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Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613986973810688 |
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| access_status_str | Open Access |
| author | Hollocks, Stephanie Anne |
| author2 | Snoep, Jacob Leendert |
| author_browse | Hollocks, Stephanie Anne Snoep, Jacob Leendert |
| author_facet | Snoep, Jacob Leendert Hollocks, Stephanie Anne |
| author_sort | Hollocks, Stephanie Anne |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128424 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:52.037Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128424 Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line Hollocks, Stephanie Anne Snoep, Jacob Leendert Van Niekerk, Dawie D. Kouril, Theresa Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Breast -- Cancer Cancer cells -- Proliferation Glycolysis -- Inhibitors Breast -- Cancer -- Treatment -- Data processing Cell lines Breast -- Cancer -- Chemotherapy Cancer -- Molecular aspects UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Most cancer cells exhibit an augmented glycolytic flux with a concomitant increase in lactate production under aerobic conditions. This phenomenon, termed the Warburg effect or aerobic glycolysis, is a well-established hallmark of cancer and supports the uncontrolled proliferation and metastatic dissem- ination of tumour cells. Accordingly, pharmacological inhibition of glycolytic proteins may hold great promise as a novel chemotherapeutic strategy. How- ever, rational drug design requires knowledge of the chief flux-controlling steps of glycolysis in both normal and tumour cells. Previous studies have yielded conflicting results regarding the degree of control exerted by hexokinase (HK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) over the rate of aer- obic glycolysis. This study aimed to investigate the glycolytic flux control of HK and GAPDH in the non-invasive MCF-7 breast cancer cell line. Flux control coefficients were experimentally assessed by titrating cell cultures with the irreversible glycolytic inhibitors iodoacetic acid (IAA), a specific GAPDH inhibitor, and 3-bromopyruvate (3-BrPA). While 3-BrPA is principally char- acterised as a HK inhibitor, titrations in whole cells and cytosolic extracts revealed that both HK and GAPDH are direct targets of 3-BrPA in MCF-7 cells. Additionally, a cell line-specific kinetic model of glycolysis was used to obtain theoretical predictions of flux control and pathway dynamics for com- parison to the experimental results. For GAPDH, IAA titrations and model analysis similarly yielded glucose and lactate flux control coefficients near zero, indicating that this enzyme exerts minimal control over glycolytic flux. As more than 85% inhibition was required to induce an appreciable decrease in glycolytic flux, GAPDH was deemed an unsuitable target for novel anticancer drugs. While 3-BrPA markedly suppressed GAPDH activity, its effects on pathway flux and HK activity were not large enough to resolve the individual contributions of each targeted enzyme to the global pathway response, pre- cluding further calculation of the flux control coefficient of HK. Nonetheless, model analysis indicated that HK exerts high control over glycolytic flux in MCF-7 cells. More broadly, this research contributes to our understanding of the Warburg effect and may distinguish alternative metabolic targets for clinical cancer treatment. AFRIKAANSE OPSOMMING: Die meeste kankerselle vertoon ’n verhoogde glikolitiese fluksie met ’n gepaard- gaande toename in laktaatproduksie onder aërobiese toestande. Hierdie ver- skynsel, wat die Warburg-effek of aërobiese glikolise genoem word, is ’n goed- gevestigde kenmerk van kanker en ondersteun die onbeheerde proliferasie en metastatiese verspreiding van tumorselle. Gevolglik kan farmakologiese inhibi- sie van glikolitiese proteïene groot belofte inhou as ’n nuwe chemoterapeutiese strategie. Rasionele geneesmiddelontwerp vereis egter kennis van die belangrik- ste fluksiekontrolestappe van glikolise in beide normale en tumorselle. Vorige studies het teenstrydige resultate opgelewer met betrekking tot die mate van kontrole wat deur heksokinase (HK) en gliseraldehied-3-fosfaatdehidrogenase (GAPDH) uitgeoefen word oor die tempo van aërobiese glikolise. Hierdie stu- die het ten doel gehad om die glikolitiese fluksiekontrole van HK en GAPDH in die nie-indringende MCF-7 borskankersellyn te ondersoek. Fluksiekontro- lekoëffisiënte is eksperimenteel bepaal deur selkulture te titreer met die on- omkeerbare glikolitiese inhibitors jodoasynsuur (IAA), ’n spesifieke GAPDH- inhibitor en 3-broompiruvaat (3-BrPA). Terwyl 3-BrPA hoofsaaklik as ’n HK- inhibitor gekenmerk word, het titrasies in heel selle en sitosoliese ekstrakte aan die lig gebring dat beide HK en GAPDH direkte teikens van 3-BrPA in MCF-7-selle is. Daarbenewens is ’n sellyn-spesifieke kinetiese model van glikolise gebruik om teoretiese voorspellings van fluksiekontrole en paddina- mika te verkry vir vergelyking met die eksperimentele resultate. Vir GAPDH het IAA-titrasies en model-analise op soortgelyke wyse glukose- en laktaat- fluksiekontrolekoëffisiënte naby nul opgelewer, wat aandui dat hierdie ensiem minimale kontrole oor glikolitiese fluksie uitoefen. Aangesien meer as 85% in- hibisie nodig was om ’n aansienlike afname in glikolitiese fluksie te veroorsaak, is GAPDH as ’n ongeskikte teiken vir nuwe teenkankermiddels beskou. Terwyl 3-BrPA GAPDH aktiwiteit merkbaar onderdruk het, was die effekte daarvan op pad fluksie en HK aktiwiteit nie groot genoeg om die individuele bydraes van elke geteikende ensiem tot die globale pad respons te bepaal nie, wat ver- dere berekening van die fluksiekontrolekoëffisiënt van HK uitsluit. Nietemin, modelanalise het aangedui dat HK hoë kontrole uitoefen oor glikolitiese fluksie in MCF-7 selle. Meer in die breë dra hierdie navorsing by tot ons begrip van die Warburg-effek en die onderskeiding van alternatiewe metaboliese teikens vir kliniese kankerbehandeling. Masters 2023-03-05T07:10:05Z 2023-08-30T13:07:46Z 2023-03 2023-08-31T09:18:40Z 2023-08-31T09:18:40Z Thesis https://scholar.sun.ac.za/handle/10019.1/128424 en_ZA Stellenbosch University application/pdf xiii, 92 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Breast -- Cancer Cancer cells -- Proliferation Glycolysis -- Inhibitors Breast -- Cancer -- Treatment -- Data processing Cell lines Breast -- Cancer -- Chemotherapy Cancer -- Molecular aspects UCTD Hollocks, Stephanie Anne Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title | Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title_full | Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title_fullStr | Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title_full_unstemmed | Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title_short | Metabolic control analysis using glycolytic inhibitor titrations in the MCF-7 breast cancer cell line |
| title_sort | metabolic control analysis using glycolytic inhibitor titrations in the mcf 7 breast cancer cell line |
| topic | Breast -- Cancer Cancer cells -- Proliferation Glycolysis -- Inhibitors Breast -- Cancer -- Treatment -- Data processing Cell lines Breast -- Cancer -- Chemotherapy Cancer -- Molecular aspects UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128424 |
| work_keys_str_mv | AT hollocksstephanieanne metaboliccontrolanalysisusingglycolyticinhibitortitrationsinthemcf7breastcancercellline |