Gerda Smets

All-trans-retinoic acid metabolites significantly inhibit the proliferation of MCF-7 human breast cancer cells in vitro.

All-trans-retinoic acid (ATRA) is well known to inhibit the proliferation of human breast cancer cells. Much less is known about the antiproliferative activity of the naturally occurring metabolites and isomers of ATRA. In the present study, we investigated the antiproliferative activity of ATRA, its physiological catabolites 4-oxo-ATRA and 5,6-epoxy-ATRA and isomers 9-cis-RA and 13-cis-RA in MCF-7 human breast cancer cells by bromodeoxyuridine incorporation. MCF-7 cells were grown in steroid- and retinoid-free medium supplemented with growth factors. Under these culture conditions, ATRA and its naturally occurring catabolites and isomers showed significant antiproliferative activity in MCF-7 cells in a concentration-dependent manner (10[-11] M to 10[-6] M). The antiproliferative activity of ATRA catabolites and isomers was equal to that of the parent compound ATRA at concentrations of 10(-8) M and 10(-7) M. Only at 10(-6) M were the catabolites and the stereoisomer 13-cis-RA less potent. The stereoisomer 9-cis-RA was as potent as ATRA at all concentrations tested (10[-11] M to 10[-6] M). In addition, we show that the catabolites and isomers were formed from ATRA to only a limited extent. Together, our findings suggest that in spite of their high antiproliferative activity the catabolites and isomers of ATRA cannot be responsible for the observed growth inhibition induced by ATRA.

Fluorescein-labeled Tyramide Strongly Enhances the Detection of Low Bromodeoxyuridine Incorporation Levels

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.

The antiproliferative activity of all-trans-retinoic acid catabolites and isomers is differentially modulated by liarozole-fumarate in MCF-7 human breast cancer cells

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

Synthesis and anti-angiogenic activity of 6-(1,2,4-thiadiazol-5-yl)-3-amino pyridazine derivatives

General screening for inhibitors of microvessel growth in vitro in the rat aortic ring assay led to the discovery of a novel series of thiadiazole pyridazine compounds with potential anti-angiogenic activity. Chemical optimization produced orally active compounds with potent in vitro and in vivo anti-angiogenesis and anti-tumor activities.

125I-Tyr0-hCRH Labelling characteristics of corticotropin-releasing hormone receptors: differences between normal and adenomatous corticotrophs

The presence of corticotropin-releasing hormone (CRH) receptors has been previously demonstrated in corticotrophs from normal pituitaries using a method combining immunocytochemistry and liquid emulsion autoradiography. The aim of this study was to compare the characteristics of the 125I-Tyr0-hCRH binding in corticotrophs from normal pituitaries (three obtained at autopsy and one obtained at surgery) with corticotrophs from pituitary adenomas (six corticotroph adenomas responsible for Cushings disease and two silent corticotroph adenomas secreting a biologically inactive ACTH molecule). In normal corticotrophs, the larger part of the 125I-Tyr0-hCRH binding was localised in patchy conglomerates at the centre of the cell and, to a much lesser degree, in a diffuse pattern at the cell periphery. In adenomatous corticotrophs, CRH receptor expression is disturbed both quantitatively and qualitatively. Except for a minority of cells in one adenoma, all adenomatous corticotrophs showed only peripherally bound 125I-Tyr0-hCRH and no centrally localised binding. Furthermore, adenomatous corticotrophs revealed a statistically significant lower signal intensity when compared to normal corticotrophs and a strongly negative correlation was found between the labelling area in adenomatous corticotrophs and both the basal and CRH-stimulated plasma ACTH levels. These findings suggest defective processing of CRH receptors and could be relevant to the sustained ACTH secretion by adenomatous corticotrophs in Cushings disease and, more generally, provide an explanation to its pathology. The silent corticotrophs secreting a biologically inactive ACTH molecule were characterised by a very faint signal intensity, although present on almost every cell.