Karl-Heinz Storbeck

11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: Quantification of steroid intermediates and end products in H295R cells

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

11-Oxygenated C19 Steroids Are the Predominant Androgens in Polycystic Ovary Syndrome

Context: Androgen excess is a defining feature of polycystic ovary syndrome (PCOS), but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to androgen excess in women with PCOS. Methods: One hundred fourteen women with PCOS and 49 healthy control subjects underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. Twenty-four–hour urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for homeostatic model assessment of insulin resistance. Baseline demographic data, including body mass index, were recorded. Results: As expected, serum concentrations of the classic androgens testosterone (P < 0.001), androstenedione (P < 0.001), and dehydroepiandrosterone (P < 0.01) were significantly increased in PCOS. Mirroring this, serum 11-oxygenated androgens 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone, and 11-ketotestosterone were significantly higher in PCOS than in control subjects, as was the urinary 11-oxygenated androgen metabolite 11β-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in patients with PCOS compared with control subjects [53.0% (interquartile range, 48.7 to 60.3) vs 44.0% (interquartile range, 32.9 to 54.9); P < 0.0001]. Obese (n = 51) and nonobese (n = 63) patients with PCOS had significantly increased 11-oxygenated androgens. Serum 11β-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance. Conclusions: We show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.

Advances in the analytical methodologies: Profiling steroids in familiar pathways-challenging dogmas.

The comprehensive evaluation of the adrenal steroidogenic pathway, given its complexity, requires methodology beyond the standard techniques currently employed. Advances in LC-MS/MS, coupled with in vitro cell models that produce all the steroid metabolites of the mineralo-, glucocorticoid and androgen arms, present a powerful approach for the comprehensive evaluation of adrenal steroidogenesis in response to compounds of interest including bioactives, drug treatments and endocrine disrupting compounds. UHPLC-MS/MS analysis of steroid panels in forskolin, angiotensin II and K(+) stimulated H295R cells provides a snapshot of their effect on intermediates and end products of adrenal steroidogenesis. The impact of full steroid panel evaluations by LC- and GC-MS/MS extends to clinical profiling with the characterization of normal pediatric steroid reference ranges in sexual development and of disease-specific profiles improving diagnosis and sub classification. Comprehensive analyses of steroid profiles may potentially improve patient outcomes together with the application of treatments specifically suited to clinical subgroups. LC-MS/MS and GC-MS/MS applications in the analyses of comprehensive steroid panels are demonstrated in clinical conditions such as congenital adrenal hyperplasia in newborns requiring accurate diagnoses and in predicting metabolic risk in polycystic ovary syndrome patients. Most notable perhaps is the impact of LC-MS/MS evaluations on our understanding of the basic biochemistry of steroidogenesis with the detection of the long forgotten adrenal steroid, 11β-hydroxyandrostenedione, at significant levels. The characterization of its metabolism to androgen receptor ligands in the LNCaP prostate cancel cell model, specifically within the context of recurring prostate cancer, lends new perspectives to old dogmas. We demonstrate that UHPLC-MS/MS has enabled the analyses of novel metabolites of the enzymes, SRD5A, 11βHSD and 17βHSD, in LNCaP cells. Undoubtedly, the continuous advances in the analytical methodologies used for steroid profiling and quantification will give impetus to the unraveling of the remaining enigmas, old and new, of both hormone biosynthesis and metabolism.

Cytochrome b5: Novel roles in steroidogenesis

Cytochrome b(5) (cyt-b(5)) is essential for the regulation of steroidogenesis and as such has been implicated in a number of clinical conditions. It is well documented that this small hemoprotein augments the 17,20-lyase activity of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1). Studies have revealed that this augmentation is accomplished by cyt-b(5) enhancing the interaction between cytochrome P450 reductase (POR) and CYP17A1. In this paper we present evidence that cyt-b(5) induces a conformational change in CYP17A1, in addition to facilitating the interaction between CYP17A1 and POR. We also review the recently published finding that cyt-b(5) allosterically augments the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD), a non cytochrome P450 enzyme, by increasing the enzymes affinity for its cofactor, NAD(+). The physiological importance of this finding, in terms of understanding adrenal androstenedione production, is examined. Finally, evidence that cyt-b(5) is able to form homomeric complexes in living cells is presented and discussed.

The Identification of Two CYP17 Alleles in the South African Angora Goat

South African Angora goats (Capra hircus) are susceptible to cold stress, due to the inability of the adrenal cortex to produce sufficient levels of cortisol. Two CYP17 isoforms were identified, cloned and characterized in this study. Sequence analysis revealed three amino acid differences between the two CYP17 isoforms, which resulted in a significant difference in 17,20 lyase activity of the expressed enzymes in both the presence and absence of cytochrome b5. Furthermore, cotransfections with 3βHSD revealed that one CYP17 isoform strongly favours the Δ5 steroid pathway. Our data implicates CYP17 as the primary cause of the observed hypoadrenocorticoidism in the South African Angora goat.

11-ketotestosterone and 11-ketodihydrotestosterone in castration resistant prostate cancer : potent androgens which can no longer be ignored

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). Under castrate conditions, DHT is produced from the metabolism of the adrenal androgen precursors, DHEA and androstenedione. Recent studies have shown that the adrenal steroid 11β-hydroxyandrostenedione (11OHA4) serves as the precursor to the androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). In this study we comprehensively assess the androgenic activity of 11KT and 11KDHT. This is the first study, to our knowledge, to show that 11KT and 11KDHT, like T and DHT, are potent and efficacious agonists of the human androgen receptor (AR) and induced both the expression of representative AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines, LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells, while in vitro conversion assays showed that 11KT and 11KDHT are metabolized at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT, respectively. Our findings show that 11KT and 11KDHT are bona fide androgens capable of inducing androgen-dependant gene expression and cell growth, and that these steroids have the potential to remain active longer than T and DHT due to the decreased rate at which they are metabolised. Collectively, our data demonstrates that 11KT and 11KDHT likely play a vital, but overlooked, role in the development and progression of CRPC.

11β-hydroxyandrostenedione: Downstream metabolism by 11βHSD, 17βHSD and SRD5A produces novel substrates in familiar pathways

11β-Hydroxyandrostenedione (11OHA4), a major C19 steroid produced by the adrenal, was first reported in the 1950s. Initially the subject of numerous studies, interest dwindled due to the apparent lack of physiological function and, by the end of the century, 11OHA4 was no longer considered as an adrenal C19 steroid. Our recent studies, however, showed that 11OHA4 is the precursor to novel active androgens which include 11-ketodihydrotestosterone (11KDHT) which has been implicated in prostate cancer, thereby renewing interest in 11OHA4. In this paper we review the biosynthesis and downstream metabolism of 11OHA4. We discuss the extra-adrenal biosynthesis of 11OHA4 in humans and in other species, highlighting the well-documented role of 11OHA4 in the testes of male fish in which the steroid functions as an active androgen. Finally, we discuss the physiological relevance of 11OHA4 metabolism in castration resistant prostate cancer and outline future prospects.

A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids.

The abundant adrenal C19 steroid 11β-hydroxyandrostenedione (11OHA4) has been written off as a dead-end product of adrenal steroidogenesis. However, recent evidence has demonstrated that 11OHA4 is the precursor to the potent androgenic 11-oxygenated steroids, 11-ketotestosterone and 11-ketodihydrotestosterone, that bind and activate the human androgen receptor similarly to testosterone and DHT. The significance of this discovery becomes apparent when considering androgen dependent diseases such as castration resistant prostate cancer and diseases associated with androgen excess, e.g. congenital adrenal hyperplasia and polycystic ovary syndrome. In this review we describe the production and metabolism of 11-oxygenated steroids. We subsequently discuss their androgenic activity and highlight the putative role of these androgens in disease states.

High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.