activity as reversal agents of drug resistance in MCF-7 MDR cells. The peptides potently inhibit phosphorylation of the PKC-α substrates P-glycoprotein (P-gp), raf kinase and PKC-α itself in MCF-7 MDR cells in association with a severalfold induction of intracellular uptake of P-gp substrate chemotherapeutics and a statistically significant twofold increase in cellular chemosensitivity. We now report that the N-myristoylated PKC-α pseudosubstrate peptide N-myristoyl-RFARKGALRQKNV (P3) is not a P-gp substrate in MCF-7 MDR cells based on a comparison of the cellular uptake of [¹²⁵I]-radiolabeled P3 in MCF-7 MDR vs MCF-7 WT cells. The extent of cellular uptake of the radiolabeled peptide in the drug-resistant cell line MCF-7 MDR was either greater than or equivalent to the uptake in the parental drug-sensitive MCF-7 WT cell line over a time course of 30 min to 6 h, and across a peptide concentration range of 25 – 100 μM. Additionally, treatment of the MCF-7 MDR cells with verapamil (VPL), a known P-gp efflux inhibitor, had no effect on the cellular accumulation of radiolabeled P3. Our results provide direct evidence that the N-myristoylated pseudosubstrate peptide is taken up equivalently by drug-sensitive and MDR cancer cells and therefore has potential value as an MDR reversal agent that operates by a novel mechanism. Received: 27 August 1996 / Accepted: 14 January 1997     

Polyamine profiles and growth properties of ornithine decarboxylase overexpressing MCF-7 breast cancer cells in culture

Summary To determine the direct influence of the polyamine (PA) pathway on breast cancer phenotype, we employed a transfection approach to induce overexpression of the PA biosynthetic enzyme ornithine decarboxylase (ODC) in the hormone-responsive MCF-7 breast cancer cell line. Using a modified calcium phosphate method and an ODC cDNA coding for a truncated and more stable enzyme, we were able to achieve a moderate to marked degree of ODC overexpression (up to 150-fold) in a transient transfection system. ODC-overexpressing MCF-7 cells exhibited a selective increase in cellular putrescine content, while the levels of spermidine and spermine remained unaffected. Under defined culture conditions, overexpression of ODC resulted in a consistent but modest increase in [³H]thymidine incorporation into DNA which was similar in the presence and absence of 17--estradiol, TGF-, and IGF-I. In the presence of serum, the effect of ODC overexpression on basal [³H]-thymidine incorporation into DNA was inconsistent, possibly as a result of subtle differences in culture conditions. Overall, our results support the hypothesis that activation of the PA biosynthetic pathway may confer a growth advantage to breast cancer cells.     

Estradiol stimulates synthesis of a major intracellular protein in a human breast cancer cell line (MCF-7)

Summary Previous studies have shown that synthesis of a 24,000 molecular weight (24k) protein (7) and the mRNA activity coding for a 24k protein (8) are selectively stimulated by estradiol in MCF-7 human breast cancer cells. Utilizing a double-label electrophoresis technique to measure the relative rates of specific protein synthesis, the present study relates further characterization of this estrogen response. The 24k protein was found to be stimulated by estradiol provided cells were preincubated with antiestrogen. Under these conditions, antiestrogens inhibit cell growth and addition of estradiol reverses growth inhibition (estrogen growth rescue). As a control experiment, specific protein synthesis was measured in another growth rescue model produced by withdrawing serum from the medium to inhibit growth and then stimulating cells by readdition of serum (serum growth rescue). This failed to stimulate synthesis of a 24k protein, implying that the effect observed following estrogen rescue was not a non-specific result of growth stimulation. Increased 24k synthesis was found to be both time and estrogen-dose dependent and required specifically those steroid hormones which interact with the estrogen receptor. Moreover, the dose response curve for 24k stimulation paralleled closely the dose response for estrogen stimulation of nuclear estrogen receptor processing, an event correlated with another estrogenic response in MCF-7 cells, induction of progesterone receptor. These findings suggest that estradiol itself directly stimulates synthesis of the 24k protein through interaction with the estrogen receptor system. Further separation of double-label cytosols by two-dimensional electrophoresis resolved the 24k protein into two isoelectric species with pI's in the range of 6.7–6.9 and 6.4–6.7, the latter being the most abundant or rapidly labeling protein. Based on the percentage of total cytosol incorporation contained in individual two-dimensional gel spots, the major species of 24k represented in the fully stimulated cell (³H-counts) about 1.6% of newly synthesized protein. It also represented about 0.7% of newly synthesized protein in the unstimulated (¹⁴C-counts) cell, which indicates that 24k is synthesized constitutively. Because of its relative abundance, the 24k protein may be a suitable end product for investigating the mechanisms of estrogen action in human breast cancer.Abbreviations used are PgR, progesterone receptor ER estrogen receptor - DHT dihydrotestosterone - Hepes N¹-2-hydroxyethylpiperazine-N¹-ethane sulfonic acid - MEM Eagle's minimum essential medium - TCA trichloroacetic acid - SDS sodium dodecylsulfate; nafoxidine, (1-(2-[p-(3,4-dihydro-6-methoxy-2phenyl-1-naphthyl) Phenoxy]ethyl) pyrrolidine hydrochloride; Upjohn); tamoxifen, (1-p--dimethylaminoethyoxyphenyl-trans-1,2-diphenylbut-1-ene; ICI-46474); CI-628,1-[2-[p[-(p-methoxyphenyl)--nitrostyryl]-phenoxy]ethyl]pyrrolidine monohydrate monocitrate Address for reprints: William L. McGuir, M.D., Department of Medicine, University of Texas Health Science Center 7703 Floyd Curl Drive. San Antonio, TX 78284.     

Reduced expression of p27 is a novel mechanism of docetaxel resistance in breast cancer cells

Introduction  

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. However, apoptosis and cell cycle regulation are key mechanisms by which most chemotherapeutic agents exert their cytotoxic effects.     

Prevention of development of dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat by the new nonsteroidal antiestrogen EM-800 (SCH57050)

The effect of EM-800, a new non-steroidal antiestrogen having pure antiestrogenic activity, was studied on chemical carcinogenesis induced by dimethylbenz(a)anthracene (DMBA) as well as on serum lipids and bone mass in the rat. Treatment with EM-800 orally, once daily, for 282 days (9 months), starting 3 days before DMBA administration, decreased the incidence of tumors from 95% in control animals to 60% (p < 0.01), 38% (p < 0.01), and 28% (p < 0.01) at the daily doses of 25 g, 75 g, and 250 g, respectively. The average number of tumors per animal decreased from 4.5 ± 0.5 tumors in the control group to 0.9 ± 0.2 (p < 0.01), 0.5 ± 0.2 (p < 0.01), and 0.3 ± 0.1 (p < 0.01) tumors in the rats treated with the above-indicated doses of the antiestrogen. In addition, treatment with the increasing doses of EM-800 reduced serum cholesterol levels to 64%, 56%, and 48% of control, while serum triglycerides decreased to 31%, 28%, and 30% of control. Bone mineral content (BMC) and bone mineral density (BMD) of total skeleton, femur, and lumbar spine were not significantly affected following 282 days of treatment with EM-800. However, treatment with EM-800 inhibited the urinary ratio of hydroxyproline to creatinine (HP/Cr) from 14.0 ± 3.90 mol/mmol in controls to 7.6 ± 0.8 (p < 0.05), 6.8 ± 0.8 (p < 0.01), and 6.8 ± 1.1 (p < 0.01) mol/mmol, respectively, while the same treatment had no effect on serum total alkaline phosphatase (tALP) activity or urinary calcium and phosphorus excretion. The 25 g, 75 g, and 250 g daily doses of EM-800 inhibited uterine weight by 35% (p < 0.01), 62% (p < 0.01), and 66% (p < 0.01), while vaginal weight was reduced by 8% (p < 0.05), 30% (p < 0.01), and 38% (p < 0.01), respectively. In agreement with the 27% increment (p < 0.05) in ovarian weight at the highest antiestrogen dose used, serum androstenedione (p < 0.05), androst-5-ene-3,17-diol (p < 0.01), testosterone (p < 0.05), and estradiol (p < 0.01) levels were increased. The present data show that EM-800 prevents the development of DMBA-induced mammary tumors while simultaneously inhibiting uterine and vaginal weight, reducing serum cholesterol and triglyceride levels, and having no adverse effect on bone mass following 9 months of treatment in the rat.This revised version was published online in October 2005 with corrections to the Cover Date.