Browsing by Author "Johnstone, Euan"

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    Comparative secretome analysis of normal prostate and prostate cancer cell models
    (Stellenbosch : Stellenbosch University, 2017-03) Johnstone, Euan; Storbeck, Karl-Heinz; Vlok, Mare; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH SUMMARY: Prostate cancer is the second most common cancer among men. The prostate specific antigen (PSA) was the first biomarker identified for the diagnosis of this prevalent disease. Although this biomarker is in routine use it has been extensively criticised and discredited due to the large number of false positives identified from its usage, which result from benign conditions such as benign prostatic hyperplasia and prostatitis. Several studies have therefore made use of genomic, transcriptomic and proteomic methods to identify novel candidate biomarkers for prostate cancer, which can replace PSA. The aim of this study was to contribute to this search by using state-of-the-art mass spectrometry based proteomics to characterise the proteome and secretome of benign (BPH-1), cancerous (LNCaP and PC-3) and normal (PNT2C2) prostate cell lines. The seeding densities of the four prostate cell lines were optimised for maximum protein secretion and reduced cell death in a chemically defined medium, with lower seeding densities yielding the best results. Acetone precipitation was subsequently found to yield the best protein recoveries from the conditioned media when compared to three other methods, which included ammonium sulphate, methanol chloroform and PTAmediated acetone precipitation. The proteome and secretome pro les of the four cell lines were subsequently characterised by mass spectrometer based proteomics. A total of 3576 and 1106 proteins were positively assigned from the proteome and secretome samples, respectively. This data was subsequently analysed using Ingenuity Pathway Analysis, several upregulated molecular pathways, upstream regulators, molecular and cellular processes, disease states and potential biomarkers were identified. The data showed that pathways involved in the regulation of a Adherens junctions, oxidative phosphorylation and mitochondria were significantly upregulated in the prostate cancer cell lines and may therefore be useful avenues to pursue when searching for candidate biomarkers or therapeutic targets for prostate cancer. Furthermore, a total of 157 candidate biomarkers which could distinguish the prostate cancer cell lines from the benign prostatic hyperplasia cell line were identified. Despite a number of limitations this study further demonstrates the use of secretome based proteomics as a tool to complement biomarker discovery.