afimoxifene and triphenylethylene

Estrogen therapy was used to treat advanced breast cancer in postmenopausal women for decades until the introduction of tamoxifen. Resistance to long-term estrogen deprivation (LTED) with tamoxifen and aromatase inhibitors used as a treatment of breast cancer inevitably occurs, but unexpectedly low-dose estrogen can cause regression of breast cancer and increase disease-free survival in some patients. This therapeutic effect is attributed to estrogen-induced apoptosis in LTED breast cancer. Here, we describe modulation of the estrogen receptor (ER) liganded with antiestrogens (endoxifen and 4-hydroxytamoxifen) and an estrogenic triphenylethylene (TPE), ethoxytriphenylethylene (EtOXTPE), on estrogen-induced apoptosis in LTED breast cancer cells. Our results show that the angular TPE estrogen (EtOXTPE) is able to induce the ER-mediated apoptosis only at a later time compared with planar estradiol in these cells. Using real-time polymerase chain reaction, chromatin immunoprecipitation, western blotting, molecular modeling, and X-ray crystallography techniques, we report novel conformations of the ER complex with an angular estrogen EtOXTPE and endoxifen. We propose that alteration of the conformation of the ER complexes, with changes in coactivator binding, governs estrogen-induced apoptosis through the protein kinase regulated by RNA-like endoplasmic reticulum kinase sensor system to trigger an unfolded protein response.

Topics: Breast Neoplasms; Cell Proliferation; Cell Survival; Crystallography, X-Ray; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Receptors, Estrogen; Stilbenes; Tamoxifen

The anti-estrogenic drug tamoxifen, which is used therapeutically for treatment and prevention of breast cancer, can lead to the development of thrombosis. We found that tamoxifen rapidly increased intracellular free calcium [Ca2+]i in human platelets from both male and female donors. Thus 10 microM tamoxifen increased [Ca2+]i above the resting level by 197 +/- 19%. Tamoxifen acted synergistically with thrombin, ADP, and vasopressin to increase [Ca2+]i. The anti-estrogen ICI 182780 did not attenuate the effects of tamoxifen to increase [Ca2+]i; however, phospholipase C inhibitor U-73122 blocked this effect. 4-hydroxytamoxifen, a major metabolite of tamoxifen, also increased [Ca2+]i, but other tamoxifen metabolites and synthetic derivatives did not. Three hydroxylated derivatives of triphenylethylene (corresponding to the hydrophobic core of tamoxifen) which are transitional structures between tamoxifen (Ca agonist) and diethylstilbestrol (Ca antagonist) increased [Ca2+]i slightly (6% to 24%) and partially inhibited thrombin-induced [Ca2+]i elevation (68% to 79%). Therefore the dimethylaminoethyl moiety is responsible for tamoxifen being a Ca agonist rather than antagonist. 4-Hydroxytamoxifen and polymer-conjugated derivatives of 4-hydroxytamoxifen increased [Ca2+]i, with similar efficacy. The ability of tamoxifen to increase [Ca2+]i in platelets, leading to platelet activation, and its ability to act synergistically with other platelet agonists may contribute to development of tamoxifen-induced thrombosis.

Topics: Adenosine Diphosphate; Adult; Blood Platelets; Calcium; Calcium Signaling; Diethylstilbestrol; Drug Synergism; Estradiol; Estrenes; Estrogen Antagonists; Ethamoxytriphetol; Female; Fulvestrant; Humans; Male; Middle Aged; Molecular Structure; Phosphodiesterase Inhibitors; Pyrrolidinones; Stilbenes; Structure-Activity Relationship; Tamoxifen; Thrombin; Vasopressins

Estradiol induces vascular endothelial growth factor (VEGF) expression in the rat uterus and this may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue. Triphenylethylene antiestrogens such as tamoxifen have mixed agonist/antagonist activity and their specific effects are tissue and gene specific. These drugs exhibit primarily antiestrogenic actions in mammary tissue and are thus used for the treatment of breast cancer. These drugs are also suggested to be inhibitors of angiogenesis. However, uterine side effects of tamoxifen are thought to stem largely from the agonist activity of the drug in this tissue. Since side effects of tamoxifen such as uterine bleeding and endometrial cancer seem likely to have an angiogenic component, we have examined the effects of this drug, its metabolite, 4-hydroxy-tamoxifen and two additional triphenylethylene antiestrogens, nafoxidine and clomiphene, on the expression of VEGF and another estrogen regulated gene, c-fos, using the rat uterus as an experimental system. All four compounds increase uterine VEGF and c-fos mRNA levels indicating that the triphenylethylene class of antiestrogens are predominantly agonists for the induction of these genes in the uterus.

Topics: Animals; Blotting, Northern; Clomiphene; Dose-Response Relationship, Drug; Endothelial Growth Factors; Estradiol; Estrogen Antagonists; Estrogens; Female; Lymphokines; Nafoxidine; Ovariectomy; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger; Stilbenes; Tamoxifen; Time Factors; Uterus; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Calmodulin; Estradiol; Estrogen Antagonists; Nafoxidine; Stilbenes; Structure-Activity Relationship; Tamoxifen

The Ca2+- and phospholipid-dependent phosphotransferase activity of protein kinase C was inhibited by the triphenylethylene compounds clomiphene [drug concentration causing 50% inhibition (IC50) = 25 microM], 4-hydroxytamoxifen (IC50 = 25 microM), and N-desmethyltamoxifen (IC50 = 8 microM). The Ca2+- and phospholipid-independent phosphorylation of protamine sulfate, which is catalyzed by protein kinase C, was not inhibited by the triphenylethylenes, suggesting that they do not interact directly with the active site of protein kinase C. The inhibitory potency of each triphenylethylene was reduced when the phospholipid concentration was increased, providing evidence that these drugs inhibited protein kinase C by interacting with phospholipids. The potencies of the effects of the triphenylethylenes on protein kinase C in the lipid environment of intact cells were evaluated by determining their efficacies in the inhibition of [3H]phorbol 12,13-dibutyrate (PDBu) binding to mouse embryo C3H/10T1/2 cells. Micromolar concentrations of each drug inhibited [3H]PDBu binding in these cells. N-Desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen inhibited protein kinase C with the same order of potency as that which has been reported for their inhibition of MCF-7 cell growth by Reddel et al. (1983). N-Desmethyltamoxifen and 4-hydroxytamoxifen were also more potent than tamoxifen in the inhibition of the growth of mouse embryo fibroblast C3H/10T1/2 cells. These correlations suggest that the mechanism of growth inhibition by tamoxifen and its metabolites includes interactions with protein kinase C.

Topics: Animals; Brain; Cells, Cultured; Estrogen Antagonists; Fibroblasts; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Rats; Stilbenes; Structure-Activity Relationship; Tamoxifen