Molecular Characterization of the Microsomal Tamoxifen Binding Site

Abstract

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ERindependent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration-and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5␣-cholest-8-en-3␤-ol for tamoxifen treatment and 5␣-cholest-8-en-3␤-ol and cholesta-5,7-dien-3␤-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3␤-hydroxysterol-⌬ Tamoxifen is a selective estrogen receptor modulator (SERM) widely used for the treatment and the prevention of breast cancer (1). More than 20 years ago, Sutherland et al. (2) discovered that tamoxifen bound to a high affinity binding site that was different from the estrogen receptor (2). This site has been named the microsomal antiestrogen binding site (AEBS) 1 because it is localized in the microsomes of cells; it binds principally aryl aminoethoxy antiestrogens and has no affinity for estrogens (3). Two classes of selective ligands have been developed so far to selectively target the AEBS. The first class includes diphenylmethane derivatives of tamoxifen (DPPE) and N-pyrrolidino-2-[4-(benzyl)-phenoxy-ethanamine⅐HCl (PBPE) (4 -6), while the second class includes oxygenated derivatives of cholesterol such as 7-ketocholestanol (7-9). We and others (5, 10 -14) have shown that AEBS ligands inhibit the growth of tumor cell lines in vitro and in vivo, demonstrating that the AEBS was involved in the mediation of the effects of these structural classes of its cognate ligands. These compounds represent not only specific tools to study AEBS function but are also anticancer drug candidates because the selective AEBS ligand DPPE (Tesmilifene) was brought up to phase II and III clinical trials for the treatment of breast and prostate cancer in association with doxorubicin (15-17). Two principal points remained unsolved: the first is the precise molecular nature of the AEBS and the second the explanation of the difference observed between the nanomolar affinity of AEBS ligands and their micromolar effectiveness for growth control and cytotoxicity.We have been involved in the identification of the AEBS for several years. The AEBS can be found in most tissues in mammals and is abundant in microsomes of liver that contained 20 -30ϫ the amount found in tumor cell lines (18). For this reason, the liver have been chosen for the purification of the AEBS but the pharmacological profiles of the AEBS found in the ...

Microsomal antiestrogen-binding site ligands induce growth control and differentiation of human breast cancer cells through the modulation of cholesterol metabolism

“…These data, coupled with the fact that the AEBS bound oxygenated derivatives of sterols such as 7-ketocholesterol, 6-ketocholestanol, and 7-ketocholestanol with high affinity (10,11), opened up the possibility of a link between the binding to the AEBS and the oxidative metabolism of cholesterol. We confirmed this hypothesis by showing that AEBS ligands induced a major modification of cholesterol metabolism in tumor cells and afforded the molecular identification of the AEBS (2). We showed that, when tumor cells were exposed to tamoxifen and PBPE at concentrations that induced growth control, the neosynthesis of cholesterol was stopped and cells accumulated cholesterol precursors that were not found in cells before the treatments.…”

“…Zymostenol was purified as described before and was 99% pure by highperformance liquid chromatography (2). Commercial sterols were purified by high-performance liquid chromatography and stored under argon before use.…”

“…Cells were maintained in RPMI 1640 supplemented with 2 g/L sodium bicarbonate, 1.2 mmol/L glutamine (pH 7.4), 5% fetal bovine serum for MCF-7 cells, ZR75-1 and TSA or 10% fetal bovine serum for other cell lines, and both penicillin and streptomycin (50 units/mL) in a humidified atmosphere with 5% CO 2 at 37jC. MCF-7 ws were grown as described before (2). For the measurement of the antiproliferative indices, 10 5 cells were plated onto 6-well plates and treated for 48 h after plating with 10, 15, and 20 Amol/L PBPE or 1, 2.5, 5, or 10 Amol/L tamoxifen or the solvent vehicle (0.1% ethanol) for 5 days.…”

“…We showed that this accumulation was due to a noncompetitive inhibition of the two enzymes, the 3h-hydroxysterol-D 8 -D 7 -isomerase (D8D7I) and the 3h-hydroxysterol-D 7 -reductase (DHCR-7) that use zymostenol and DHC, respectively, as substrate. The coexpression of the D8D7I and DHCR-7 was found necessary and sufficient for the reconstitution of the AEBS in mammalian cells, indicating that the AEBS consisted of both enzymes (2).…”

“…1). Structure-affinity studies revealed that the AEBS, as opposed to the ER, does not bind estrogens and antiestrogens devoid of a protonable amino-ethoxy side chain but binds selective ER modulators (SERM) such as tamoxifen and raloxifene (2,3). The identification of diphenylmethane derivatives of tamoxifen that selectively target the AEBS, such as PBPE and DPPE, enabled the functions and pharmacology of the AEBS to be studied (4,5).…”