All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-transretinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC 50 -value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg 71 . In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.
The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378–2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14α-methyl-ergosta-8,24(28)-diene-3β,6α-diol (3,6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14α-methyl-ergosta-5,7,22,24(28)-tetraene-3βol, and 14α-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 andCDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans andHistoplasma capsulatum but has not been reported inCandida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in theseERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.
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. Images Figure 4
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