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Biomedical subjects

G Leclercq

Publications and source records attributed to G Leclercq.

At least 145 records · Page 8Linked to original sources
PubMed1987

Endocrine effects of Trilostane: in vitro and in vivo studies.

Trilostane (4-alpha-5-epoxy-17 beta-hydroxy-3-oxo-5-alpha-androstan-2-carbonitrile) is a modified steroidal molecule. In vitro and in vivo studies in rats have shown that it inhibits adrenal, ovarian and placental steroid synthesis. It seems to act by exerting a selective blockade on 3 beta-hydroxysteroid dehydrogenase. In this study, we investigated whether this molecule interacts with hormone receptors for estrogen, androgen or progesterone. We also tried to demonstrate the effect which Trilostane may have on cellular cultures of human mammary carcinoma (MCF-7 Evsa-T). We also studied hormonal modifications in a series of 12 patients treated with different doses of Trilostane, since this drug is supposed to inhibit the production by the adrenal glands of mineralocorticoids, of glucocorticoids and of the precursors of estrogens. Our results indicate that Trilostane does not react with any of the main hormonal sex steroid receptors, nor does it interfere with cultures of human mammary cancer cells either containing estrogen receptors and therefore allegedly hormone-dependent (MCF-7 line), or estrogen receptor-negative cells, presumably independent of hormonal manipulations (Evsa-T cell line). Finally, endocrine studies on postmenopausal women with advanced breast cancer show that Trilostane significantly reduces the plasma levels of estrone and of its major androgen precursor (androstenedione). However, the latter inhibition is no different from that exerted by hydrocortisone acetate administered alone at a dose of 40 mg/day. The results of clinical trials comparing hydrocortisone alone with hydrocortisone plus Trilostane are awaited.

Antineoplastic Agents↗
PubMed1987

Synthesis, conformational considerations, and estrogen receptor binding of diastereoisomers and enantiomers of 1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylbutane (dihydrotamoxifen).

As part of a study into nonisomerizable antiestrogens, the diastereoisomeric dihydrotamoxifens 7 and 8 were prepared by catalytic transfer hydrogenation of (Z)- and (E)-tamoxifen and were shown by NMR spectrometry to exist in preferred conformations with hydrogen atoms in an antiperiplanar relationship. The corresponding 4-hydroxy derivatives 9 and 10 were prepared from hydrogenated precursors of (Z)- and (E)-4-hydroxytamoxifen. The relative binding affinities (RBA) of the compounds to estrogen receptors are consistent with the assigned conformations and parallel reported data on derivatives of the nonsteroidal estrogen hexestrol. The growth-inhibitory activity against the MCF-7 human breast cancer cell line in vitro was for 10 comparable to that of 4-hydroxytamoxifen, although increasing the concentration from 10(-8) to 10(-6) M did not significantly improve the growth inhibition. The derivative 9 analogous to (E)-4-hydroxytamoxifen antagonized the growth-stimulating effect of added estradiol and is therefore also an antiestrogen but at low concentration (10(-8) M) in the absence of estradiol, MCF-7 cell growth was stimulated, indicating an estrogenic influence. The enantiomers of the dihydrotamoxifen 8 were individually prepared from the resolved enantiomers of 2-phenylbutanoic acid, the key reaction step being a lithium-ammonia reduction of the 1-(4-methoxyphenyl)-1,2-diphenyl-1-butanol to generate the triphenylbutane. The enantiomers of 8 gave identical RBA values in cytosol.

Animals↗
PubMed1987

A subpopulation of immunoglobulin G in man selectively interacts with the hormone-binding sites of estrogen receptors.

Since the binding sites of hormone receptors may be similar to those of antihormone antibodies, we wondered whether the former might not be recognized by the idiotypic network. To test this hypothesis we investigated the interaction of plasma immunoglobulin G (IgG) with the binding sites of estrogen receptors (ER) from uterine or mammary tissue. Using ER isolated from uterine cytosol we found that IgG from normal subjects shifted the position of purified receptor in sucrose gradients and displaced [3H]estradiol (E2) from its receptor-binding sites. Equilibrium studies revealed competitive inhibition by IgG of E2 binding to the ER. IgG isolated by adsorption on a rat uterine cytosol-Blue B matrix gel column also bound to the ER, and this binding was inhibited by an excess of E2. After an 18-h exposure of MCF-7 mammary carcinoma cells in monolayer culture to IgG (2 mg/ml), Scatchard analysis of [3H]E2 binding revealed a reproducible decrease in the available receptor sites from 2.52 +/- 0.56 (+/- SEM) to 0.68 +/- 0.48 fmol/microgram DNA (n = 10). This effect was selective, since enriched anti-ER IgG obtained by adsorption on purified receptor was 20 times more potent than total IgG, whereas IgG identically prepared but not retained by affinity chromatography had no activity. Exposure of the cells to the IgG for 45 min also revealed, as with isolated ER, specific competition of the IgG with E2 for the E2-binding sites; the Kd increased from 10.5 +/- 1.6 to 27.5 +/- 7.2 X 10(-11) M (n = 7). Enriched antireceptor IgG was a 20 times more effective competitor, and the IgG not retained by affinity chromatography had no activity. In conclusion, our observations indicate the presence of ER on the cell surface, interaction of ER with IgG from plasma of normal subjects, and competitive antagonism of these IgG with E2 uptake leading to a decrease in effective ER concentrations.

Binding, Competitive↗
PubMed1987

NK activity in neuroblastoma bearing mice: in vivo versus in vitro propagated tumor variants.

Two days after inoculation of A/J mice with in vivo propagated C1300 neuroblastoma (NB) cells, a 4-5 fold increase of the splenic natural killer (NK) activity is noticed. A similar rise of NK activity is also displayed by the host lymph node, bone marrow and peritoneal cells, reflecting a general stimulation of NK cells. These effector cells only lyse classical NK target cells. They are of low specific gravity, nylon wool non-adherent and insensitive to anti-Thy treatment, suggesting that they are indeed NK cells. In spite of the ability of in vivo maintained NB cells to increase the NK activity, they are not lysed by NK cells, nor do they have NK-recognizable target structures as demonstrated by competition studies. In contrast with the previously mentioned experiments, NB cells that have been cultured in vitro for at least one week, do not increase the NK activity. During further growth of both tumor variants, the NK activity gradually decreases. In vivo experiments revealed that the presence of a large NB tumor load exerts a negative effect on the induction of active NK effectors. This effect is not due to the intervention of splenic suppressor cells.

Animals↗
PubMed1986

Competitive binding assay for estrogen receptor in monolayer culture: measure of receptor activation potency.

MCF-7 cells were incubated with [3H]estradiol, unlabeled estradiol and various estrogens or antiestrogens to measure their relative binding affinity (whole-cell assay). Comparison of the values with those previously established on uterine cytosol with a DCC assay revealed a good parallelism for both steroid and diphenolic diethylstilbestrol based estrogens. On the contrary, in the whole-cell assay, antiestrogens and weak estrogens of the triphenyl- and gem-diphenylethylene categories always displayed low values which were in the order of magnitude found with weak steroid estrogens. This property was not due to a reduction of binding capacity, nor to the presence in some compounds of an ethoxyaminoalkyl side-chain (source of antiestrogenicity). In view of all these data, it was concluded that the present test would provide an estimate of the ability of a given compound to transform the receptor in a form which interacts with genomic sites involved in the regulation of "estrogenic-induced products" ("activation").

Binding, Competitive↗
PubMed1986

Induction of progesterone receptor in an estrogen, progesterone receptor-negative breast cancer cell line.

In MCF-7 cell culture, some sera endow estradiol-17 beta with strong growth promoting properties ("active" sera) while other fail to display this property ("inactive" sera). Passage from "inactive" to "active" sera are shown here to induce the appearance of a progestin binding capacity in the receptor negative line Evsa-T. Competition with various unlabeled steroids established the specificity of this binding reaction. The induction of progesterone receptor required neither estrogens, nor ER and failed to confer major growth sensitivity to hormonal steroids: only medroxyprogesterone acetate was slightly inhibitory at high concentration. These observations disclose the influence of seric factors independent of estrogens and of ER-related mechanisms on PgR induction.

Breast Neoplasms↗
PubMed1986

Aromatase, 17 beta-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normal glandular breast tissue in postmenopausal women.

In a study of the origin of estrogens in patients with breast cancer, the concentrations of estrogens and their androgen precursors, and aromatase and 17 beta-hydroxysteroid dehydrogenase (E2DH) activities were determined in normal glandular and cancerous breast tissue. The correlation between tissue estrogens, precursor concentrations, enzyme activities and plasma levels and/or receptor status were calculated. In both normal glandular and carcinomatous breast tissue, the concentrations of androstenedione (A), dehydroepiandrosterone (DHEA), 5 androstene-3 beta, 17 beta-diol (5-Adiol), estrone (E1), estradiol (E2) and progesterone (P) were significantly higher than plasma concentrations. While testosterone (T) concentrations were similar, dehydroepiandrosterone (DHCA) and estrone sulphate (E1S) concentrations were lower in tissue than in plasma. In carcinomatous tissue androgen concentrations were lower, but estrogen concentrations were higher than in glandular breast tissue. Estradiol (E2) concentration was positively correlated with the receptor concentration with the mean E2 concentration corresponding to an estimated receptor occupancy of about 25%, probably sufficient for a submaximal biological response. Aromatase and E2DH (E2----E1) activities were observed in all breast cancer and glandular breast tissues, activities being higher in carcinoma than in glandular breast tissues; nevertheless, aromatase activity accounts probably only for a small fraction of tissue estrogen concentration. E2DH, but not aromatase activity, was significantly higher in estrogen receptor positive than in estrogen receptor negative tissues and was negatively correlated with tissue dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) concentration; the latter two steroids are non competitive inhibitors of E2DH which inactivates E2 to E1. This effect of DHEA(S) may constitute a mechanism by which these androgens stimulate cancer growth and a rationale (besides suppression of estrogen precursors) for medical or surgical adrenalectomy in hormone sensitive metastatic mammary cancer. E2DH activity might constitute an additional marker of hormone dependency of mammary cancer.

17-Hydroxysteroid Dehydrogenases↗
PubMed1986

Morphology of MXT mouse mammary tumors. Correlation with growth characteristics and hormone sensitivity.

The transplantable MXT mouse mammary tumor has been a useful tool for studying endocrine mechanisms underlying mammary tumor growth. It is our experience, however, that this model is unstable during serial transplantation. This paper analyses this variability from the viewpoints of histology and estrogen receptor content and indicates that these parameters should always be checked before planning experimental work. It is advised that a more homogeneous material is needed and that this goal should be achieved by clonal selection before transplantation.

Animals↗
PubMed1986

Sensitivity of the hormone dependent MXT-mouse mammary carcinoma to estradiol during tumoral growth. An autoradiographic study.

The MXT transplantable mammary tumor is an ovarian sensitive carcinoma of the B6D2F1 mouse strain. The present work documents the mitogenic effect of a single near-physiological dose (pulse) of estradiol (E2) in castrated mice bearing MXT neoplasms 2-7 weeks after their inoculation. Histological sections of tumors and uteri (as control E2 target organs) were processed and nuclear thymidine labeling indices (TLI) were determined by autoradiography. Tumor estrogen receptors contents were evaluated in parallel. The results indicate that the MXT tumor keeps a constant sensitivity to E2 6 weeks after implantation.

Animals↗
PubMed1986

Synthesis and estrogen receptor binding of 6,7-dihydro-8-phenyl-9-[4-[2-(dimethylamino)ethoxy] phenyl]-5H-benzocycloheptene, a nonisomerizable analogue of tamoxifen. X-ray crystallographic studies.

Syntheses of the title compound (5), a novel nonisomerizable antiestrogen containing a seven-membered ring, are described. In one method, 6,7-dihydro-9-(4-methoxyphenyl)-5H-benzocycloheptene was brominated at the 8-position and the bromine displaced by phenylzinc chloride with palladium complex catalysis to introduce the 8-phenyl substituent. Alternatively, benzosuberone was alpha phenylated with tricarbonyl(eta 6-fluorobenzene)chromium (0) and the product treated with the appropriate aryllithium reagent to introduce the 9-aryl group last. The relative binding affinities for estrogen receptors in cell cytosol and whole cells and growth inhibitory activity against the MCF-7 human breast tumor cell line in vitro were for 5 comparable to those of tamoxifen (1) and the corresponding six-membered ring analogue (7). X-ray crystallographic analyses of 10 and 15, which are methoxy derivatives of 5 and 7, show that in some respects 5 bears a closer structural relationship to tamoxifen than does nafoxidine (3) or 7. Thus, the aromatic ring, which is fused in the cyclic analogues, was twisted 64, 45, 20, and 19 degrees out of the plane of the double bond for 1, 10, 3, and 15, respectively. Low-temperature NMR studies indicate that 5 is more rigid than tamoxifen; interconversion between enantiomeric conformers is slow on the NMR time scale at -75 degrees C.

Animals↗
PubMed1986

Abbott monoclonal enzyme immunoassay measurement of estrogen receptors in human breast cancer: a European multicenter study.

A new enzyme immunoassay (Abbott ER-EIA Monoclonal) for the determination of estrogen receptor in cytosols from breast tumor specimens has been developed by Abbott Laboratories. To establish the correlation of the results from this new technique with currently existing steroid binding methods, a multicenter study was conducted in eight European laboratories. All participants followed the same protocol consisting of a familiarization phase, a proficiency evaluation, and a comparison of existing steroid binding methods with the new immunoassay using panel samples and clinical specimens. ER-EIA was compared with the multipoint dextran coated charcoal assay in six laboratories, four of which followed the EORTC protocol; of the remaining two laboratories, one used a single saturating dose assay, the other an isoelectric focusing assay. The results show no significant difference between reducing agents when used in the ER-EIA. Reproducibility for the immunoassay (interassay coefficient of variation, 6%, interlaboratory coefficient of variation, 11-19%) was somewhat better than that for the steroid binding methods (interlaboratory coefficient of variation, 12-32%). The correlation between the methods was dependent on the origin of the lyophilized specimens. In breast tumor samples, an excellent correlation, (not statistically different from 1) was found between the ER-EIA and the steroid binding method in six laboratories. One laboratory showed a slope of 1.1 for the correlation line; the laboratory using isoelectric focusing showed a slope of 1.9. The mean value determined by the enzyme immunoassay in premenopausal women was 74 fmol/mg cytosol protein, and in postmenopausal women it was 187 fmol/mg cytosol protein with no significant difference in the slope of the correlation line. Results suggest the usefulness of the new standardized enzyme immunoassay for routine use in the clinical laboratory.

Antibodies, Monoclonal↗
PubMed1986

Immune response to murine neuroblastoma: effects of in vitro culture of the tumor. Mitogen and mixed lymphocyte culture responses, interleukin-2 production, suppression and natural killer activity.

The cellular immune response in A/J mice inoculated with in vitro cultured neuroblastoma (NB) cells is completely different from the response obtained after inoculation of in vivo passaged NB cells. Indeed, inoculation of in vivo grown tumor cells leads to a strongly decreased MLC and PHA response of the host spleen cells. This depressed response is not reversed by in vitro addition of indomethacin, but correlates with a low IL-2 production by the spleen cells. In addition, non-specific suppressor cells are present in the spleen and in the peritoneal cavity. The splenic suppressor cells are of low specific gravity and they are nylon wool adherent. The suppressor capacity is not reduced by anti-Thy plus rabbit complement treatment. Another effect of in vivo maintained NB cells is that the natural killer (NK) activity of the spleen cells of the host is increased 4-5 fold 2 days after inoculation of the tumor cells. On the contrary, in vitro passaged NB cells do not induce a reduced MLC and PHA responsiveness, and the IL-2 production of the host spleen cells remains nearly normal. No non-specific suppression can be demonstrated and the NK activity is not augmented 2 days after inoculation. We can conclude that it is very important in which condition tumor cells are maintained when used to study anti-tumor immunity.

Animals↗
PubMed1986

Modification of the basic side chain in tamoxifen: effects on microsomal metabolism and in vitro biological activity.

The effect of reducing the basicity of the side chain in tamoxifen (1) on in vitro microsomal metabolism and biological potency has been investigated. The N-methyl-N-(2,2,2-trifluoroethyl) analogue 10 and its 4-hydroxy derivative 18 have been synthesised. The side chain of 10 was not metabolised detectably by rat liver microsomes but a small amount of 18 was formed; there was no evidence of metabolic switching. The lack of metabolism of 10 is unlikely to reflect steric effects since the N,N-diethyl analogue 9 had a metabolism profile similar to that of tamoxifen, but could reflect the reduction in the basicity of the side chain. Compounds 10 and 18 had relative binding affinities (RBA) to oestrogen receptors in rat uterine cytosol and MCF-7 whole cells approximately 5 times lower than the values for tamoxifen (1) and 4-hydroxytamoxifen (3), respectively. Also 10 was similarly less potent than 1 as an inhibitor of the growth of MCF-7 cells in culture although antioestrogenicity was maintained. There was no evidence for improved cell penetration by 10 relative to 1. Of the N-acyl analogues (15-17) of tamoxifen synthesised, the N-trifluoroacetyl analogue 15 had a very low RBA. The N-acetyl (16) and N-formyl (17) analogues had RBA values comparable to that of 1 in cell cytosol or whole cell systems, but did not inhibit growth of MCF-7 cells. Thus, although metabolism of the side-chain in tamoxifen can be inhibited, reduction of biological potency results.

Animals↗
PubMed1986

Analogues of tamoxifen: the role of the basic side-chain. Applications of a whole-cell oestrogen-receptor binding assay to N-oxides and quaternary salts.

Derivatives of tamoxifen (1) and 4-hydroxy-2-methyltamoxifen (2) in which the basic side chain has been modified by N-oxidation or by quaternization have been investigated with respect to the effects on affinity for the oestrogen receptor and on cytostatic activity towards the MCF-7 cell line in vitro. In addition to the conventional cytosol assay for receptor binding affinity (RBA) a recently developed whole-cell assay was employed. N-oxidation (e.g. 2----3) produced no significant alteration in RBA value either in cytosol or in whole cells, nor in activity towards the MCF-7 line. Quaternization with methyl iodide (1----4, 2----6) or ethyl bromide (1----5, 2----7b: the cis isomer 7a also formed) did not alter receptor binding in the cytosol assay but almost abolished binding in the whole cell and cytostatic activity. The whole-cell RBA values for 2 (0.45) and 3 (0.5) were lower than those for 4-hydroxytamoxifen (2.9), suggested to be due to the lower oestrogenicity of the 2-methyl derivatives since activity against MCF-7 cells was unimpaired. The even lower values of whole-cell RBA (0.01-0.02) for the quaternary ethyl bromide derivatives 7a and 7b were ascribed to poor penetration into the cell since these compounds had minimal cytostatic activity.

Animals↗