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Comparative secretome analysis of normal prostate and prostate cancer cell models
Thesis (MSc)--Stellenbosch University, 2017.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2017
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| _version_ | 1867614133411643392 |
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| access_status_str | Open Access |
| author | Johnstone, Euan |
| author2 | Storbeck, Karl-Heinz |
| author_browse | Johnstone, Euan Storbeck, Karl-Heinz |
| author_facet | Storbeck, Karl-Heinz Johnstone, Euan |
| author_sort | Johnstone, Euan |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2017. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/101437 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:47:10.728Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/101437 Comparative secretome analysis of normal prostate and prostate cancer cell models Johnstone, Euan Storbeck, Karl-Heinz Vlok, Mare Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Prostate -- Cancer Biomarkers Proteomics Secretomes Mass spectrometry Ingenuity Pathway Analysis Prostate-specific antigen Acetone -- Precipitation UCTD Thesis (MSc)--Stellenbosch University, 2017. 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. AFRIKAANSE OPSOMMING: Prostaatkanker is die tweede mees algemene kanker onder mans. Die prostaatspesifieke antigeen (PSA) was die eerste biomerker geidentifiseer vir die diagnose van hierdie algemene siekte. Alhoewel hierdie biomerker in roetinegebruik is, is dit gekritiseer en gediskrediteer as 'n biomarker vir prostaatkanker as gevolg van die groot aantal vals positiewes wat geidentifiseer is met die gebruik daarvan, wat as gevolg van toestande soos nie-kwaadaardige prostaat hiperplasie en prostatitis is. Verskeie studies het dus gebruik gemaak van genomiese, transkriptomiese en proteomiese metodes om nuwe kandidaat biomerkers vir prostaatkanker, wat PSA kan vervang, te identfiseer. Die doel van hierdie studie was om by te dra tot hierdie soektog deur die gebruik van massaspektrometrie-gebaseerde proteomika. Die proteoom en secretoom van nie-kwaadaardige (BPH-1), kanker (LNCaP en PC-3) en normale (PNT2C2) prostaat sellyn was ontleed. Die lotingdigthede van die vier prostaat sellyne is geoptimiseer vir maksimum proteien sekresie en minimale seldood. 'n Laer lotingdigthede het die beste resultate gelewer. Asetoonpresipitasie het die beste proteienherwinning van die gekondisioneerde media gelewer. Die ander metodes wat getoets was, was die ammoniumsulfaat, metanol chloroform en PTA-bemiddelde asetoonpresipitasie metodes. Die proteoom en secretoom profiele van die vier sellyne is daarna met behulp van massaspektrometer bemiddelde proteomika ontleed. 'n Totaal van 3576 en 1106 proteiene is positief uit die proteoom en secretome monsters onderskeidelik, geintifiseer. Hierdie data is verder ontleed met behulp van \Ingenuity Pathway Analysis" sagteware. Verskeie veranderinge in molekulêre paaie, stroomop reguleerders, molekulêre en sellullêre prosesse, siektetoestande en potensile biomerkers is geidentifiseer. Die data het getoon dat paaie wat betrokke is by die regulering van 'n Adherens aansluitings, oksidatiewe fosforilering en mitochondria is opgereguleer in die prostaatkanker sellyne en is dus nuttig paaie om te ondersoek vir kandidaatbiomerkers of terapeutiese middels vir prostaatkanker. Verder is 'n totaal van 157 kandidate biomerkers wat die prostaatkanker sellyne van die nie-kwaadaardige prostaat hiperplasie sellyn kan onderskei, geidentifiseer. Ten spyte van 'n aantal beperkings in hierdie studie toon die resultate dat sekretoom-gebaseerde proteomika gebruik kan word om biomerker ontdekking aan te vul. 2017-02-22T17:06:49Z 2017-03-29T21:00:55Z 2018-02-21T03:00:07Z 2017-03 Thesis http://hdl.handle.net/10019.1/101437 en_ZA Stellenbosch University xiv, 111 pages : maps, illustrations application/pdf application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Prostate -- Cancer Biomarkers Proteomics Secretomes Mass spectrometry Ingenuity Pathway Analysis Prostate-specific antigen Acetone -- Precipitation UCTD Johnstone, Euan Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title | Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title_full | Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title_fullStr | Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title_full_unstemmed | Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title_short | Comparative secretome analysis of normal prostate and prostate cancer cell models |
| title_sort | comparative secretome analysis of normal prostate and prostate cancer cell models |
| topic | Prostate -- Cancer Biomarkers Proteomics Secretomes Mass spectrometry Ingenuity Pathway Analysis Prostate-specific antigen Acetone -- Precipitation UCTD |
| url | http://hdl.handle.net/10019.1/101437 |
| work_keys_str_mv | AT johnstoneeuan comparativesecretomeanalysisofnormalprostateandprostatecancercellmodels |