Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

Abstract

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide's various therapeutic uses will begin to resolve the pleotropic nature of this compound.

Fig 1

A) In vitro inhibition of recombinant mouse BCO1 activity by HPR, its metabolites, EPR and A1120. Inhibitor (0–200 μM) was added from a 10 mM stock in DMSO (4-HPR) or 20 mM stock in ethanol (A1120, EPR, MPR, 3-keto-HPR) to an in vitro reaction mixture prior to addition of substrate. The final concentration of ethanol in all samples was 1%. The enzyme was preincubated with the inhibitor for 10 min at 4°C in the standard reaction buffer and was then added to 20 μM β-carotene and activity determined as described in Methods. Data plotted as means±standard deviation; n = 3; B) Accumulation of total A2E bisretinoid in the eyes of aged (1 year old) mice. Analysis was done in C57BL/6 wt (3 animals) and Bco1^-/- mice (13 animals).

Fig 2. Effect of fenretinide and its metabolites on SCD expression and activity.

A) SCD1 protein expression is decreased after treatment with fenretinide, and its metabolite 3-keto-HPR. ARPE-19 cells were treated with the indicated concentration of fenretinide, its metabolites, A1120, or vehicle for 24 h. Cell lysates were analyzed by Western blotting using mouse monoclonal antibody against SCD1 (upper panel) in comparison with immunoreactivity to α-tubulin which was employed as a loading control (lower panel) and densitometry for each lane was performed and analyzed by ImageJ to provide the SCD1/α-tubulin ratio for each lane. B) SCD1 protein activity is decreased after treatment with fenretinide, and its metabolite 3-keto-HPR. ARPE-19 cells were treated with the indicated concentration of fenretinide, its metabolites, A1120, or vehicle for 16 h, then incubated an additional 5 h in the presence of methyl D3-palmitic acid (50 μM) before harvesting. Free fatty acids present in lipid extracts were analyzed by LC/MS and SCD1 activity, measured by the conversion of methyl-D3 palmitic acid to methyl-D3 palmitoleic acid, was determined. Fenretinide and 3-keto-HPR are effective inhibitors of SCD1 activity, whereas MPR and A1120 have no inhibitory effect on SCD1 activity at all. Lipids were extracted from two 100 mm Petri dishes and combined for mass spectrometry analysis. Data plotted as means ± standard deviation; n = 3.

Fig 3. Effect of fenretinide and its metabolites on DES1 activity.

ARPE-19 cells were treated with fenretinide, its metabolites, A1120 (all at 10 μM) or vehicle for 24 h, and endogenous C16 and C18 ceramides measured by LC/MS. Suppression of DES1 activity by fenretinide and its metabolites is confirmed by the higher substrate/product (dihydroceramide/ceramide) ratio relative to the DMSO control. The presence of fenretinide or 3-keto-HPR led to a greater accumulation of C16 and C18 dihydroceramide than did MPR or A1120. Lipids were extracted from one 100 mm Petri dish for each biological replicate and subjected to mass spectrometry analysis. Data plotted as means±standard deviation; n = 3.

Fig 4. Phenylretinamides and A1120 do not compete or compete poorly with 1 nM at-RA in RARbeta antagonist assay.

Fenretinide is the only tested compound that appears to compete, exhibiting an IC₅₀ = 10 μM. Fluorescence was measured at excitation (ex) 409 nm/emission (em) 460 and at ex 409nm/em 530 nm and the ratio was determined and plotted against a range of concentrations (10⁻⁵ to 10⁻⁹ M) of the tested compounds.