Abstract
Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.
Highlights
We describe the induction of the oxidative catabolism of alltrans-retinoic acid (RA) by different retinoids in MCF-7 cells and further characterize the induced pathway by its sensitivity to inhibitors
All-trans-retinoic acid was obtained from Serva (Heidelberg, Germany). 4-Keto-all-transretinoic acid, 4-keto- 13-cis-retinoic acid and 9-cis-retinoic acid were a generous gift from Hoffmann-La-Roche (Basle, Switzerland)
We studied the induction of the oxidative catabolism of all-trans-RA by different retinoids in MCF-7 cells and examined the effects of the inhibitor liarozole-fumarate
Summary
IH-benzimidazole (E)-2-butenedioate(2:3)}, synthesized at the Janssen Research Foundation (Beerse, Belgium), was dissolved at a concentration of 10-2 M in ethanol. All-trans-retinoic acid was obtained from Serva (Heidelberg, Germany). CH55 ((E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-l-propenyl]benzoic acid) was obtained through the generosity of Dr Shudo (Tokyo, Japan). All the retinoids described above were dissolved at an initial concentration of 4 x 10-3 M in ethanol. Retinol and 13-cis-retinoic acid were purchased from Eastman Kodak (Rochester, NY, USA) and were dissolved at an initial concentration of 10-2 M in ethanol. 3H(N)]All-trans-retinoic acid (1875.9 GBq mmol-1, 50.7 Ci mmol-1) was obtained from NEN (Dupont de Nemours, Brussel, Belgium). Retinoid stock solutions were regularly checked for purity using high-performance liquid chromatography (HPLC) analysis. Final solvent concentrations during incubations were always < 0.5% (v/v). All other chemicals and solvents were of the highest purity available
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