Binding Sites of Droloxifene in the Cytosol of 7,12-Dimethylbenz[α]anthracene-induced Rat Mammary Tumor Cells

The binding sites, other than the estrogen receptor (ER), of the antiestrogens droloxifene (DROL, (E)-α-[ p [2-(dimethylamino)ethoxy]-phenyl]-α'-ethyl-3-stilbenol) and tamoxifen (TAM), and estradiol-17β (E₂) in the cytosol of 7,12-dimethylbenz[α]anthracene-induced rat mammary ER-positive tumor cells were studied using a high-performance liquid chromatography (HPLC) gel filtration assay. The cytosol was incubated with ³H-labeled drug with or without unlabeled drug, and separated by HPLC gel filtration. ³H-E₂ produced two major peaks of radioactivity at fractions No. 40 and No. 70. The peak at fraction No. 70 was identified as the ER in an ER-enzyme-immuno assay. This peak was dose-dependently inhibited by unlabeled DROL or TAM, DROL being a more potent inhibitor than TAM. The peak at fraction No. 40 was also inhibited by co-incubation with unlabeled DROL or TAM. ³H-DROL or ³H-TAM provided only one peak at fraction No. 43. This peak was thought to be an antiestrogen binding site (AEBS), because it was inhibited by unlabeled antiestrogen hut not by E₂. The results suggest that the antiestrogens DROL and TAM have a higher affinity for the AEBS than for the ER in the absence of E₂, while in the presence of E₂ both have an affinity for the ER and inhibit E₂ binding to the ER.