Antiestrogen Binding Sites Research Articles

Fluorescent tamoxifen derivatives as biophotonic probes for the study of human breast cancer and estrogen-receptor directed photosensitizers

Currently, breast cancer is the most common type of cancer worldwide, being the so-called estrogen receptor (ER) positive the subtype with highest prevalence. For decades, the most successful strategy to prevent recurrence of ER + breast cancers is tamoxifen coadjuvant therapy. However, the involvement of the different estrogen receptors (ERα, ERβ and GPER) and the interaction in their signaling pathways, the evidence for side-effects in chronic tamoxifen treatments, as well as the appearance of tamoxifen-resistant breast cancers, have encouraged the need for novel receptor-targeted fluorescent probes. In line with this, multifunctional antiestrogen conjugates, exhibiting fluorescent properties while retaining the ability to antagonize estrogen actions, have been synthesized and proven to be particularly useful in the study of the molecular biology of ER + breast cancers. These novel fluorescent tamoxifen derivatives (FTDs) exhibit pharmacological features of pure antiestrogens, with similar or even greater affinity for ERα than tamoxifen, inhibit ER-dependent gene transcription and cell proliferation, and are devoid of uterotrophic effects. In the present study we have aim at providing a detailed view of their biophotonic potential, including their spectroscopic properties, their usefulness for fluorescent labelling of cellular compartments and intracellular targets, their application in the identification of non-ER antiestrogen binding sites thought fluorescence competition assays, and finally, in their ability to function as efficient ER-targeted photosensitizers.

Elucidating Binding Sites and Affinities of ERα Agonists and Antagonists to Human Alpha-Fetoprotein by In Silico Modeling and Point Mutagenesis.

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth, which can be attributed to its estrogen-binding ability. Despite AFP having long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP–ligand interaction remains obscure. In our study, we constructed a homology-based 3D model of human AFP (HAFP) with the purpose of molecular docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol), and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on the ligand-docked scoring functions, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity binding sites were located (i) in a tunnel formed within HAFP subdomains IB and IIA and (ii) on the opposite side of the molecule in a groove originating from a cavity formed between domains I and III, while (iii) the third low-affinity binding site was found at the bottom of the cavity. Here, 100 ns molecular dynamics (MD) simulation allowed us to study their geometries and showed that HAFP–estrogen interactions were caused by van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP–antiestrogen binding. Molecular mechanics/Generalized Born surface area (MM/GBSA) rescoring method exploited for estimation of binding free energies (ΔGbind) showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP–ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues, along with two disulfide bonds (Cys224–Cys270 and Cys269–Cys277), have key roles in both HAFP–estrogen and HAFP–antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein–ligand interactions and anticancer therapy strategies based on ERα-binding ligands.

From tamoxifen to dendrogenin A: The discovery of a mammalian tumor suppressor and cholesterol metabolite.

Tamoxifen (Tam) was developed as a ligand and modulator of estrogen receptor α (ERα) and is one of the main drugs used globally for the hormonotherapy of breast cancers. Besides ERα, Tam also binds with high affinity to the microsomal antiestrogen binding site (AEBS). The AEBS is a hetero-oligomeric proteinaceous complex with cholesterol-5,6-epoxide hydrolase (ChEH) activity that is associated with an intracellular histamine (HA) binding site. The enzymatic activities of the ChEH subunits control developmental programs in mammals and transform cholesterol-5,6-epoxides (5,6-EC) into cholestane-3β,5α,6β-triol. Inhibition of the ChEH activity by pharmacological agents such as Tam induce cancer cell re-differentiation through the accumulation of 5,6-EC. A few years ago, the putative chemical reactivity of the 5,6-EC epoxide group towards nucleophiles led our group to hypothesize that 5,6-EC could react with HA that was co-localized at the AEBS to give a new molecule involved in cell differentiation. This hypothesis was chemically tested and the conjugation of 5,6α-EC: with HA was found possible but only under catalytic conditions. It gave a stereo-selective single product of transformation which was named dendrogenin A (DDA). DDA was found to display potent cancer cell differentiation and anticancer properties invitro and invivo, suggesting that it was a tumor suppressor metabolite. The presence of DDA was then established in several mammalian tissues, providing the first evidence of a steroidal alkaloid metabolite in mammals. The discovery of DDA highlights a new metabolic pathway in mammals which lies at the crossroads of cholesterol and histamine metabolism and produces this tumor suppressor metabolite.

Antiestrogen-binding site ligands induce autophagy in myeloma cells that proceeds through alteration of cholesterol metabolism

Multiple myeloma (MM) is a malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Despite extensive efforts to design drugs targeting tumoral cells and their microenvironment, MM remains an incurable disease for which new therapeutic strategies are needed. We demonstrated here that antiestrogens (AEs) belonging to selective estrogen receptor modulators family induce a caspase-dependent apoptosis and trigger a protective autophagy. Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3. Moreover, autophagy was inhibited by drugs such as bafilomycin A1 and 3-methyladenosine. Autophagy was mediated by the binding of AEs to a class of receptors called the antiestrogen binding site (AEBS) different from the classical estrogen nuclear receptors. The binding of specific ligands to the AEBS was accompanied by alteration of cholesterol metabolism and in particular accumulation of sterols: zymostenol or desmosterol depending on the ligand. This was due to the inhibition of the cholesterol-5,6-epoxide hydrolase activity borne by the AEBS. We further showed that the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathway mediated autophagy signaling. Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells. Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.

Abstract 1884: 6-oxo-cholestan-3β,5α-diol (OCDO) is a metabolite produced in tumors by cholesterol epoxide hydrolase activity and a tumor promoter: OCDO inhibition contributes to the anti-tumor activity of tamoxifen and dendrogenin A.

Abstract Cholesterol Epoxide Hydrolase (ChEH) selectively catalyses the hydrolysis of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC) into cholestane-3β,5α,6β-triol (CT). We recently reported that the ChEH activity is carried out by the anti-estrogen binding site (AEBS), formed by 3β-hydroxysterol-Δ7-reductase (DHCR7) and 3β-hydroxysterol-Δ8-Δ7 isomerase (D8D7I) (de Medina et al, PNAS, 2010). In the early 70’s, 5,6-ECs were reported to be carcinogenic and mutagenic, but these data were then refuted, and recently we confirmed that 5,6-ECs are stable and non-alkylating substances. Interestingly, it was reported that CT was genotoxic under strong oxidative stress conditions in yeast. This suggested to us that the inhibition of ChEH may protect cells against cytotoxic insults and may contribute to the anti-proliferative and chemopreventive properties of AEBS/ChEH inhibitors such as tamoxifen (Tam), raloxifene or docosahexaenoic acid that accumulated 5,6-ECs at therapeutic doses. These different data suggested a potential role of ChEH activation in cancer. We have explored this hypothesis in the present study. We show here that the activity of ChEH generated an unexpected metabolite from CT in a large panel of cancer cells. By using RP-HPLC and GC-MS, we determined this unknown metabolite to be the oxysterol: 6-oxo-cholestan-3β,5α-diol OCDO). We showed that OCDO stimulated the growth and invasiveness of different tumor cell lines (1.4-fold and 6-fold respectively for MCF7 cells) and the growth and invasiveness of thyroid tumors (TT) or breast cancers (TS/A, MCF7) grafted into mice. Immunohistology of OCDO-treated tumors showed there was an increase in the expression of the proliferation marker Ki67 compared with control tumors. Interestingly, OCDO formation in tumor cells and tumors grafted into mice was strongly inhibited by ChEH inhibitors known to have anti-tumor activity such as Tam, PBPE or Dendrogenin A (DDA), a steroidal alkaloid discovered in our laboratory. The anti-tumor activity of Tam or DDA against breast tumors implanted into mice was completely reversed by co-treatment with OCDO, indicating that inhibition of ChEH and OCDO production contribute to the anti-tumor mechanism of action of these molecules. In conclusion, the present study identifies OCDO as being an oxidative metabolite of CT with an uncharacterized role of tumor promoter. The inhibition of OCDO production contributes to the anti-tumor activity of Tam and Dendrogenin A. Thus, the use of these ChEH inhibitors appears as a relevant strategy to block the production of this tumor promoter that may be produced during oxidative stress conditions and inflammation driven carcinogenesis. Citation Format: Philippe de Medina, Michael R. Paillasse, Maud Voisin, Marc Poirot, Sandrine Silvente-Poirot. 6-oxo-cholestan-3β,5α-diol (OCDO) is a metabolite produced in tumors by cholesterol epoxide hydrolase activity and a tumor promoter: OCDO inhibition contributes to the anti-tumor activity of tamoxifen and dendrogenin A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1884. doi:10.1158/1538-7445.AM2013-1884

Cholesterol epoxide hydrolase and cancer

Cholesterol epoxide hydrolase (ChEH) catalyzes the hydration of cholesterol-5,6-epoxides (5,6-EC) into cholestane-3β,5α,6β-triol. ChEH is a hetero-oligomeric complex called the anti-estrogen binding site (AEBS) comprising 3β-hydroxysterol-Δ(8)-Δ(7)-isomerase (D8D7I) and 3β-hydroxysterol-Δ(7)-reductase (DHCR7). D8D7I and DHCR7 regulate cholesterol biosynthesis, fetal development and growth, tumor cell differentiation and death. The un-reactivity of 5,6-EC toward nucleophiles has recently been demonstrated indicating that 5,6-EC are not alkylating and carcinogenic agents as first postulated. Here we discuss recent advances in the molecular characterization of ChEH, its potential role in cancer progression and resistance as well as the interest of inhibiting ChEH and to accumulate 5,6-EC which may contribute to the anti-tumor and chemopreventive action of ChEH inhibitors used in the clinic such as tamoxifen.

Abstract 956: The liver-X-receptor-α is involved in the induction by Tamoxifen of breast cancer cell differentiation and death

Abstract Hormonotherapy with Tamoxifen (Tam) is one of the main strategies used world-wide for the treatment of estrogen receptor positive breast cancer. However, partial responses, tumor recurrence and acquired resistance to Tam may involve mechanisms unrelated to Estrogen Receptors (ER). Studies on non-ER related mechanisms of action of Tam led us to find that it induced breast cancer cell differentiation and death through the modulation of sterol metabolism and the stimulation of sterol oxidation (de Medina et al, Cell Death Differ, 2009), and that Tam inhibited cholesterol epoxide hydrolase (ChEH). ChEH catalyzes the trans hydration of cholesterol-5,6-epoxides (CE) and this activity is a property of the microsomal antiestrogen binding site (AEBS) (de Medina et al, PNAS, 2010). In the present study we report that Tam and AEBS ligands stimulated the oxidation of cholesterol into CE and induced the accumulation of CE in MCF-7 cells through the inhibition of ChEH at therapeutic concentrations. Since CE has been reported to be a modulator of Liver-X-Receptors (LXR), we investigated the impact of Tam and other AEBS ligands on LXR-dependent gene expression: Tam and AEBS ligands modulated the expression LXR-dependent genes in MCF-7 cells. This effect had a 24 to 48 hour lag time while the effect of direct LXR modulators was observable after 6 hours, suggesting an indirect action of AEBS ligands. The involvement of CE in the control of transcription by Tam and AEBS ligands was supported by the observation that vitamin E, which inhibited the formation and the accumulation of CE, totally blocked this transcriptional modulation. We found that knocking down LXR-α using interfering RNAs in MCF-7 cells blocked the transcriptional modulation of LXR-controlled genes induced by CE, Tam and AEBS ligands. Finally, we found that the knock down of LXR-α reproduced the vitamin E inhibition of the induction of differentiation and death of MCF-7 cells. Altogether, these data showed that LXR-α is implicated in the anticancer action of Tam and identified a new signaling pathway that could explain sensitivity and resistance to Tam. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 956. doi:1538-7445.AM2012-956

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

Identification and pharmacological characterization of cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS ligands

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

Abstract 2918: Molecular identification of the cholesterol-5,6-epoxide hydrolase: A new target for selective estrogen receptor modulators and unsaturated fatty acids

Abstract The cholesterol-5,6-epoxide hydrolase (ChEH) is the last epoxyde hydrolase which coding gene remains to be identified. The ChEH catalyzes the hydration of 5,6α-epoxy-cholestanol (α-CE) and 5,6β-epoxy-cholestanol (β-CE), two autoxydation products of cholesterol, into cholestane-3β,5α,6β-triol (CT). We earlier reported that the antiestrogen binding site (AEBS) was a hetero-oligomeric complex made of two subunits: the 3β-hydroxysterol-Δ7-reductase (DHCR7) and 3β-hydroxysterol- Δ8- Δ7-isomerase (D8D7I), two enzymes involved in post-lanosterol cholesterol biosynthesis (Kedjouar et al, J Biol Chem, 2004). We have recently reported that AEBS ligands induced differentiation and apoptosis of human breast cancer cell lines through the intracellular accumulation of autoxydation products of sterols such as α-CE and β-CE (Payre et al, Mol Cancer Ther, 2008; de Medina et al, Cell Death &amp; Differ, 2009). Because the ChEH and AEBS displayed some biochemical similitudes, we have investigated, in the present study, the potential relationship that may exist between the ChEH and AEBS. We established that α-CE and β-CE (the substrates of the ChEH) and CT (the product of the catalytic activity of the ChEH), were high-affinity and competitive ligands of AEBS. AEBS ligands include selective estrogen receptors modulators (SERMs) such as tamoxifen and raloxifene, poly unsaturated fatty acids such as docosahexaenoic acid (DHA), oxysterols such as 7-hydroxycholesterol and 7-ketocholesterol, and diphenylmethane compounds such as N-Pyrrolidino-4-(phenylmethyphenoxyl)-ethanamine (PBPE) and tesmilifene. We established that all AEBS ligands were inhibitors of the ChEH at pharmacological concentrations. We demonstrated a positive correlation between the binding to AEBS and the inhibition of the ChEH catalytic activity by AEBS ligands (r2 = 0.95, n = 39, p &amp;lt; 0.0001) and showed that AEBS and the ChEH had the same pharmacological profile. Finally, the structural similarity between the ChEH and AEBS was established by co-expression or genetic invalidation in mammalian cells of the D8D7I and the DHCR7, which respectively allowed the reconstitution or the impairment of both AEBS and the ChEH activity. Collectively, these data demonstrate that AEBS and the ChEH are molecularly and pharmacologically similar. These results provide the first evidence of the molecular identification of the ChEH, which coding gene had never been reported. In conclusion, we have identified a mechanism by which AEBS ligands induce the accumulation of sterol autoxidation products that were previously shown to induce breast cancer cell differentiation and apoptosis. The ChEH constitutes a new target for the chemoprevention and the treatment of breast cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2918.

Effects of steroid hormones and antihormones in cultured cells.

Mouse L-929 fibroblasts contain androgen (AR), estrogen (ER) and glucocorticosteroid receptors (GR). The addition of androgens and estrogens (10 to 50 nM) to the culture medium stimulated cell growth and this growth stimulation was inhibited when the corresponding anti-hormones (cyproterone acetate or tamoxifen, 100 nM) were present at the same time. Glucocorticosteroids (10 to 100 nM) had negative effects on cell growth leading even to cell death over 1 microM concentrations. The addition of a new antiglucocorticosteroid RU 486, inhibited the negative effects of glucocorticosteroids. RU 486 displays also anti-progesterone activity, but as L-929 cells have no progesterone receptor, only the anti-glucocorticosteroid action of this new compound was studied. Cell adhesiveness to culture plates was increased by androgen or estrogen, but decreased by glucocorticosteroid and again, the corresponding anti-hormones abolished these positive or negative effects. All these observations can be made in serum-free culture medium supplemented with insulin but not with other hormones, proteins or growth factors. Cyproterone acetate, tamoxifen and RU 486 have specific affinity for AR, ER and GR, respectively. After incubation of whole cells with 3H-tamoxifen or 3H-RU 486, accumulation of either estradiol or glucocorticosteroid receptor complexes in the nuclear fraction of the cell was observed. A tamoxifen-resistant clone of L-929 cells was selected in serum-free medium. This variant had about 50% less ER than tam-sensitive L-929 cells and also less specific antiestrogen binding sites which were found in the particulate fraction of the cells. Shionogi SC-115 mammary cancer cells contain AR and ER. In the presence of testosterone (50 nM) a considerable difference of cell growth was observed. Testosterone increased cell proliferation, and the anti-androgen cyproterone acetate (100 nM) completely inhibited this growth stimulation. RU 486 (100 nM) showed also some antagonistic effect on androgen induced cell growth. The shape of cells cultured with or without androgens was completely different. In the presence of testosterone, clonal growth of the cells was observed with no contact inhibition, whereas in the absence of hormone, cells formed a monolayer. No clonal growth was observed when cyproterone acetate (100 nM) was present at the same time as testosterone in the culture medium. Both testosterone and cyproterone acetate were bound to AR. In the murine thymoma cell lines W7TB and T1M1b, RU 486 is a strong antagonist of the glucocorticosteroid induced cytolytic response.(ABSTRACT TRUNCATED AT 400 WORDS)

Ligands of the antiestrogen-binding site induce active cell death and autophagy in human breast cancer cells through the modulation of cholesterol metabolism

We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol- and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was sterol- and Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators.

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

The microsomal antiestrogen-binding site (AEBS) is a high-affinity membranous binding site for the antitumor drug tamoxifen that selectively binds diphenylmethane derivatives of tamoxifen such as PBPE and mediates their antiproliferative properties. The AEBS is a hetero-oligomeric complex consisting of 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase. High-affinity AEBS ligands inhibit these enzymes leading to the massive intracellular accumulation of zymostenol or 7-dehydrocholesterol (DHC), thus linking AEBS binding to the modulation of cholesterol metabolism and growth control. The aim of the present study was to gain more insight into the control of breast cancer cell growth by AEBS ligands. We report that PBPE and tamoxifen treatment induced differentiation in human breast adenocarcinoma cells MCF-7 as indicated by the arrest of cells in the G0-G1 phase of the cell cycle, the increase in the cell volume, the accumulation and secretion of lipids, and a milk fat globule protein found in milk. These effects were observed with other AEBS ligands and with zymostenol and DHC. Vitamin E abrogates the induction of differentiation and reverses the control of cell growth produced by AEBS ligands, zymostenol, and DHC, showing the importance of the oxidative processes in this effect. AEBS ligands induced differentiation in estrogen receptor-negative mammary tumor cell lines SKBr-3 and MDA-MB-468 but with a lower efficiency than observed with MCF-7. Together, these data show that AEBS ligands exert an antiproliferative effect on mammary cancer cells by inducing cell differentiation and growth arrest and highlight the importance of cholesterol metabolism in these effects.

Contraceptive and hormonal properties of the stem bark of Dysoxylum binectariferum in rat and docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction

Background This study was aimed to investigate the pregnancy interceptive activity of the stem bark of Dysoxylum binectariferum Hook. f. administered during the pre- and peri-implantation periods and immediately after implantation by oral route in adult female Sprague–Dawley rats. Study Design Ethanolic extract and its fractions were administered to female rats on Days 1–10, Days 1–7, Days 1–5 or Day 1 postcoitum by oral route. At autopsy on Day 12, the number and status of corpora lutea and implantations were recorded. For estrogenic activity, ovariectomized immature rats received the test extract or the vehicle once daily for 3 days and at autopsy on Day 4, uterine weight and status of vaginal opening and extent of vaginal cornification were recorded. For antiestrogenic activity, the extract was administered along with ethinyl estradiol. Docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction, to estrogen receptor (ERα) was conducted using AutoDock 3.0.5 on a Linux workstation. Results The ethanolic extract intercepted pregnancy in rats at a daily dose of 500 mg/kg on Days 1–7 postcoitum. On fractionation, the activity was localized in the chloroform fraction, which inhibited pregnancy in all females at the 35-mg/kg dose on Days 1–7, at the 50-mg/kg dose on Days 1–5 or at the single 300-mg/kg dose on Day 1 postcoitum. Chromatography of this fraction yielded an alkaloid, rohitukine, which prevented pregnancy at the 10-mg/kg dose administered on Days 1–7 but was partially (45%) effective at this dose when administered during the entire preimplantation period and ineffective even at 10 times this dose when administered only on Day 1 postcoitum, except that there was a significant reduction in implantation number in pregnant females. While the active chloroform soluble fraction was devoid of any estrogen agonistic or antagonistic properties, a mild uterotropic effect without induction of premature opening of vagina or cornification of vaginal epithelium was observed in rohitukine at the 10-mg/kg dose. Rohitukine, with an almost similar molecular size (mol. wt. 305) as 17β-estradiol, fits ideally into the hydrophobic pocket of ER. While it does not appear to simultaneously interact with GLU353, ARG394 and HIS524 as estradiol to elicit frank estrogenic response, different conformations of the ligand or its metabolite(s) might acquire geometry with phenolic groups at C-3′, C-5 and C-7 positions disposed in a fashion to interact with active site(s) of ER, which might be responsible for its contraceptive and/or weak uterotropic effects. The absence of a basic side chain directed toward the antiestrogen binding site (ASP351) on the receptor appears to be responsible for the lack of any estrogen antagonistic activity. Conclusions Findings demonstrate the antifertility activity of the ethanolic extract of D. binectariferum, its chloroform soluble fraction and rohitukine. Efforts are being made to enhance the anti-implantation activity of rohitukine by structural modifications.

Farnesyl-transferase inhibitor R115,777 enhances tamoxifen inhibition of MCF-7 cell growth through estrogen receptor dependent and independent pathways.

IntroductionWe have previously shown that FTI-277, a farnesyl transferase inhibitor (FTI), enhances the efficacy of tamoxifen (Tam) in inhibiting the proliferation of the estrogen dependent MCF-7 cell line. As the cellular response to Tam is the result of an inhibition of both estrogen receptor-dependent and -independent pathways, we have used the estrogen receptor selective anti-estrogen ICI182,780 and N-pyrrolidine(-phenylmethyl-phenoxy)-ethanamine-HCl (PBPE), a selective ligand of anti-estrogen binding site (AEBS), to dissect out the mechanism(s) associated with the observed additivity resulting from combination treatment with FTI-277 and Tam. Moreover, for these studies, FTI-277 has been replaced by R115,777, a FTI currently in phase III clinical trials.MethodsThe quantitative sulphorhodamine B (SRB) colorimetric assay was used to determine the growth inhibitory effect of agents on MCF-7 cells. Dose response interactions between R115,777-Tam, R115,777-ICI182,780 and R115,777-PBPE were evaluated, at the IC50 point, using the isobologram method. Apoptotic cell death (DNA fragmentation, nucleus condensation and cytokeratin 18 cleavage) and inhibition of the mevalonate pathway (western blot) were also determined.ResultsCombinations of the specific FTI R115,777 with either ICI182,780 or PBPE exhibit a synergistic effect on MCF-7 cell growth inhibition, while its combination with Tam is additive, as previously reported for FTI-277. Apoptosis is detected after treatment with combinations of R115,777 with either Tam or PBPE but not with ICI182,780, suggesting that each combination inhibits cell proliferation by different mechanisms. Even though the ER pathway has not yet been deciphered, it is shown here that the AEBS pathway is able to interfere with the mevalonate pathway at the level of protein farnesylation.ConclusionOverall, this work reveals that combinations of R115,777 with either selective ER ligands or a selective AEBS ligand are able to induce large increases in their anti-proliferative activities on MCF-7 cells. Moreover, these results suggest that it may be of definite interest to evaluate combinations of R115,777 with different anti-estrogens in the treatment of ER positive breast tumours. Based on these experimental data, such combinations may prove beneficial in different clinical scenarios or when used in specific sequences; studying the combination of R115,777 with ICI182,780 for early treatment and reserving combinations with either Tam or a selective AEBS ligand, such as BMS-217380-01, for more resistant disease.

Antiestrogen Binding Site and Estrogen Receptor Mediate Uptake and Distribution of 4-Hydroxytamoxifen-Targeted Doxorubicin−Formaldehyde Conjugate in Breast Cancer Cells

The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-TAM, was found to possess superior cell growth inhibition characteristics relative to clinical doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines, three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of 4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding site and estrogen receptor.

Multiple Targeting by the Antitumor Drug Tamoxifen: A Structure-Activity Study

Tamoxifen is a well-known antiestrogen used for the hormonotherapy of estrogen receptor positive breast cancer. In addition to its high affinity binding to the estrogen receptor (ER), tamoxifen binds with comparable affinity to the microsomal antiestrogen binding site (AEBS), and inhibits with a micromolar efficiency, protein kinase C (PKC), calmodulin (CaM)-dependent enzymes and Acyl CoenzymeA: Cholesterol Acyl Transferase (ACAT). Each of these tamoxifen targets might explain the genomic as well as non-genomic effects of tamoxifen. In this review, we will report current knowledge about the structural features of tamoxifen involved in this multiple targeting. These data provide a useful guide for the conception of selective ligands of ERs, AEBS, PKC, CaM or ACAT based on the chemical structure of tamoxifen.

Molecular Characterization of the Microsomal Tamoxifen Binding Site

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 ER-independent 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 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.

Design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells.

The anthracycline antitumor drug doxorubicin (DOX) has been utilized for decades as a broad-spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design and 11-step synthesis of doxorubicin-formaldehyde conjugates targeted to the estrogen receptor, which is commonly overexpressed in breast cancer cells, are reported. The formaldehyde is incorporated in a masked form as an N-Mannich linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release the doxorubicin-formaldehyde active metabolite, is tethered via derivatized ethylene glycols to an E and Z mixture of 4-hydroxytamoxifen. The targeting group, E/Z-4-hydroxytamoxifen, was selected for its ability to tightly bind the estrogen receptor and antiestrogen binding sites. The targeted doxorubicin-formaldehyde conjugates' estrogen receptor binding and in vitro growth inhibition were evaluated as a function of tether length. The lead compound, DOX-TEG-TAM, bearing a triethylene glycol tether, binds the estrogen receptor with a binding affinity of 2.5% relative to E/Z-4-hydroxytamoxifen and inhibits the growth of four breast cancer cell lines with 4-fold up to 140-fold enhanced activity relative to doxorubicin.

Insulin and estrogen receptor ligand influence the FGF-2 activities in MCF-7 breast cancer cells

From the MCF-7 cell line we have developed, a human mammary cancer cell subline with the same karyotype as the mother strain and named MCF-7 SF, able to grow in serum-free chemically defined medium. This cell subline was firstly used to analyze the effect of basic fibroblast growth factor (FGF-2) in estrogen-receptor-positive human breast cancer cells. FGF-2 like estradiol is able to increase cell proliferation and pS2 expression but was also found to inhibit progesterone receptor (PR) expression. The anti-estrogen tamoxifen partly counteracts the effects of FGF-2 and to discriminate between its two main mediators (estrogen receptor vs. anti-estrogen binding site, AEBS) we compare the efficacies of pure anti-estrogen (ICI 182,780) and AEBS ligand (PBPE). It appears that pure anti-estrogen counteracts cell growth and pS2 effects of FGF-2 since AEBS ligand inhibits the cell growth but has no activity on pS2 expression. Secondly, adding insulin (10 −6 M) in the culture medium induces a strong increase in cell proliferation, which then elicits an inhibitory effect of FGF-2 and addition of anti-estrogens, are less efficient to further decrease growth, since the effects of FGF-2 and anti-estrogens on pS2 expression are conserved.