Biomedical subjects

Guy Leclercq

Publications and source records attributed to Guy Leclercq.

7 recordsLinked to original sources

PubMed2003

Tamoxifen derivatives for delivery of the antitumoral (DACH)Pt group: selective synthesis by McMurry coupling, and biochemical behaviour.

The goal of our study was to potentiate the effects of the ((R,R)-trans-1,2-diaminocyclohexane)-platinum(II) fragment [(DACH)Pt], known for its cytotoxic properties, either with tamoxifen (Tam), the most widely used antiestrogen in the treatment of hormone-dependent breast cancers, or with its active metabolite hydroxytamoxifen (hydroxy-Tam). We coupled Tam or hydroxy-Tam derivatives bearing a malonato group at the para position of the beta aromatic ring with the (DACH)Pt fragment. The malonato-Tam and malonato-hydroxy-Tam compounds were prepared through McMurry coupling of the appropriate ketones. The presence of the malonate group resulted in a pronounced stereospecificity in the reaction, since malonato-Tam was obtained only as the Z isomer, while malonato-hydroxy-Tam was obtained as an 80/20 E/Z mixture. Attribution of the isomeric structures was achieved by 2D NMR spectroscopy. The platinum complexes (DACH)Pt-malonato-Tam and (DACH)Pt-malonato-hydroxy-Tam were then prepared by coupling the barium salts derived from the malonato-Tam and malonato-hydroxy-Tam with the nitrate derived from (DACH)PtCl(2). Study of the biochemical properties of these two platinum complexes showed that, while the hydroxy-Tam complex is satisfactorily recognized by the estrogen receptor (relative binding affinity, RBA=6.4 %), the Tam complex is less well recognized (RBA=0.5 %). The effects of these complexes on two hormone-dependent breast cancer cell lines (MCF7 and MVLN) were studied in vitro. Both complexes showed an antiproliferative effect on MCF7 cells, and an antiestrogenic effect on MVLN cells. The observed effects appear to be essentially antihormonal, since incorporation of the (DACH)Pt fragment into the tamoxifen skeleton did not cause an increase in the cytotoxicity of the complexes.

Antineoplastic Agents, Hormonal↗

PubMed2003

Synthesis, characterization and biological evaluation of 7 alpha-perfluoroalkylestradiol derivatives.

Linkage of a long CH(2 )side chain ('spacer') onto C-7 alpha of estradiol-17 beta (E(2)) does not abrogate the binding affinity of this hormone for its receptor. Our purpose was to assess whether the linkage of a CF(2 )side chain, which is more bulky and rigid, could also be accommodated by the estrogen receptor (ER). We describe here the synthesis of 7 alpha-perfluorohexylestradiol 7 by perfluoroalkylation of a key silylenolether 2 with FITS-6 (perfluorohexyl-phenyliodoniurn trifluoromethanesulfonate). 7 alpha-Trifluoromethylestradiol 10a was prepared as a fluorinated control compound by UV-light induced trifluoromethylation of 2 with Umemoto reagent (S-trifluoromethyldibenzo[b,d]thiophenium trifluoromethanesulfonate). Endocrine properties of these two E(2 )derivatives were tested on the MCF-7 breast cancer cell line. Our data reveal that rigidity of the side chain of 7 affected the association of its hormone moiety with the ER to the same extent as a long alkyl side chain. Rigidity also failed to abrogate estrogenicity, as demonstrated by the ability of 7 to enhance ERE-dependent transcription and cell growth. Compound retained the capacity of inducing down regulation of the receptor. Interestingly, no evidence of antiestrogenicity was recorded since this compound behaved like a weak estrogen, exerting a mitogenic effect at high concentration. Of note, control 10a displayed a higher binding affinity than 7 for ER and consequently acted like the latter, albeit with a higher efficiency. Selection of appropriate residues to be linked at the end of a long 7 alpha alkyl side chain is known to be essential for generating strong antiestrogenicity. One may hope that such a property may also hold for perfluorinated chains to produce antiestrogens with strong metabolic stability.

Alkylation↗

PubMed2003

Decrease of estrogen receptor expression and associated ERE-dependent transcription in MCF-7 breast cancer cells after oligomycin treatment.

Oligomycin at 0.01 microM produces very rapid decrease of [3H]estradiol (E2)-binding capacity in MCF-7 cells maintained in culture in glucose- and serum-free medium. Loss of binding capacity was associated with elimination of the estrogen receptor (ER) as well as a decrease of basal expression of ERE-luciferase reporter gene. These effects were not due to major cell death as shown by MTT assay. Hence, the inhibition of ATP synthesis produced by oligomycin seems to influence ER turnover, resulting in very rapid loss of receptor. Withdrawal of oligomycin and maintenance of glucose in the medium led to only a partial reappearance of ER and failed to restore optimal ERE-dependent transcription. Oligomycin significantly down-regulated progesterone receptor (PR) level and partially abrogated E2-induced PR up-regulation, indicating that this drug also affects other nuclear receptors. Treatment of cytosol from MCF-7 cells with acid and alkaline phosphatases decreased [3H]E2-binding capacity, indicating the requirement of ER phosphorylation for optimal hormone binding. On the other hand, oligomycin-induced ER loss was partly compensated by E2 and partial anti-estrogens (AEs; 4-OH-TAM or RU 39 411); i.e. oligomycin failed to improve the E2-induced ER down-regulation and very weakly suppressed partial AE-induced receptor up-regulation. The known ability of these ligands to stabilize ER in the cell nucleus before regulating ER level may explain this phenomenon since such antagonism was not recorded with pure AE RU 58 668, which is known to impede nuclear translocation of the receptor. Interestingly, ligands able to down-regulate ER (i.e. E2 or RU 58 668) increased ER phosphorylation while 4-OH-TAM which up-regulate the receptor had little effect in this regard. Oligomycin failed to strongly affect such phosphorylation enhancements while it produced a weak decrease of basal phosphorylation level. Hence, phosphorylations/dephosphorylations of specific sites on ER and/or co-regulators seem to govern its turnover.

Binding Sites↗

PubMed2002

Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the receptor to regulate its level. ER elimination was, however, found in the presence of conditioned media (CMs) prepared from cells pre-exposed to E2-BSA conjugates, suggesting that they may produce (a) modulator(s) that may enhance receptor down-regulation when released within the medium.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Molecular forms of the estrogen receptor in breast cancer.

Estrogen receptors (ERs) of which two isoforms have been identified (alpha and beta) are subjected to phosphorylation, glycozylation, ubiquitination and other post-transcriptional conformational changes giving rise to a very high molecular heterogeneity. Partial proteolysis of these receptors, as well as their high tendency to associate within oligomeric structures, reinforces this property. Investigations demonstrated that this heterogeneity is not a biochemical artefact suggesting some biological relevance. Our purpose was to review this topic, especially with regard to ERalpha from breast cancers.

Animals↗

PubMed2002

Estrogenic effect of a series of bisphenol analogues on gene and protein expression in MCF-7 breast cancer cells.

Bisphenols constitute a family of compounds, which includes many substances that have as a common chemical structure two phenolic rings joined together through a bridging carbon. In the present study, we aimed to determine whether several events triggered by 17 beta-estradiol (E(2)) in MCF-7 breast cancer cells were also observed in response to various bisphenol-A (BPA) analogues. We studied the expression of estrogen controlled genes by measuring the induction of pS2 (mRNA and protein) and progesterone receptor (PgR) as well as the expression of a luciferase reporter gene transfected into MVLN cells. These data were compared to the cell proliferation potency and effectiveness as the latest expression of estrogen controlled functions. Bisphenols showed an agonistic effect in all our assays, suggesting that these compounds may act through all the response pathways triggered by the natural hormone. We found differences between the assays in the potency of bisphenols, defined as the minimum concentration required to produce a maximal effect. In the cell proliferation assay, all tested compounds needed a lower concentration than in the other assays to give maximal response. Our results suggest that the polarity and nature of the substituent in the central carbon determines the estrogenic potency. Presence of two propyl chains at the central carbon appears to confer the greatest potency in both gene and protein expression assays.

Benzhydryl Compounds↗

PubMed2002

Pharmacological profile of 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b] [1,4]benzothiazin-6-one, a novel human estrogen receptor agonist.

Pharmacological studies were carried out to characterize further the endocrinological profile and the binding mode to the estrogen receptor (ER) of 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one (1). Binding experiments were conducted with highly purified recombinant human estrogen receptors hERa and beta. Potent estrogenic activity of compound 1 was assessed by testing its ability to down-regulate ERs and to enhance estrogen receptor element (ERE)-dependent transcription. The latest step of our work dealt with the synthesis of the 9-fluorinated derivative 15 for ionic microscopy experiments to determine the intracellular localization of compound 1. Although 1 failed to compete with [3H]E2 for binding to both ER isoforms, evidence was reported that it interacted with hERalpha in MCF-7 cells (ER down-regulation/ERE-dependent luciferase induction). Hence, an appropriate conformation of the hormone binding domain, most probably conferred by co-regulators of ER, is required for the onset of an activity of the compound 1. Estrogenic activity was weak but on the order of magnitude of that of coumestrol (slightly weaker). The synthesis of the 9-methoxylated derivative 16 and its pharmacological evaluation led us to propose a binding mode of 1 on hERalpha. Compound 1 appears to interact with ERa mainly through interactions of its 3-methoxy substituent with the residue His-524 of the hormone binding domain.

Cell Line↗