Progesterone 5ß-reductase genes of the Brassicaceae family as function-associated molecular markers.

This study aimed to define progesterone 5ß-reductases (P5ßR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5ßR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5ßRs are ubiquitous in the Brassicaceae. The recombinant P5ßR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5ßR enzymes and homology modelling using Digitalis lanata P5ßR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5ßR genes or P5ßR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective.

The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Delta4,5-steroid 5beta-reductase activity in Arabidopsis thaliana L.

The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5beta-reductase, an enzyme considered to catalyze a key step in the biosynthesis of cardiac glycosides. A. thaliana does not accumulate cardenolides but protein extracts prepared from its leaves were capable of reducing progesterone to 5beta-pregnane-3,20-dione. A full-length cDNA clone encoding a Delta(4,5)-steroid 5beta-reductase (At5beta-StR, EC 1.1.1.145/1.3.1.23), a member of the short-chain dehydrogenase/reductase (SDR) family, was isolated from A. thaliana leaves. A SphI/SalI At5beta-StR gene fragment was cloned into the pQE vector system and transformed into Escherichia coli. The gene was functionally expressed and the recombinant His-tagged fusion protein was characterized. K(m) values and specific activities for putative 3-oxo-Delta(4,5)-steroid substrates such as progesterone, cortisol, cortexone and 4-androstene-3,17-dione, and for the co-substrate NADPH were determined. Progesterone was stereo-specifically reduced to 5beta-pregnane-3,20-dione and none of the 3-oxo-Delta(5,6)-steroids tested were accepted as a substrate. The gene encoding At5beta-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5beta-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5beta-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5beta-StR when compared to the activity of the recombinant form of the D. lanata enzyme.

Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells.

The effects of vinorelbine and paclitaxel on the activity of extracellular signal-regulated protein kinase2 (ERK2), a member of MAP kinase, and its role in the induction of bcl-2 phosphorylation and apoptosis were evaluated in MCF-7 cells. We demonstrated that ERK2 was activated rapidly by vinorelbine, and was inhibited by either paclitaxel or estramustine. A 3-fold increase of ERK2 kinase activity was observed within 30 min when MCF-7 cells were treated with 0.1 microM vinorelbine. In contrast, the same treatment with paclitaxel resulted in a significant decrease of ERK2 kinase activity. We also demonstrated that elevated bcl-2 phosphorylation induced by vinorelbine is paralleled by decrease of a complex formation between bcl-2 and bax, cleavage of poly (ADP) ribose polymerase (PARP) protein, activation of caspase-7, and apoptosis. The levels of bcl-2 phosphorylation, bax, and PARP were not significantly affected by 2'-amino-3'-methoxyflavone (PD 98059), an ERK kinase specific inhibitor. Thus, our data suggest that the apoptosis induced by vinorelbine in MCF-7 cells is mediated through the bcl-2 phosphorylation/bax/caspases pathways, and that activation of ERK2 by vinorelbine does not directly lead to the drug-mediated apoptosis. Since decrease of PARP occurred quickly following the treatment of MCF-7 cells with either 0.1 microM of vinorelbine or paclitaxel, this protein may serve as an early indicator of apoptosis induced not only by DNA damaging agents, but also by antimicrotubule drugs.

In vitro effects of dexrazoxane (Zinecard) and classical acute leukemia therapy: time to consider expanded clinical trials?

Anthracyclines have been the backbone of acute leukemia therapy in the adult for many years, but little attention has been paid to the long-term toxicity of these agents in this disease because of the poor survival of this population of patients. Recent studies have examined dose-intensified daunorubicin with dosages as high as 95 mg/m2 daily x 3 in this population with the attendant concerns of both acute and chronic toxicity. We have examined three human leukemia cell lines in vitro, treated with either daunorubicin, mitoxantrone, with or without cytosine arabinoside in the presence of dexrazoxane to determine whether such treatment would be synergistic or antagonistic. AML-193, CRF-SB, and Molt-4 cell lines were grown to confluence, plated into microtiter dishes and incubated for 72 h with varying concentrations of the above drugs. Cytotoxicity was determined by the MTT assay, and synergy or antagonism by median effect analysis. Dexrazoxane demonstrated additive or synergistic cytotoxic effects (CI <1) under most conditions. The triplet of daunorubicin, cytosine arabinoside, and dexrazoxane showed profound synergy in all three cell lines. These effects occurred at clinically achievable levels. If high dosages of anthracyclines are contemplated in this population, these preclinical data suggest that the addition of dexrazoxane to classical therapy is not antagonistic and thus may allow an investigation of the role of dexrazoxane as a cardiac protectant.

Phase I and pharmacokinetic study of LY309887: a specific inhibitor of purine biosynthesis.

PURPOSE: In this phase I trial in humans the safety and pharmacology of LY309887 on a weekly schedule combined with daily oral 5-mg doses of folic acid were evaluated. BACKGROUND: LY309887 is an inhibitor of folate-dependent enzymes involved in de novo purine biosynthesis and has a broad preclinical antitumor activity. In murine systems, combining this agent with exogenous folic acid results in an enhanced therapeutic index. METHODS: This study was a single-institution, open-label, clinical trial of dose escalation with toxicity and pharmacokinetic parameters determined. The dose range studied was 0.5-4 mg/m2 per week x6 and then a modified schedule weekly x3 every 6 weeks. RESULTS: Dose-limiting toxicities were of delayed onset and associated with hematologic, neurologic, and mucosal effects. Pharmacokinetic parameters revealed dose linearity for AUC and Cmax. Low circulating levels of drug persisted for over 200 h. Urinary excretion accounted for approximately 50% of the parent drug but was highly variable. The urinary excretion was near maximal within 24 h of dosing. CONCLUSIONS: The modified dosing schedule allowed repetitive dosing in patients. Further evaluation of the 2 mg/m2 per week x3 every 6 weeks with daily oral folate supplement as a potential phase II dose may be warranted.

A reexamination of PSC 833 (Valspodar) as a cytotoxic agent and in combination with anticancer agents.

BACKGROUND: The cyclosporins have been thought as being mainly immunosuppressive agents which interfere with the function of the MDR pump and thus play a role in resistance to drug anticancer effects. We reexamined their cytotoxicity in defined cell lines both as single agents and in combination with agents which may be of value in human malignant disease. METHODS: Cells were grown to confluence following inoculation at 5,000-8,000 cells/well in 96-well dishes, and growth patterns and death were determined by an MTT assay. Median effect analysis was used to look for synergy, additive effects, or antagonism between the cyclosporins and drugs with antitumor effects in humans. RESULTS: Cyclosporin A and PSC 833 were found to have cytotoxic activity at clinically achievable concentrations in breast, leukemia, and prostate cell lines. Synergistic or additive effects were demonstrated in all three prostate cell lines when PSC 833 was combined with estramustine, etoposide, ketoconazole, suramin, or vinorelbine in the prostate cancer cell lines. Cell line-selective additive effects or synergism were also identified with bicalutamide, carboplatin, cisplatinum, cis-retinoic acid, dexamethasone, 5-fluorouracil, liarozole, and trans-retinoic acid. CONLCLUSIONS: PSC 833 or cyclosporin alone or in combination with other agents may have an anticancer effect independently of their modulatory action on MDR. Several of the synergistic combinations which are not mediated by the MDR pump need to be tested in vivo for efficacy.

Synergism of cytotoxic effects of vinorelbine and paclitaxel in vitro.

Empiric combinations of vinca alkaloids with taxanes have been recently used in clinical oncology. To enhance the activity of these two classes of agents, we evaluated the sequence and duration of exposure, looking for synergistic effects. Cell lines DU 145, PC 3, LnCaP, LL 86, MCF7wt, and MCF7/ADR (NCI/ADR-RES) were incubated with varying concentrations of paclitaxel or vinorelbine. Cytotoxicity was evaluated by a semiautomated MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) method. Synergism or antagonism of these two agents either sequentially or in combination was determined by median effect analysis. Prolonged exposure of cells to either drug enhanced cytotoxic effect. Synergism or antagonism with vinorelbine and paclitaxel were both sequence dependent and cell line specific. In the case of MCF7wt, synergism was seen when a 48-hr exposure to vinorelbine preceded paclitaxel, whereas antagonism was noted when both agents were applied simultaneously or when the sequence was reversed. Concurrent vinorelbine and paclitaxel were synergistic in four of six cell lines when the exposure was extended to 96 hr but not for shorter durations of exposure. Sequential exposure of vinorelbine preceding paclitaxel or prolonged exposure to both agents concurrently needs to be tested clinically to determine whether the antitumor activity of this combination can be enhanced. In addition, these studies suggest concurrent administration of these two agents may lead to a less than optimal cytotoxic result.

Survival of patients who had salvage castration after failure on bicalutamide monotherapy for stage (D2) prostate cancer.

Patients with hormone-naive stage D2 prostate cancer often benefit from castration. This treatment, however, frequently produces many unacceptable physical and psychological side effects, especially in younger and sexually active patients. Bicalutamide is an oral antiandrogen with excellent tolerance and preservation of sexual function. Three institutions participated in phase II and III trials of bicalutamide monotherapy (50 mg daily) as primary therapy in hormone-naive patients with stage D2 prostate cancer. Upon bicalutamide failure, all patients underwent castration and were followed until death. Fifty-four patients received bicalutamide 50 mg orally once a day. One patient (2%) had complete response, 9 patients (17%) had partial response, and 27 patients (50%) had stable disease. Seventeen patients (31%) had progressive disease. The median time to bicalutamide failure was 47.4 weeks, 70.5 weeks for the responders vs. 25.4 weeks for the nonresponders (p < 0.001). The median survival time after the sequential use of bicalutamide and castration was 119.2 weeks for all 54 patients, 162.0 weeks for the responders, and 73.5 weeks for nonresponders (p < 0.0001). The median survival time after initiation of castration was 71.1 weeks for all 54 patients, 91.4 weeks for bicalutamide responders, and 42.1 weeks for nonresponders (p < 0.01). In hormone-naive patients with stage D2 prostate cancer, sequential treatment with bicalutamide monotherapy followed by castration upon failure may produce survival time within the range reported for initial treatment with castration. Thus, considering the favorable quality of life profile of bicalutamide, further studies are needed to define the role of sequential hormonal therapy in younger sexually active patients.

Structure-specificity relationship of cardiac glycosides as a substrate for glucohydrolase II.

Cardenolide glucohydrolase II (CGH II) is a cardenolide-specific glucohydrolase obtained from Digitalis lanata leaves. We investigated the structure-specificity relationship of several cardenolide disaccharides as a substrate for CGH II. Conformation analysis of the substrates was performed using molecular mechanics calculations. The sugar chain conformation of two inert glycosides was significantly different from that of the other glycosides. The other two glycosides, which were weak substrates of CGH II, were suggested to have an intramolecular hydrogen bond between the sugar groups. It was deduced that this hydrogen bond restricts the conformational change of the sugar chain and prevents the glycosides from enzymatic recognition.

Enzymatic degradation of cichoric acid in Echinacea purpurea preparations.

Cichoric acid (2R,3R-O-dicaffeoyltartaric acid) (1) is highly susceptible to enzymatic degradation during the preparation of Echinacea purpurea products. Degradation of 1 and other caffeic acid derivatives can be inhibited by antioxidants added to the extraction solvent or in buffered protein extracts saturated with nitrogen. Inhibitor studies conducted with protein extracts prepared from dried overground parts of E. purpurea revealed that polyphenol oxidases (PPO) but not peroxidases are responsible for the oxidative degradation of exogenous and endogenous caffeic acid derivatives. With a view to stabilizing aqueous extracts with respect to their content of 1, the effects of ascorbic acid and ethanol were tested. Compound 1 was not stable under conditions where oxidative processes could almost be excluded. It was found that an esterase hydrolyzing the ester bonds between tartaric acid and caffeic acid is still active under PPO inhibitory conditions. Finally, addition of 40% ethanol and 50 mM ascorbic acid to aqueous extracts of "Echinaceae purpureae herba" resulted in a constant amount of cichoric acid over four weeks.

Daily oral estramustine and intermittent intravenous docetaxel (Taxotere) as chemotherapeutic treatment for metastatic, hormone-refractory prostate cancer.

As part of an extensive search of synergistic combinations of agents that demonstrate cytotoxic activity in tissue culture against prostate cancer cell lines, we have identified estramustine phosphate and docetaxel (Taxotere; Rhône-Poulenc Rorer, Collegeville, PA) as active. Patients with hormone-refractory metastatic prostate cancer with good performance status were entered onto a phase I trial of this combination. Estramustine phosphate was given on a daily oral dosing schedule of 14 mg/kg; docetaxel was administered intravenously over 1 hour every 3 weeks. Prophylactic corticosteroids were administered to minimize taxane side effects. Four dose levels of docetaxel were studied: 40, 60, 70, and 80 mg/m2. Actual weight was used for dosing calculations. An intermittent docetaxel dose of 70 mg/m2 with estramustine phosphate at 12 mg/kg was determined to be a phase II dose and to allow repetitive dosing. Eight additional patients were entered at this dose level of docetaxel. In all patients, the limiting side effects were fatigue and leukopenia. Thromboembolic events also were seen in 16% of patients. In the first 17 patients treated in the phase I aspect of the study, the biochemical response rate was 82%, demonstrating greater than a 50% decline in prostate-specific antigen. Twenty-four percent had normalization of prostate-specific antigen. These early studies indicate that the combination of estramustine and docetaxel has activity in this population of patients.

A phase II study of docetaxel (Taxotere), estramustine, and low-dose hydrocortisone in men with hormone-refractory prostate cancer: preliminary results of cancer and leukemia group B Trial 9780.

Combinations of estramustine with other antimitotic agents, such as docetaxel (Taxotere; Rhône-Poulenc Rorer, Collegeville, PA), are synergistic in vitro and show significant clinical activity in hormone-refractory prostate cancer (HRPC). We have studied intravenous docetaxel 70 mg/m2, oral estramustine, and low-dose daily hydrocortisone in men with HRPC who demonstrated progression after initial hormone therapy. Of the 47 men who were enrolled, 40 are evaluable for response and/or toxicity. One patient (3%) has had a complete response and eight (20%) have had a partial response, yielding a total objective response rate of 23%. Of 39 patients with elevated pretreatment prostate-specific antigen levels who have had at least one posttreatment prostate-specific antigen measurement, 27 (69%) have had > or =50% decreases and 21 (54%) have had > or =75% decreases in prostate-specific antigen levels. Toxicity is modest but manageable. This therapy is efficacious and well tolerated in HRPC and should be compared in phase III trials with other drugs active in HRPC, such as mitoxantrone and hydrocortisone.

The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review.

PURPOSE: Microtubules are important cytoskeletal components involved in many cellular events. Antimicrotubule agents including polymerizing agents (paclitaxel and docetaxel) and depolymerizing drugs (vincristine, vinorelbine, and estramustine phosphate) are widely used either alone or in combination with other anticancer drugs. These antimicrotubule agents are promoters of apoptosis in cancer cells. In this review, we discuss the role of bcl-2 family genes in the regulation of apoptosis, and summarize effects of microtubule targeting agents on apoptotic signal transduction pathways. CONCLUSION: Disruption of microtubule structure by antimicrotubule drugs results in induction of tumor suppressor gene p53 and inhibitor of cyclin-dependent kinases, p21WAF1/CIP1 (p21), and activation/inactivation of several protein kinases including Ras/Raf, PKC/PKA I/II, MAP kinases, and p34cdc2. These protein kinases are associated directly or indirectly with phosphorylation of bcl-2. Phosphorylation of bcl-2 and the elevations of p53 and p21 lead to apoptosis. New pathways of antitumor agents could be directed at this p53, p21 and bcl-2/bax function, and may enhance the effect of existing agents.

Synergistic effect of estramustine and [3'-keto-Bmtl]-[Val2]-cyclosporine (PSC 833) on the inhibition of androgen receptor phosphorylation in LNCaP cells.

Estramustine phosphate has been used frequently alone or in combination with other drugs for the treatment of hormone-refractory prostate cancer. Estramustine is one of the major active metabolites of estramustine phosphate in vivo. We recently demonstrated that estramustine acts as an androgen antagonist, and the combination of estramustine with [3'-keto-Bmtl]-[Val2]-cyclosporine (PSC 833) results in synergistic cytotoxicity. Unlike other regulators of microtubules, such as paclitaxel, the present study demonstrated that estramustine alone or in combination with PSC 833 did not induce bcl-2 phosphorylation in LNCaP cells. No synergism between estramustine and PSC 833 in the induction of bcl-2 phosphorylation was obtained in MCF-7 cells exposed for 16 hr to estramustine (5-15 microM) and PSC 833 (5 microM). A significant synergistic antiandrogenic effect as measured by the inhibition of dihydrotestosterone-induced reporter gene luciferase expression in both wild-type and mutated androgen receptor (AR) cDNA-transfected HeLa cells was observed when the cells were exposed to estramustine and PSC 833. Treatment of LNCaP cells with estramustine alone (5-15 microM) resulted in a decrease of AR expression and phosphorylation. This effect was enhanced markedly by PSC 833. A strong correlation between AR phosphorylation and expression of the AR target gene PSA was obtained in dihydrotestosterone-stimulated LNCaP cells. The up-regulated PSA expression is a function of the level of the phosphorylated AR (r = 0.9814), but not the dephosphorylated form of the receptor protein (r = 0.4808). Thus, our studies suggest that the synergism between estramustine and PSC 833 in LNCaP cells is a consequence of inhibition of AR expression and phosphorylation, thus leading to interruption of AR-mediated gene expression.

Phosphorylation/dephosphorylation of androgen receptor as a determinant of androgen agonistic or antagonistic activity.

Protein phosphorylation/dephosphorylation is an important posttranslational modification that plays a critical role in signal transduction. The androgen receptor (AR) is under such control. We demonstrate that androgen receptor phosphorylation determines whether or not AR ligands perform as agonists or antagonists in LNCaP cells. Androgen receptor ligands (such as dihydrotestosterone and beta-estradiol) stimulate receptor expression and phosphorylation and, as a result, they act as agonists or partial agonists. In contrast, agents such as bicalutamide and estramustine inhibit the receptor phosphorylation and act as antagonists. This model is supported by gene expression and transactivation assays. Significant increases in levels of both mRNA and protein of prostate-specific antigen (PSA), a natural AR target gene, occur following the treatment of LNCaP cells with DHT, beta-estradiol, or hydroxyflutamide. In contrast, exposure of LNCaP cells to bicalutamide or estramustine results in a sharp decrease of PSA expression. Agonistic or antagonistic effect of these compounds on PSA expression parallels the level of phosphorylated, but not dephosphorylated androgen receptors. These agonistic or antagonistic effects are also observed in HeLa cells transfected with wild-type AR expression plasmid (pAR0) and AR-driven luciferase expression plasmid GRE-tk-LUC in the presence of different groups of AR blockers. Our data indicate that the functional status of androgen receptors is strongly correlated with the phosphorylation status of the receptors, and that the phosphorylated androgen receptor is the form of the receptor transcriptionally active in regulation. Thus the androgen receptor phosphorylation/dephosphorylation may serve as a new molecular target for screening androgen antagonists for the treatment of prostate cancer.

Phase I trial of the combination of daily estramustine phosphate and intermittent docetaxel in patients with metastatic hormone refractory prostate carcinoma.

BACKGROUND: To apply our preclinical findings of cytotoxic synergy with the combination of estramustine phosphate (EP) and docetaxel as the basis of treatment of hormone refractory metastatic prostate cancer in man. To determine the optimal dosage and the toxicities of these two agents for future trials. PATIENTS AND METHODS: Seventeen patients with hormone refractory metastatic prostate cancer who were ambulatory with performance status < or = 2, normal marrow, renal and hepatic function were entered. Prior exposure to EP or a taxane were exclusion factors. EP was given orally at a dose of 14 mg/kg of body weight daily with concurrent docetaxel administered every 21 days as an intravenous infusion over 1 hour with dexamethasone 8 mg. PO BID for five days. EP dosages were kept static; docetaxel dosages were explored in a minimum of three patients per level for dosages of 40, 60, 70, and 80 mg/m2. Patients were evaluated weekly. Prostate specific antigen (PSA) was measured every three weeks. RESULTS: Five patients were entered at a docetaxel dose of 40 mg/m2, three at 60 mg/m2, six at 70 mg/m2, and three at 80 mg/m2. Only one patient had received prior chemotherapy. Grades 1 or 2 hypocalcemia and hypophosphatemia were seen at all dosage levels. Other grade 2 or less toxicities not related to dosage included alopecia, anorexia, stomatitis, diarrhea, and epigastric pain. Dose limiting toxicities (DLT) as grade 4 leukopenia and grade 4 fatigue were seen at 80 mg/m2. The phase II dose was defined at 70 mg/m2 with rapidly reversible leukopenia and minor liver function abnormalities. At this dosing level, dose intensity was 88% and 86% over consecutive cycles for docetaxel and EP, respectively. Two vascular events occurred at this dose level (70 mg/m2): one arterial and the other venous. PSA decreases greater than 50% from baseline were seen in 14 of 17 patients at all dosage levels. Four of the 17 patients demonstrated a complete biochemical response (PSA < or = 4 ng/ml). One patient had a partial response with measurable lung and liver lesions. CONCLUSION: EP given continuously with every three-week docetaxel at a dose of 70 mg/m2 is tolerable with evidence of antitumor activity based upon significant declines in PSA in the majority of patients and improvement of lung metastasis in one patient. Larger phase II studies of this combination in a homogenous population are warranted.

In vitro evaluation of synergism or antagonism with combinations of new cytotoxic agents.

New cytotoxics with significant activity both in preclinical and clinical situations continued to be applied in the clinic by empiric means. The use of defined cell lines allows unanticipated antagonism between agents to be identified and to suggest synergistic combinations which need to be tested. By means of a semi-automated MTT assay and median effect analysis, we have identified antagonism in two couplets being evaluated in the clinic: etoposide with paclitaxel and vinorelbine with gemcitabine. Optimal use of these agents in man may require spacing these agents in time to prevent an adverse drug interaction between the agents which may diminish the potential response rate.

Synthesis of cardenolide glycosides and putative biosynthetic precursors of cardenolide glycosides.

A rapid and efficient procedure for glycosylation of steroids was established using a modified Koenigs-Knorr procedure. Peracetylated beta-glycosides were synthesized by reaction of cardenolides, various pregnanes and 23-nor-5,20(22)E-choldienic acid at room temperature with the peracetylated 1-bromo derivatives of D-glucose, D-galactose, D-fucose and cellobiose. Subsequent deprotection was performed by alkaline hydrolysis with sodium methoxide. Structures of the respective glycosides were established by NMR techniques. The complete protocol was shown to be non-destructive at all stages to the sugar moiety and the steroidal nucleus. The gamma-unsaturated lactone ring of the cardenolides was shown to remain intact and no formation of C-14 unsaturated compounds was observed.

Androgen antagonistic effect of estramustine phosphate (EMP) metabolites on wild-type and mutated androgen receptor.

Estramustine phosphate is used frequently, alone or in combination with other antitumor agents, for the treatment of hormone-refractory prostate cancer. Estramustine phosphate is metabolically activated in vivo, and its metabolites, estramustine, estromustine, estrone, and beta-estradiol inhibit the assembly of microtubules [for review see: Kreis W, In: Concepts, Mechanisms, and New Targets for Chemotherapy (Ed. Muggia FM), pp. 163-184. Kluwer Academic Publishers, Boston, 1995]. We investigated, by displacement of [3H]methyltrienolone in the presence of 2.5 mM of triamcinolone acetonide, the binding of estramustine phosphate and its metabolites, estramustine, estromustine, estrone, and beta-estradiol, as well as other antiandrogen agents including alpha-estradiol, bicalutamide, and hydroxyflutamide, to the mutated androgen receptor (m-AR) in LNCaP cells and to the wild-type androgen receptor in wild-type AR cDNA expression plasmid (w-pAR0) cDNA-transfected HeLa cells. Analogous to the antiandrogens, bicalutamide and hydroxyflutamide, binding of estramustine phosphate metabolites to the androgen receptor was observed. The EC50 values (in microM) were: estramustine phosphate, > 10; estramustine, 3.129 +/- 0.312; estromustine; 2.612 +/- 0.584; estrone, 0.800 +/- 0.090; alpha-estradiol, 1.051 +/- 0.096; beta-estradiol, 0.523 +/- 0.028; bicalutamide, 4.920 +/- 0.361; and hydroxyflutamide, 0.254 +/- 0.012. The transactivation assay demonstrated that, analogous to bicalutamide, estramustine could not induce luciferase activity in either w-pAR0 or m-pARL transfected HeLa cells. In contrast, a strong induction of the reporter activity by dihydrotestosterone was observed. Down-regulation of prostate-specific antigen (PSA) expression, an AR-target gene, by estramustine and bicalutamide was accompanied by the blockade of the mutated androgen receptor. Exposure of LNCaP cells to estramustine for 24 hr caused transcriptional inhibition of PSA in a concentration-dependent manner. The levels of PSA mRNA decreased 56 and 90% when LNCaP cells were treated with 5 and 10 microM of estramustine, respectively (IC50 = 10.97 +/- 1.68 microM). Binding of hydroxyflutamide to m-AR in LNCaP cells resulted in a concentration-dependent stimulation of PSA expression, suggesting that hydroxyflutamide acted as an agonist of the m-AR. Our data indicate that estramustine phosphate metabolites perform as androgen antagonists of AR, an additional mechanism involved in the therapeutic effect of estramustine phosphate in patients with prostate cancer.