Browsing by Author "Muvirimi, Takudzwa Kevin"
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- ItemComparative secretome analysis of benign prostate hyperplasia and prostate cancer cell models(Stellenbosch : Stellenbosch University, 2017-12) Muvirimi, Takudzwa Kevin; Storbeck, Karl-Heinz; Vlok, Mare; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Prostate cancer is the second most prevalent non-cutaneous malignancy affecting men in the world. The current method for screening and diagnosis of prostate cancer relies on the measurement of serum kallikrein 3 (KLK-3) in conjunction with the digital rectal exam. However, serum KLK-3 or prostate specific antigen (PSA) as it is better known, is also elevated by benign prostate hyperplasia (BPH). Consequently, a major challenge in the diagnosis of prostate cancer is the ability to discriminate between prostate cancer and BPH. Misdiagnosis of prostate cancer entails unnecessary treatment for patients which results in psychological stress and physical discomfort. Despite the ineffectiveness of PSA as a biomarker, it is still the primary diagnostic tool. This is largely due to lack of an alternative and it is therefore crucial to find a new biomarker which can distinguish malignant prostate cancer from BPH. In this study, the secretome and proteome of four prostate cell lines (BPH-1, LNCaP, PC3 and PNT2) were investigated to identify novel biomarkers for BPH using a MS based proteomic approach. The analysis of the secretome necessitated that the growth conditions of prostate cell lines were optimized to reduce cell death and maximize secreted proteins. Optimal seeding densities were selected following measurements of total protein, LDH and the LDH/protein ratio. Subsequent optimization of protein precipitation methods identified acetone precipitation as the most suitable method for isolating protein from the conditioned media of prostate cell lines. The cell proteome of the four prostate cell lines was comparatively analysed by LC-MS/MS. The total number of proteins identified per cell line were as follows: 2079 in BPH-1, 2081 in LNCaP, 1853 in PC3 and 2137 proteins in the PNT2 cell line. A literature search of the proteins unique to BPH-1 identified mesencephalic astrocyte-derived neurotrophic factor (MANF) as a potential biomarker for BPH. Ingenuity pathway analysis was subsequently used to analyse and identify aberrant pathways in BPH-1. The following pathways were significantly altered in BPH-1: antiproliferative role of transducer of ERB2 in T cell signalling, pyrimidine ribonucleotide de novo biosynthesis, cell signalling and vitamin and mineral metabolism. The optimised secretome samples could not be analysed due to technical difficulties beyond our control. As a proof of concept preliminary analysis of the cell secretome from PC3 and BPH-1 cell lines yielded forty-six and ninety-nine proteins which were unique to each cell line, respectively. The identification of MANF and the antiproliferative role of transducer of ERB2 in T cell signalling during the proteomic analysis both pointed towards an inflammatory response in BPH. Differences in inflammation could therefore be explored in future in order to develop biomarkers unique to BPH and prostate cancer.