An investigation into some mechanistic aspects of estrogen biosynthesis

The mechanistic aspects of the 19-hydroxy1ation and aromatization of androgens were investigated. Fungal, bacterial and mammalian enzymatic activities were studied in this regard . The fungus Pell i cular~ fi1amentosa metabolized androst-4-ene-3 , 17-dione to the corresponding 110<' , 11 f and 14 0( hydroxylated derivatives. No ~19- hydroxylated products were isolated, although this transformation was previously observed for the C21-steroids . The intestinal bacterium Clostridi um paraputrific~ had been reported to aromatize androsten-4-ene-3,17-dione. In the present study, however, only the ring A reduced products , 17(3 - hydroxy-5f -andro8tane- 3-one and 5f-androstane-3,17-dione , were recovered . Human placental microsomes contain substantial aromatase activity and were employed in an effort to elucidate some of the mechanistic details of aromatization. Selectively deuterated steroidal substrates were employed as a probe in order to distinguish b'!tween certain of the mechanisms proposed for aromatization . Retention of deuterium at C4 and C6 was observed. It was concluded that no free intermediates allowing for loss of hydrogen from either of these two positions are implicated in this process . The involvement of a Schiff base enzyme-sup strate complex in aromatization was examined using the substrate 17f - hydroxyandrost-4-ene-3-one- 3_ 1BO. Since no loss of label was ob~erved, the implication of a Schiff base was discounted . Mixed label1ir~ studies were performed in order to determine if hydroxylation at C19 is a rate-determining process in aromatization . Isotope effects of 2 .1 and 1.7 were determined for the conversion of 17f - hydroxyandrost-4-ene-J-one-19,19,19-dJ and -19-dl respectively to estrogens. It was concluded from this that 19-hydroxylation is at l east a partially rate-determinjng process in aromatization. A homoenb~ation mechanism for 19-hydroxylation was not supported by the data obtained in this s tudy. In vitro 1JC NMR monitoring using l7f-hydroxyandrost-4-ene-Jone- 19-l3C was found not to be a successful approach in the study of steroid transformations, owing in part t o their low solubility in the incubation medium.

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (similar to 22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.