Browsing by Author "Barnard, Monique"

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    The characterization of 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) activity towards novel C19 substrates.
    (Stellenbosch : Stellenbosch University, 2017-03) Barnard, Monique; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Castration resistant prostate cancer (CRPC) is an androgen dependent disease driven by the intratumoural metabolism of adrenal androgen precursors to potent androgens. The alternative 5α-dione pathway converts the adrenal steroids DHEA and androstenedione (A4) to the potent androgen DHT, while the recently identified 11β-hydroxyandrostenedione (11OHA4) pathway converts the adrenal steroid 11OHA4 into the potent androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). Two 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes catalyse vital reactions in both pathways. 17βHSD2 catalyses the oxidation of androgens to their less active form, while 17βHSD5, which is better known as AKR1C3, catalyses the reduction of weak androgens to more potent androgens. The relative activity and expression levels of these enzymes are therefore vital in regulating the amount of active androgen produced. The aim of this project was to characterise the activity of 17βHSD2 towards 11-oxygenated steroids from the 11OHA4 pathway and to investigate the effect of different ratios of AKR1C3 and 17βHSD2 on the flux through the alternative 5α-dione and 11OHA4 pathways. 17βHSD2 activity towards the 11-oxygenated steroids 11KT and 11KDHT were characterised for the first time using HEK293 cells transiently transfected to express 17βHSD2. The results showed that 17βHSD2 efficiently catalysed the conversion of 11KT and 11KDHT to their respective products. Interestingly the catalytic efficiency tended to be higher for the conversion of testosterone (T) to A4 than the conversion of 11KT to 11KA4, although this difference was not significant. The effect of increasing ratios of AKR1C3:17βHSD2, which occur during CRPC, were subsequently investigated. HEK293 cells, which do not endogenously express either AKR1C3 or 17βHSD2, were transiently transfected to express each enzyme, and the cells subsequently combined in different ratios. PC3 cells, which endogenously express 17βHSD2, were transfected with increasing amounts of AKR1C3 to obtain different AKR1C3:17βHSD2 ratios. Collectively the results showed that increased expression of AKR1C3 had a significant influence on the flux through the 11OHA4 pathway, leading to the production of more potent androgens, but had little or no effect on the classical pathways. Increasing AKR1C3:17βHSD2 expression in both HEK293 and PC3 cells lead to increased levels of 11KA4 being converted to 11KT while the conversion of A4 to T remained low. A mathematical model was subsequently constructed and confirmed the experimental findings. These results were further validated in three prostate cancer cell lines each expressing different AKR1C3:17βHSD2 ratios. Taken together, the results from this study show that 17βHSD2 likely plays an important role in regulating intratumoural androgen levels and that increased AKR1C3:17βHSD2 ratios favour the flux through the 11OHA4 pathway.