Identification of dihydroceramide desaturase as a direct in vitro target for fenretinide

Abstract

The dihydroceramide desaturase (DES) enzyme is responsible for inserting the 4,5-trans-double bond to the sphingolipid backbone of dihydroceramide. We previously demonstrated that fenretinide (4-HPR) inhibited DES activity in SMS-KCNR neuroblastoma cells. In this study, we investigated whether 4-HPR acted directly on the enzyme in vitro. N-C8:0-d-erythro-dihydroceramide (C(8)-dhCer) was used as a substrate to study the conversion of dihydroceramide into ceramide in vitro using rat liver microsomes, and the formation of tritiated water after the addition of the tritiated substrate was detected and used to measure DES activity. NADH served as a cofactor. The apparent K(m) for C(8)-dhCer and NADH were 1.92 ± 0.36 μm and 43.4 ± 6.47 μm, respectively; and the V(max) was 3.16 ± 0.24 and 4.11 ± 0.18 nmol/min/g protein. Next, the effects of 4-HPR and its metabolites on DES activity were investigated. 4-HPR was found to inhibit DES in a dose-dependent manner. At 20 min, the inhibition was competitive; however, longer incubation times demonstrated the inhibition to be irreversible. Among the major metabolites of 4-HPR, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) showed the highest inhibitory effect with substrate concentration of 0.5 μm, with an IC(50) of 1.68 μm as compared with an IC(50) of 2.32 μm for 4-HPR. N-(4-Methoxyphenyl)retinamide (4-MPR) and 4-Oxo-N-(4-methoxyphenyl)retinamide (4-oxo-4-MPR) had minimal effects on DES activity. A known competitive inhibitor of DES, C(8)-cyclopropenylceramide was used as a positive control. These studies define for the first time a direct in vitro target for 4-HPR and suggest that inhibitors of DES may be used as therapeutic interventions to regulate ceramide desaturation and consequent function.

FIGURE 1.

Kinetic parameters for in vitro DES assay. A, conversion of C₈-dhCer into C₈-Cer. This scheme depicts the oxidation of C₈-dhCer into C₈-Cer by the enzyme dihydroceramide desaturase. The formed water is radiolabeled, and it was the target for measurements in our assays. B and C, Michaelis-Menten plots of DES activity for determination of K_m and V_max for C₈-dhCer and NADH. B, determination of K_m and V_max for C₈-dhCer. A fixed amount of labeled C₈-dhCer and increasing amounts of unlabeled C₈-dhCer starting from 0.05 μm to 10 μm were used. C, determination of K_m and V_max for NADH. Increasing amounts of NADH were used and [C₈-dhCer] was 0.25 K_m. Assays were performed in vitro using rat liver microsomes with an incubation time of 20 min. Values represent the mean ± S.D. of at least two separate determinations performed in triplicate. K_m and V_max values are shown in Table 1. D, table showing K_m and V_max values for NADH and C₈-dhCer.

FIGURE 2.

The effects of 4-HPR on DES activity and determination of K_i for 4-HPR. A, the effects of increasing concentrations of 4-HPR on DES activity in rat liver microsomes. DES activity was measured using a fixed amount of substrate (2 nm labeled and 500 nm unlabeled substrate) and increasing concentrations of 4-HPR (0, 1, 2.5, 5, 10, 25, and 50 μm). Values represent the mean ± S.D. of three independent experiments performed in triplicate. B, the effects of increasing amounts of substrate on 4-HPR inhibition of DES activity in rat liver microsomes. DES activity was measured using a fixed concentration of 4-HPR (10 μm) and increasing amounts of substrate (between 0.25 and 3 K_m). Values represent % inhibition as compared with controls and the mean ± S.D. of two independent experiments performed in triplicate. C, Lineweaver-Burke plot for K_i demonstrating DES activity with increasing concentrations of 4-HPR. For these experiments, the [substrate] was fixed and ranged from 0.25, 0.5, 1, 2, to 3 K_m, and the [4-HPR] used ranged from 0, 1, 2.5, 5, 10, 25, and 50 μm. Values represent the mean ± S.D. of five independent experiments performed twice in triplicate.

FIGURE 3.

Effects of 4-HPR and its metabolites on in situ desaturase activity and endogenous sphingolipids. A, structures of 4-HPR and its metabolites. B, desaturase activity was measured using the in situ assay. SMS-KCNR cells were treated with increasing concentrations of 4-HPR, 4-oxo-4-HPR, 4-MPR, or 4-oxo-4-MPR for 6 h. C₁₂-dhCCPS was added at the same time as 4-HPR or its metabolites. Cells were collected after 6 h, and the conversion to C₁₂-CCPS was determined by LC/MS. Total endogenous levels of ceramides (Cer) and dihydroceramides (dhCer) in SMS-KCNR cells treated with 4-HPR (C), 4-oxo-4-HPR (D), 4-MPR (E), or 4-oxo-4-MPR (F) were measured by LC/MS as described under “Experimental Procedures.” Sphingolipid levels were normalized to total lipid phosphate. The data presented are representative of the mean of two independent experiments ± S.D. The error bars represent the S.D., and when not seen, they are smaller than the thickness of the lines on the graphs.

FIGURE 4.

The effects of 4-HPR, 4-HPR metabolites, and C₈-CPPC on desaturase activity in vitro. A, the effects of increasing concentrations of 4-HPR and 4-HPR metabolites on DES activity in rat liver microsomes. In these experiments, [C₈-dhCer] was 0.25 K_m, and the inhibitor concentrations were between 1 and 7.5 μm. The values represent the mean ± S.D. of two independent experiments performed in triplicate. B, the effects of increasing concentrations of C₈-CPPC on DES activity in rat liver microsomes. In these experiments, [C₈-dhCer] was 0.25 K_m, and C₈-CPPC concentrations were 0.5, 0.75, and 1 μm. The values represent the mean ± S.D. of two independent experiments performed in triplicate. C, the effects of increasing concentrations of 4-oxo-HPR on DES activity in rat liver microsomes. DES activity was measured using a fixed amount of substrate (2 nm labeled and 500 nm unlabeled substrate) and increasing concentrations of 4-oxo-HPR (0, 1, 2.5, 5, 10, 25, and 50 μm). The values represent the mean ± S.D. of two independent experiments performed in triplicate, D, the effects of increasing amounts of substrate on 4-oxo-HPR inhibition of DES activity in rat liver microsomes. DES activity was measured using a fixed concentration of 4-oxo-HPR (10 μm) and increasing amounts of substrate (from 0.25 to 3 K_m). The values represent % inhibition as compared with controls and the mean ± S.D. of two independent experiments performed in triplicate. E, Lineweaver-Burke plot for K_i demonstrating DES activity with increasing concentrations of 4-oxo-HPR. For these experiments, the [substrate] was fixed and ranged from 0.25, 0.5, 1, 2, to 3 K_m, and the [4-oxo-HPR] used ranged from 0, 1, 2.5, 5, 10, 25, and 50 μm. Values represent the mean ± S.D. of five independent experiments performed twice in triplicate. F, table of IC₅₀ values for 4-HPR, 4-oxo-4HPR, and C₈-CPPC. Values represent the mean ± S.D. of three independent experiments performed in triplicate.

FIGURE 5.

The effects of 4-HPR on the reversibility of inhibition of desaturase activity in rat liver microsomes or SMS-KCNR cells. A, the effects of inhibition by 4-HPR over time. Rat liver microsomes were treated with 1, 2.5, and 5 μm of 4-HPR, and the in vitro assay was performed for either 20, 60, or 120 min along with controls. Values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate. B, the effects of dilution of 4-HPR on DES activity. Rat liver microsomes were preincubated with either 0.5 or 5 μm 4-HPR for 60 min at 37 °C. After preincubation, the microsomes were centrifuged and washed twice. The in vitro enzyme assay was performed with 100 μg of either “control microsome” or “preincubated and washed microsomes” for 60 or 120 min. Control microsomes were not washed or preincubated with 4-HPR. The values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate. C, the effect of 4-HPR inhibition on SMS-KCNR cells. Cells were treated with 5 μm of 4-HPR for 2 h. After 2 h, cells were washed with PBS, and the medium was replaced by fresh medium. Total cell homogenates were prepared from SMS-KCNR cells after 2 h of 4-HPR treatment, and at 24, 48, and 72 h after 4-HPR treatment. Determination of DES activity was performed at these time points, using the in vitro assay with 400 μg of total cell homogenate for 20 min. Values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate.