ANDROGEN ANTAGONISTIC EFFECT OF ESTRAMUSTINE PHOSPHATE (EMP) METABOLITES ON WILD-TYPE AND MUTATED ANDROGEN RECEPTOR

Estramustine phosphate is used frequently, alone or in combination wit h other antitumor agents, for the treatment of hormone-refractory pros tate cancer. Estramustine phosphate is metabolically activated in vivo , and its metabolites, estramustine, estromustine, estrone, and beta-e stradiol inhibit the assembly of microtubules [for review see: Kreis W , In: Concepts, Mechanisms, and New Targets for Chemotherapy (Ed. Mugg ia FM), pp. 163-184. Kluwer Academic Publishers, Boston, 1995]. We inv estigated, by displacement of [H-3]methyltrienolone in the presence of 2.5 mM of triamcinolone acetonide, the binding of estramustine phosph ate and its metabolites, estramustine, estromustine, estrone, and beta -estradiol, as well as other antiandrogen agents including alpha-estra diol, bicalutamide, and hydroxyflutamide, to the mutated androgen rece ptor (m-AR) in LNCaP cells and to the wild-type androgen receptor in w ild-type AR cDNA expression plasmid (w-pAR0) cDNA-transfected HeLa cel ls. Analogous to the antiandrogens, bicalutamide and hydroxyflutamide, binding of estramustine phosphate metabolites to the androgen recepto r was observed. The EC50 values (in mu M) were: estramustine phosphate , >10; estramustine, 3.129 +/- 0.312; estromustine; 2.612 +/- 0.584; e strone, 0.800 +/- 0.090; alpha-estradiol, 1.051 +/- 0.096; beta-estrad iol, 0.523 +/- 0.028; bicalutamide, 4.920 +/- 0.361; and hydroxyflutam ide, 0.254 +/- 0.012. The transactivation assay demonstrated that, ana logous to bicalutamide, estramustine could not induce luciferase activ ity in either w-pAR0 or m-pARL transfected HeLa cells. In contrast, a strong induction of the reporter activity by dihydrotestosterone was o bserved. 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 ce lls 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 mu M of estram ustine, respectively (IC50 = 10.97 +/- 1.68 mu M). Binding of hydroxyf lutamide 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 phospha te metabolites perform as androgen antagonists of AR, an additional me chanism involved in the therapeutic effect of estramustine phosphate i n patients with prostate cancer. (C) 1998 Elsevier Science Inc.