The characterization of 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) activity towards novel C19 substrates.

Barnard, Monique (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OSOMMING: Kastrasie weerstandige prostaat karsinoom (CRPC) is 'n sieke wat aangedryf word deur die teenwoordigheid van kragtige androgene afkomstig van swakker adrenal voorlopers. Die alternatiewe 5α-dione padweg skakel die adrenale voorloper steroide DHEA(S) en androsteendioon (A4) om na die meer potente androgen dihidroksitestosteroon (DHT), terwyl die 11β-hidroksiandrosteendione (11OHA4) padweg die adrenal steroïed 11OHA4 omskakel na potente androgene soos 11-keto-testosteroon (11KT) and 11-keto-5α dihidroksitestosteroon (11KDHT). Twee 17β-hidroksisteroïed dehidrogenase ensieme kataliseer belangrike reaksies in beide van bogenoemde padwee. Die ensiem 17βHSD2 kataliseer die oksidasie van potente androgene na swakker steroïede terwyl 17βHSD5, beter bekend as AKR1C3, die reduksie van potente androgene omskakel na swakker steroiede. Dus is die uitdrukkings vlakke van hierdie twee ensieme belangrik vir die regulering van potente androgene in die ligaam. Die doelwit van hierdie studie was dus om die aktiwiteit van die ensim 17βHSD2 te karakteriseer vir nuwe 11- geoksideerde steroiede geproduseer deur die 11OHA4 padweg en die effek van verskillende verhoudings van AKR1C3:17βHSD2 op die vloed deur die alternatiewe 5α-dione en 11OHA4 padweg te ondersoek. Die aktiwiteit van 17βHSD2 tot die 11-geoksideerde steroiede 11KT and 11KDHT was gekarakteriseer vir die eerste keer deur die uitdrukking van 17βHSD2 in HEK293 selle. Die resultate het getoon dat 17βHSD2 effektief 11KT sowel as 11KDHT omskakel na hul onderskeie produkte. Dis was interessant om waar te neem dat 17βHSD2 T meer effektief omskakel na A4 in vergelyking met die effektiwiteit van die omskakeling van 11KT tot 11KA4, alhoewel die verskil nie statisties relevant is nie. Die effek van 'n toename in die verhouding tussen AKR1C3:17βHSD2, soos waargeneem in die geval van CRPC, was ondersoek. HEK293 selle wat geen van die twee bogenoemde ensieme uitdruk nie, was gemanipuleer om die ensiem van belang uit te druk waarna die selle gemeng is volgens die verhouding van AKR1C3:17βHSD2 verlang. PC3 cells wat reeds 17βHSD2 uitdruk was ook gemanipuleer om toenemende vlakke van AKR1C3 uit te druk. Saamgevat, bewys die resultate dat 'n toename in die uitdrukking van AKR1C3 'n merkwaardige invloed op die vloed deur die 11OHA4 padweg gehad het wat tot gevolg lei tot die produksie van meer potente androgene, terwyl die teenoorgestelde is geldig vir die vloed deur die klassieke padweg. Toenemende AKR1C3:17βHSD2 uitdrukking in beide die HEK293 en die PC3 selle het gelei tot 'n toename in die produksie van 11KT vanaf 11KA4 terwyl die produksie van T vanaf A4 konstant laag gebly het. 'n Wiskundige model afgelei van die resultate wat bekom is, bevestig hierdie eksperimentele bevindinge. Verder was die resultate ook bevestig deur die drie PCa sellyne wat verskillende verhoudings van AKR1C3:17βHSD2 uitdruk. Opgesom, bewys die resultate dat 17βHSD2 moontlik „n belangrike rol speel in die regulering van intratumorale androgeen vlakke en dat die toeneming in AKR1C3:17βHSD2 verhouding die vloed deur die 11OHA4 padweg bevoordeel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101459
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