Chemical synthesis and biochemical characterization of a biotinylated derivative of 17beta-estradiol with a long side chain covalently attached to its C-7alpha position

High-affinity biotinylated derivatives of 17beta-estradiol (E(2)) are of value for isolation of various estrogen-binding proteins (including estrogen receptors) and also for studying protein-protein interactions involving these proteins. In this study, we developed a simplified route for the chemical synthesis of a biotinylated derivative of E(2) (compound 7) with a side chain attached to its C-7alpha position. Compound 7, i.e., 7alpha-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-keto-estradiol-3,17beta-di-tetrahydropyranyl ether (compound 2, which can be prepared from E(2)), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized affinity ligand has a high binding affinity for both human estrogen receptor alpha and beta. When the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it effectively bound human estrogen receptor alpha, and the receptor protein could be selectively eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase beta-subunit (also known as protein disulfide isomerase) was identified as one of the high-affinity E(2)-binding proteins in the whole cytosolic protein mixture prepared from MCF-7 human breast cancer cells. Computational molecular modeling analysis showed that compound 7 can bind to human estrogen receptor alpha in a similar manner as ICI-182,780 and raloxifene, and their binding energy values are also similar.     

Synthesis of 7alpha-substituted derivatives of 17beta-estradiol

Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alternatives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of postmenopausal, receptor-positive human breast cancers. Structurally, these pure antagonists contain the basic core structure of 17beta-estradiol (E(2)) with a long side chain attached to its C-7alpha position. We explored and compared in this study various synthetic routes for preparing a number of C-7alpha-substituted derivatives of E(2), which are highly useful for the design and synthesis of high-affinity ER antagonists, ER-based imaging ligands, and other ER-based multi-functional agents. Using E(2) as the starting material and 1-iodo-6-benzyloxyhexane as a precursor for the C-7alpha side chain, a seven-step synthetic procedure afforded 3,17beta-bis(acetoxy)-7alpha-(6-hydroxyhexanyl)-estra-1,3,5(10)-triene (one of the derivatives prepared) in an overall yield of approximately 45% as compared to other known procedures that afforded substantially lower overall yield (8-27%). The synthetic steps for this representative compound include: (1) protection of the C-3 and C-17beta hydroxyls of E(2) using methoxymethyl groups; (2) hydroxylation of the C-6 position of the bismethoxymethyl ether of E(2); (3) Swern oxidation of the C-6 hydroxy to the ketone group; (4) C-7alpha alkylation of the C-6 ketone derivative of E(2); (5) deprotection of the two methoxymethyl groups; (6) reprotection of the C-3 and C-6 free hydroxyls with acetyl groups; (7) removal of the C-6 ketone and the benzyl group on the side chain by catalytic hydrogenation in acetic acid. As predicted, two of the representative C-7alpha-substituted derivatives of E(2) synthesized in the present study retained strong binding affinities (close to those of E(2) and ICI-182,780) for the human ERalpha and ERbeta subtypes as determined using the radioligand-receptor binding assays.     

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concepts for the syntheses of biotinylated steroids. Part II: 17beta-estradiol derivatives as immunochemical probes

Biotinylated 17beta-estradiol (E2) derivatives are helpful probes for a better understanding of biospecific E2 interactions with steroid-binding proteins such as the estrogen receptor and anti-steroid antibodies. We describe synthetic strategies for the biotinylation of E2 toward the 3, 6alpha, and 7alpha positions using biotinyl-N-hydroxysuccinimide esters with different spacers, varying in structure and chain length. Key reaction for biotinylation at the 3 position is the regioselective ether formation of the phenolate E2 anion with a linker mesylate without protecting the 17beta-hydroxyl group. The 6alpha position is accessible via a 3,17beta protected 6alpha-hydroxy E2, prepared by stereospecific sodium borohydride reduction of 6-oxo E2. Direct cyanoethylation of the alcohol followed by reduction to the amine allows the biotinylation to 6alpha-O-coupled cyanoethyloxy linker E2 derivatives. Alternatively, 6alpha-O-coupled cyanoalkyloxy polyether linker E2 probes are obtained by a Williamson ether synthesis of the alcohol precursor with omega-t-butyl-dimethylsilyloxy-5-oxa-nonylmesylate. Cyanoethylation of the desilylated compound and further reduction of the nitrile led to the terminal amine. Reductive amination of the 3, 17beta acetylated 6-oxo E2 compound with 6-cyanoethyloxyhexyl ammonium acetate yields in a mixture of 6alpha/beta-N-alkylated E2 nitriles. The epimers are separated by reversed-phase HPLC and the 6alpha-compound subsequently reduced to the terminal amine. The 7alpha-biotinylated E2 compound is derived from 7alpha-(11'-undecyl-N-methyl-N-butylamide) E2, which is already known from literature. Subsequently, the 3 and 17beta positions are protected, and the amide is reduced to the 7alpha-(11'-undecanol) compound. Further cyanoethylation and reduction led to the 11'-amino-ethyloxyundecyl E2. Using (1)H NMR analysis, it could be shown that the biotin moiety of the biotinylated 6alpha- and 7alpha-E2 derivatives has an axial position which results in a vertical orientation of the substituent toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 6alpha- and 7alpha-derivatives as optimal tracers in competitive immunoassays.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell growth and the secretion of the estrogen-induced 34-, 52- and 160-kDa proteins in human breast cancer cells

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth of estrogen-responsive MCF-7 human breast cancer cells in the presence of 17 beta-estradiol was determined. After treatment with 17 beta-estradiol (1 nM), TCDD (10 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) the cells were monitored daily for cell growth and DNA content for 7 days. The results showed that TCDD inhibited cell proliferation and DNA content of untreated cells and inhibited the 17 beta-estradiol-stimulated cell proliferation and increase in cellular DNA content. In contrast, TCDD did not effect the growth of estrogen non-responsive MDA-MB-231 human breast cancer cells. TCDD (0.1-10 nM) also caused a concentration-dependent decrease in the 17 beta-estradiol-induced proliferation in MCF-7 cells. The effects of TCDD on the 17 beta-estradiol-induced secretion of the 52-kDa protein (i.e. procathepsin D), the 34-kDa (cathepsin D) and 160-kDa proteins were also determined in the MCF-7 and MDA-MB-231 human breast cancer cell lines. The levels of the proteins were determined by autoradiographic analysis of the incorporation of [35S]methionine into the secreted proteins which were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of MCF-7 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in levels of the 52-kDa protein which were 497, 63.6, 98.1 and 66.3%, respectively, of the corresponding levels observed in control (untreated) cells. Using the same concentrations, the levels of the 34-kDa protein secreted into the media were 372, 42.3, 64.0 and 43.8% of control values, respectively, and the corresponding levels of the 160-kDa protein were 381, 52.9, 71.2 and 76.6% of the control values, respectively. In contrast, treatment of MDA-MB-231 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in a 31-39% reduction in the secretion of the 52-kDa protein however these effects were not statistically different from the control values. In addition, the treatments did not cause any significant effects on the secretion of the 34- and 160-kDa proteins by MDA-MB-231 cells. These results clearly confirm and extend the range of antiestrogenic effects caused by TCDD in estrogen-responsive MCF-7 cells and indicate that the MDA-MB-231 cells are not responsive to the antiestrogenic effects of TCDD.     

Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells

Metabolism of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstene-3,17-dione (delta(4)) was performed at their physiological plasma concentrations in MCF-7 cell cultures (1 microM, 10 and 2 nM, respectively). Final metabolic products of these steroids were separated by HPLC-radioactive flow detection and identified by LC/MS or MS/MS. Typical and specific mass fragmentation spectra identified the presence of estrone (E(1)), 17beta-estradiol (E(2)), delta(4), DHEA, 5-androstene-3beta,17beta-diol (delta(5)), and testosterone as principal DHEAS metabolites. Other steroids, such as androstenedione, androsterone, and DHEA fatty acid esters at very low concentrations (from pM to nM), were also obtained after steroid incubation. This highly specific method allowed us to conclude whether a metabolite and enzymatic activity of interest were present in MCF-7 cells or not. We also showed that DHEAS at its physiological plasma concentration may be converted into estrogens and estrogen-like compounds in breast cancer cells. The estrogenic action of DHEAS on breast cancer cells was also measured by bioluminescence in a stably transfected human breast cancer MCF-7 cell line with a reporter gene that allowed expression of the firefly luciferase enzyme under the control of an estrogen regulatory element.     

Estrogenic properties of isoflavones derived from Millettia griffoniana.

In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ERalpha)-dependent assays. In a yeast-based ERalpha assay, all test substances and 17beta-estradiol as endogenous agonist, showed a significant induction of beta-galactosidase activity. The test compounds at the concentration of 5 x 10(-6) M could achieve 59-121% of the beta-galactosidase induction obtained with 10(-8) M 17beta-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ERalpha. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.     

Evaluating the Potential Bioactivity of a Novel Compound ER1626

Background

ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.

Method

MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.

Results

ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC₅₀ of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.

Conclusion

In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.     

Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF

Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30. We show that 17beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, but also in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells. MDA-MB-231 breast cancer cells (ER alpha-, ER beta+) are GPR30 deficient and insensitive to Erk-1/-2 activation by 17beta-estradiol. Transfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in overexpression of GPR30 protein and conversion to an estrogen-responsive phenotype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists, including ICI 182,780, yet not by 17alpha-estradiol or progesterone. Consistent with acting through a G protein-coupled receptor, estradiol signaling to Erk-1/-2 occurred via a Gbetagamma-dependent, pertussis toxin-sensitive pathway that required Src-related tyrosine kinase activity and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon trans-activation of the epidermal growth factor (EGF) receptor via release of heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surface with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negative breast tumors that continue to express GPR30 may use estrogen to drive growth factor-dependent cellular responses.     

Synthesis and biological evaluation of a series of A,B-ring modified 16,17-secoandrostane derivatives

The starting compound for the synthesis of 16,17-secoandrostane derivatives with the 4-en-3-on, 1,4-dien-3-on, 4,6-dien-3-on, and 1,4,6-trien-3-on systems was 3β-hydroxy-17-methyl-16,17-secoandrost-5-en-16-nitrile-17-one (1), the Oppenauer oxidation of which yielded the corresponding 4-en-3-one derivative 2. Dehydrogenation of compound 2 with the aid of 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) gave the three products: 17-methyl-16,17-secoandrosta-1,4-dien-3,17-dione-16-nitrile (3), 17-methyl-16,17-secoandrosta-4,6-dien-3,17-dione-16-nitrile (4), and 17-methyl-16,17-secoandrosta-1,4,6-trien-3,17-dione-16-nitrile (5). On the other hand, epoxidation of compound 2 resulted in a mixture of α and β isomers of 4,5-epoxy-17-methyl-16,17-secoandrosta-3,17-dione-16-nitrile (6 and 7). Opening of the oxirane rings of the mixture of 6 and 7 by the action of formic acid yielded the 4-hydroxy-4-en derivative 8. Antiaromatase activity and in vitro cytotoxicity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, and prostate cancer PC3) of selected compounds were evaluated. Compound 2 exhibited a relatively strong inhibition of aromatase and extremely potent cytotoxicity against PC3 cells. Compound 8 showed satisfactory cytotoxicity against MCF-7 cells.     

Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells

Resveratrol is a natural phytoalexin compound found in grapes and other food products. In this study, the effect of resveratrol on the growth of human breast cancer cells was examined. Results show that resveratrol inhibits the growth of estrogen receptor(ER)-positive MCF-7 cells in a dose-dependent fashion. Detailed studies with MCF-7 cells demonstrate that resveratrol antagonized the growth-promoting effect of 17-beta-estradiol (E2) in a dose-dependent fashion at both the cellular (cell growth) and the molecular (gene activation) levels. At 5 x 10(-6) M, resveratrol abolished the growth-stimulatory effect mediated by concentrations of E2 up to 10(-9) M. The antiestrogenic effect of resveratrol could be observed at a concentration of 10(-6) M and above. The antiestrogenic effect of resveratrol was also demonstrated at the molecular level. Resveratrol in a dose-dependent fashion antagonized the stimulation by E2 of progesterone receptor gene expression in MCF-7 cells. Moreover, expression of transforming growth factor-alpha and insulin-like growth factor I receptor mRNA was inhibited while the expression of transforming growth factor beta2 mRNA was significantly elevated in MCF-7 cells cultivated in the presence of resveratrol (10(-5) M). In summary, our results show that resveratrol, a partial ER agonist itself, acts as an ER antagonist in the presence of estrogen leading to inhibition of human breast cancer cells.     

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.     

Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17beta-estradiol

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.