BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Prostatacancer är en cancerform som drabbar män i övre medelåldern. Cancerformen påverkar patienter både fysiskt och psykiskt genom symtom och biverkningar från behandling. Syftet med litteraturstudien var att beskriva hur patienter som behandlas för prostatacancer upplever sin situation. Studiens resultat utgick ifrån tio vetenskapliga artiklar som besvarade studiens syfte. Resultatet bearbetades och resulterade i fem kategorier: socialt stöd, brist på information, emotionell påverkan, förändrad sexualitet och upplevelse av behandlingspåverkan. I resultatet framkom det att stöd från vänner, familj och sjukvård var en viktig del i hanteringen av sjukdomen samt att patienter med prostatacancer upplevde informationsbrist från sjukvården. Patienterna upplevde en emotionell påverkan både fysiskt och psykiskt som beskrevs som oro och depression. Sexualiteten blev förändrad hos de flesta patienterna som genomgått behandling samt att de olika behandlingarna gav biverkningar som kunde vara svåra att hantera. Tidigare forskning visar att patienter upplever att de är bristfälligt informerade om prostatacancer och dess behandlingar. Därför bör ytterligare forskning fokusera på varför det är en informationsbrist från sjukvården och samhället. / Prostate cancer is a form of cancer that men in the upper middle age fall victim to. This cancer form affects the patients both physically and mentally through symptoms and side effects from the treatment. The aim of the literature study was to describe how patients, who have been treated for prostate cancer, experience their situation. The result of the study was based on ten scientific articles which answered the purpose of the study. The result was processed and it resulted in five categories: social support, lack of information, emotional impact, changed sexuality and experience of treatment. The result showed that support from family and health care was an important part in dealing with the disease and that patients with prostate cancer felt a lack of information from health services. The patients experienced an emotional effect on both their physical and mental health, described as anxiety and depression. Sexuality changed for most of the patients who went through treatment, and the different treatments caused side effects that could be difficult to deal with. Previous research show that patients are not fully informed about prostate cancer and its treatments. For this reason further research should focus on why there is a lack of information from the health services and the society.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a multifunctional protein primarily expressed in neuronal cells and involved in numerous cellular processes. UCHL1 has been linked with neurodegenerative diseases and a wide range of cancers but its specific role remains unknown. Previous UCHL1 knockdown studies have shown that UCHL1 controls the expression of pro- and anti-apoptotic genes as well as genes involved in cell cycle regulation but it is unknown how UCHL1 regulates these genes. We have shown that UCHL1 is cross-linked to DNA in DU145 but not in LNCaP or PC3 prostate cancer cells. Therefore, we hypothesized that UCHL1 regulates the expression of pro- or anti-apoptotic genes as well as the genes involved in the cell cycle through its interaction with DNA. By utilizing ChIP and ChIP-seq analyses it is possible to determine the UCHL1 target sequences on the genomic DNA. It was shown that UCHL1 is only expressed in DU145 but not in LNCaP, PC3 or C4-2 prostate cancer cell lines. Additionally, UCHL1 is expressed and cross-linked to DNA in HEK293T cells. It is believed that UCHL1 is silenced by upstream promoter methylation when it is not expressed. However, treatment with the epigenetic drugs 5-aza-2′-deoxycytidine and trichostatin A (TSA) did not result in induction of UCHL1 expression in LNCaP, PC3 or C4-2 prostate cancer cell lines. UCHL1 is also associated with p53. However, ChIP assay results have shown that UCHL1 and p53 do not bind to genomic DNA of upstream promoter regions CDKN1A and BAX genes. Additionally, through UCHL1 ChIP-seq analyses in DU145 and HEK293T cells, we discovered that UCHL1 co-localizes to the DNA with the shelterin complex shedding light on a new role of UCHL1 that has never been described before. / October 2014

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In this study, the influence of rooibos on the catalytic activity of enzymes 17β -hydroxysteroid dehydrogenase type 3 (17βHSD3), 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), 5α-reductase type 1 (SRD5A1) and 5α-reductase type 2 (SRD5A2), which catalyse prostate androgen metabolism, was investigated. The activities of both 17βHSD3 and AKR1C3 heterologously expressed in CHO-K1 and HEK293 cells were inhibited significantly by rooibos, with rooibos reducing the conversion of androstenedione (A4) and 11keto-androstenedione (11KA4) to testosterone (T) and 11ketotestosterone (11KT), respectively. The catalytic activity of 17βHSD2 towards T, 11hydroxytestosterone (11OHT) and 11KT was also significantly inhibited by rooibos in transiently transfected HEK293 cells. In transiently transfected HEK293 cells rooibos did not inhibit SRD5A1 while the rate of T conversion to dihydrotestosterone (DHT) by SRD5A2 was decreased. Analysis of steroid metabolism in PNT2 cells also suggests that rooibos does not modulate the catalytic activity of endogenously expressed SRD5A towards A4, however, the conversion of T to DHT was reduced. In addition, reductive 17βHSD activity towards A4 was inhibited in the presence of rooibos in both PNT2 and BPH-1 cells. In contrast, the conversion of 11KA4 to 11KT was inhibited in BPH-1, PC-3 and LNCaP cells, with negligible conversion of 11KA4 in PNT2 cells. Interestingly, data suggests inhibition of 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) activity in the production of androsterone (AST) from 5α–androstenedione (5α-dione), as well as the dehydrogenase reaction of T to A4 in PNT2 cells by rooibos. Androgen metabolism pathways were subsequently investigated in LNCaP cells to determine androgen metabolism by endogenous enzymes. Rooibos resulted in the reduced conversion of A4 in LNCaP cells to the same extent as indomethacin, a known AKR1C3 inhibitor. Rooibos also modulated T, DHT and AST metabolism in LNCaP cells. Furthermore, uridine diphosphate glucuronosyltransferase (UGT) activity in LNCaP cells was inhibited by rooibos, decreasing T-, DHT– and AST-glucuronide formation. These data prompted subsequent investigations into the influence of rooibos at cellular level, and prostatespecific antigen (PSA) levels were assayed in the presence of rooibos. PSA was significantly inhibited by rooibos in the absence and presence of DHT, suggesting possible interaction of rooibos with the mutated androgen receptor (AR) or estrogen receptor-β (ERβ) expressed in LNCaP cells. Taken together, rooibos inhibited the catalytic activity of key enzymes that catalyse the activation of androgens in the prostate, as well as inhibiting enzymes involved in the conjugation of androgens. At cellular level, PSA levels were also decreased by rooibos, possibly through AR or ERβ interactions – clearly indicating a modulatory role for rooibos in active androgen production. / AFRIKAANSE OPSOMMING: In hierdie studie was die invloed van rooibos ten opsigte van die katalitiese aktiwiteite van die ensieme 17β-hidroksi-steroïed-dehidrogenase tipe 2, tipe 3 en tipe 5 (17βHSD2, 17βHSD3, AKR1C3), asook 5α-reduktase tipe 1 en tipe 2 (SRD5A1, SRD5A2) ondersoek. Hierdie ensieme is betrokke in die produksie van androgene in die prostaat. Rooibos het die katalitiese aktiwiteit van 17βHSD3 en AKR1C3 in CHO-K1 en HEK293 selle beïnvloed en het vermindere omskakeling van androstenedioon (A4) en 11keto-androstenedioon (11KA4) na testosteroon (T) en 11-ketotestosteroon (11KT), afsonderlik, veroorsaak. Die katalitiese aktiwiteit van 17βHSD2 teenoor T, 11-hidroksie-testosteroon (11OHT) en 11KT was ook beïnvloed in die teenwoordigheid van rooibos in HEK293 selle. Die katalitiese aktiwiteit van SRD5A1 teenoor A4 en T is nie beïnvloed deur rooibos nie, alhoewel dit voorkom asof rooibos die omsettingstempo van T na dihidrotestosteroon (DHT) deur SRD5A2, getransfekteer in HEK293 selle, verminder het. Verdere ondersoeke is in normale prostaat epiteel selle, in die teenwoordigheid van rooibos uitgevoer. Rooibos het geen invloed op die katalitiese aktiwiteit van SRD5A teenoor A4 gehad nie, alhoewel vermindere omskakeling van T na DHT aangetoon kon word. Rooibos het ook die omskakeling van A4 na T in beide PNT2 en BPH-1 selle tot „n mate geïnhibeer. Die omskakeling van 11KA4 na 11KT was ook verminder in BPH-1, PC-3 en LNCaP selle. Die omskakeling van 11KA4 na 11KT was beduidend laer in PNT2 selle en kon die invloed van rooibos nie aangetoon word nie. Bykomende data toon dat rooibos ook die omskakeling van 5α-androstenedioon (5α-dione) na androsteroon (AST), gekataliseer deur 3α-hidroksi-dehidrogenase tipe 3 (AKR1C2), verminder, gesamentlik met die vermindere omskakeling van T na A4, deur 17βHSD2, in PNT2 selle. Hierdie studie het ook ondersoek ingestel, na die metabolisme van androgene in LNCaP selle. Vermindere A4 metabolisme is in die teenwoordigheid van rooibos asook in die teenwoordigheid van indometasien, „n bekende AKR1C3 inhibitor, gevind. Rooibos verminder dus die aktiwiteit van reduktiewe 17βHSD in LNCaP selle. Verandering in die metabolisme van T, DHT en AST in LNCaP selle, in die teenwoordigheid van rooibos, is ook gevind. Verdere ondersoek in LNCaP selle het gewys dat rooibos „n vermindering in die produksie van gekonjugeerde T, DHT en AST veroorsaak. Die studie het die invloed van rooibos op prostaat-spesifieke antigeen (PSA) ook ondersoek. Daar is vasgestel dat rooibos die vlakke van PSA verminder in die afwesigheid en teenwoordigheid van DHT in LNCaP selle. Hierdie resultaat dui op moontlike interaksie van rooibos met die androgeen (AR) of estrogeen-reseptor-β (ERβ), teenwoordig in LNCaP selle. Rooibos het die katalitiese aktiwiteit van ensieme, wat bydra tot androgeen produksie, geïnhibeer, asook die konjugasie van androgene. Op „n sellulêre vlak, het rooibos die vlakke van PSA-sekresie verminder, wat moontlike interaksie met die AR en ERβ aandui. Hierdie bevindings dui daarop dat rooibos wel n rol het om te speel in die modulasie van aktiewe androgene in die prostaat.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Parthenolide (PN), a major active component of the traditional herbal medicine feverfew, has been shown to have anti-inflammatory and anti-tumor properties. More remarkably, the cytotoxicity of PN seems selective to tumor cells but not their normal cell counterparts. In the present study, we investigate whether and how PN selectively enhances tumor sensitivity to radiation therapy by using prostate cancer cells LNCaP, DU145 and PC3, as well as normal prostate epithelial cells PrEC. Our study demonstrates that inhibition of NF-κB pathway and suppression of its downstream target MnSOD are common mechanisms for the radiosensitization effect of PN in prostate cancer cells. The differential susceptibility to PN in two radioresistant cancer cells, DU145 and PC3, is due, in part, to the fact that in addition to NF-κB inhibition, PN activates the PI3K/Akt pro-survival pathway in both cell lines. The presence of PTEN in DU145 cells enhances the radiosensitization effect of PN by suppression of the steady state level of activated p-Akt. We also demonstrate that PN selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme MnSOD and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Overall, our data support the concept that increasing oxidative stress in cancer cells, which are already under high constitutive oxidative stress, will lead to cell death, while the same stress may allow normal cells to maintain redox homeostasis through adaptive response. Thus, modulating cell redox status may be a novel approach to efficiently and selectively kill cancer cells.