Fig. 5.

VCaP cell–based washout experiments to assess the phenotypic consequence of CYP17A1 engagement after inhibitor removal. (A) DHEA formation at increasing concentrations of 17α-hydroxypregnenolone conformed to saturable Michaelis-Menten kinetics (R² = 0.974) (B) The effect of either abiraterone (20 µM) or ketoconazole (25 µM) coincubation on 17α-hydroxypregnenolone–mediated DHEA formation in VCaP cells. Cells were incubated for 2 hours at 37°C. (C) Time dependent reversal of CYP17A1 inhibition by abiraterone or ketoconazole as quantified via restoration of DHEA production. VCaP cells were preincubated for 2 hours without (control) or with abiraterone (40 µM) or ketoconazole (40 µM), washed, and further incubated with fresh medium for the indicated washout periods before final incubation with 30 μM 17α-hydroxypregnenolone for 30 minutes. DHEA oxime formation, representing residual CYP17A1 activity in (B) and recovery of CYP17A1 activity in (C) was expressed as the percentage of maximal DHEA oxime produced in the corresponding vehicle controls. In (C), an unpaired t test revealed overall significant difference between treatment arms across the 60-minute washout period (0 minutes: one-sided P value = 0.0158; 30 minutes: one-sided P value = 0.0071; 60 minutes: one-sided P value = 0.0281). Data in (A) represent the means ± S.D. from two experiments with triplicate determinations, whereas data in (B and C) represent the means ± S.D. from at least three experiments with triplicate determinations. *P < 0.05; **P < 0.01.