Sequential transformation of 4-androstenedione into dihydrotestosterone in prostate carcinoma (DU-145) cells indicates that 4-androstenedione and not testosterone is the substrate of 5α-reductase.

BACKGROUND

Although it is well recognized that 5α-reductases possess higher affinity for 4-androstenedione than testosterone, and the affinity of 4-androstenedione is higher for 5α-reductases than 17β-hydroxysteroid dehydrogenases, it is generally believed that dihydrotestosterone is necessarily produced by the transformation of testosterone into dihydrotestosterone, suggesting that the step catalyzed by 17β-hydroxysteroid dehydrogenase precedes the step catalyzed by 5α-reductase. This interpretation is in contradiction with the enzymatic kinetic law that suggests that the 5α-reduction step that catalyzes the transformation of 4-dione into 5α-androstane-3,17-dione precedes the 17keto-reduction step.

MATERIALS AND METHODS

To verify which of these two pathways is operative, we quantified mRNA expression levels of steroidogenic enzymes in prostate carcinoma DU-145 cells by real-time PCR and determined the metabolites produced after incubation with [14C]4-dione in the presence and absence of a 5α-reductase inhibitor and analyzed the metabolites produced by thin layer chromatography and HPLC.

RESULTS

Real-time PCR analysis strongly suggests that the new type 3 5α-reductase is responsible for 5α-reductase activity in DU-145 cells. Steroid profile analysis shows that in the absence of inhibitor 5α-androstanedione is first produced, followed by the production of androsterone and dihydrotestosterone. The concentration of testosterone was not detectable. In the presence of Finasteride, an inhibitor of 5α-reductase, there was no transformation of 4-androstenedione and also there was no production of testosterone. The present data clearly indicate that the biosynthesis of dihydrotestosterone in DU-145 cells does not require testosterone as intermediate, and the step catalyzed by 5α-reductase precedes the step catalyzed by 17β-hydroxysteroid dehydrogenase.