enclomiphene and Breast-Neoplasms

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.

Topics: Binding, Competitive; Breast Neoplasms; Clomiphene; Cytosol; Enclomiphene; Estrogen Antagonists; Estrogens; Humans; Kinetics; Microsomes; Nafoxidine; Tamoxifen

In order to obtain breast tumor directed agents, we have prepared mixed compounds using estradiol or (E)-clomiphene as specific vectors of the breast tissue and a DNA intercalator from the ellipticine series as the cytotoxic agent. Among the newly synthesized ellipticine derivatives, only the 2-[3-aza-5-(3,17 beta-dihydroxy-1,3,5-estratrien-17 alpha-yl)-4-oxopentamethylene]ellipticinium bromide shows the desired properties, DNA intercalation and affinity for estrogen receptor. Competition experiments with estradiol on the hormone-dependent human MCF-7 breast cancer cell line demonstrate that a transport by the estrogen receptor system is not involved in the antitumor activity of derivative 24.

Topics: Alkaloids; Animals; Breast Neoplasms; Cattle; Cell Line; DNA; Ellipticines; Female; Humans; In Vitro Techniques; Intercalating Agents; Leukemia L1210; Neoplasms, Hormone-Dependent; Receptors, Estrogen

The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor. Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth. In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor. In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected.

Topics: Breast Neoplasms; Cell Division; Cells, Cultured; Clomiphene; Enclomiphene; Estrogen Antagonists; Female; Humans; Receptors, Estrogen; Tamoxifen

The antitumor activity of three enclomiphene (trans-1- (p-beta-diethylaminoethoxyphenyl)1,2-diphenyl-2-chloroethylene) analogs and the cis isomer zuclomiphene was investigated in MCF 7 human mammary carcinoma cells in culture. Relative antitumor activity was compared with relative binding affinities (RBAs) for the nuclear estrogen receptor (RE; estradiol = 100%) and a microsomal antiestrogen-binding site (AEBS; tamoxifen = 100%) measured in cell-free extracts from MCF 7 cells. Modifications in the structure of the diethylaminoethoxy side chain influenced affinity of both RE and AEBS. Deethylation of the side chain (9599) reduced affinity for both sites by 65-70%, while a diethylaminoproproxy side chain (6866) resulted in a 3-fold increase in affinity for RE (enclomiphene = 2%; 6866 = 6%), but reduced the RBA for AEBS by 66% (enclomiphene = 140%; 6866 = 45%). Conversion of the ether linkage to an amine (10222) increased affinity for RE (10222 = 5%), but markedly reduced affinity for the AEBS (10222 = 15%). All five compounds inhibited the growth of MCF 7 cells in culture, but with markedly different potencies. At low doses (0.25-1.0 microM), where the growth-inhibitory effects were reversed by estradiol (except in the case of zuclomiphene), the relative antitumor activity (6866 greater than 10222 greater than enclomiphene greater than 9599 greater than zuclomiphene) was in the same order as RBA for RE. At higher doses (greater than 2.5 microM), where the growth-inhibitory effects were unaffected or partially reversed by estradiol, the relative potencies of these compounds as antitumor agents changed. Zuclomiphene became the most active, while the potency of enclomiphene increased relative to 6866 and 10222 (zuclomiphene greater than enclomiphene greater than 6866 greater than 10222 greater than 9599). At these doses, estrogen-irreversible antitumor activity was unrelated to affinity for RE, but showed some correlation with affinity for AEBS. It is concluded that the major effect of these compounds on mammary carcinoma growth in vitro is an estrogen-reversible growth-inhibitory effect, the magnitude of which is correlated with affinity for RE. However, studies with zuclomiphene and estrogen-irreversible doses of enclomiphene and 6866 indicate that these antiestrogens also influence cell proliferation in vitro by mechanisms that probably do not involve RE.

Topics: Antineoplastic Agents; Binding, Competitive; Breast Neoplasms; Cell Division; Cell Line; Clomiphene; Dose-Response Relationship, Drug; Enclomiphene; Humans; Receptors, Estrogen; Structure-Activity Relationship