Improving breast cancer therapy through oestrone analogue and glycolysis inhibitor synergism

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dc.contributor.advisor Cromarty, Allan Duncan
dc.contributor.coadvisor Joubert, Annie M.
dc.contributor.coadvisor Van Tonder, Alet
dc.contributor.postgraduate Anderson, Roxette Dianne
dc.date.accessioned 2017-11-07T07:45:03Z
dc.date.available 2017-11-07T07:45:03Z
dc.date.created 2017
dc.date.issued 2017
dc.description Dissertation (MSc)--University of Pretoria, 2017. en_ZA
dc.description.abstract Introduction: In South Africa, breast cancer has the highest prevalence with a life time risk of 1 in every 9 women being diagnosed annually. There are four sub-types of breast cancer and according to the stage of the cancer, various treatment regimens are prescribed. A major obstacle is that majority of cancers have developed multi-drug resistance and new treatment regimens need to be developed in order to obtain therapeutic efficacy. Cancer cells use aerobic glycolytic metabolism for energy generation and inhibition of this pathway increases sensitivity of the cells to anti-neoplasic treatments. 2-Deoxyglucose (2-DG) competes with and inhibits glucose uptake inhibiting the glycolytic pathway which can result in depolarisation of the mitochondrial membrane potential releasing cytochrome c. Two 2-Methoxyestradiol (2-ME) derivatives, ESE- 15-ol and ESE-16 have shown to be promising anti-cancer agents and combination therapy could allow the use of these compounds with a decreased side effect profile. The combination of these compounds with 2-DG was therefore investigated. Aim: To investigate combinations of two oestrone analogues and the glycolysis inhibitor 2- deoxyglucose for potential synergistic effects using a cell enumeration assay, mitochondrial membrane potential and cell cycle analysis, on breast cancer cells in an in vitro setting. Cell apoptosis, necrosis and autophagy pathways were assessed to indicate the mechanism of cytotoxicity. Methods: The breast cancer MCF-7 and non-tumorigenic MCF-12A cell line were used. Cells were exposed to ESE-15-ol, ESE-16 and 2-DG alone and in combination. Mechanistic studies were performed using the various research methodologies including the sulforhodamine B assay for cell enumeration, Annexin-V FITC and propidium iodide labeling for apoptosis/necrosis studies, PlasDIC and light microscopy for morphological analysis, propidium iodide staining for cell cycle progression, JC-1 for mitochondrial membrane potential studies, transmission electron microscopy and western blotting for the analysis of autophagy. Results: A GI50 of 34.1 nM was reported for MCF-7 cells after treatment with ESE-15-ol, 141 nM for ESE-16 and 1.3 mM 2-DG. The GI50 of ESE-15-ol treated MCF-12A cells was 141 nM, 140.1 nM for ESE-16 treated cells and 1.7 mM for 2-DG. ESE-16 had the greatest effect on cell viability in MCF-7 cells and a shift from an inhibitory effect to the initiation of cell death was evident after treatment of 100 nM of ESE-15-ol and ESE-16. 2-DG had a lower cytotoxic effect than the oestrone analogues. The MCF-12A cell line was less susceptible to the experimental compounds. The combination of the oestrone analogues with 2-DG elicited a greater effect on cell enumeration than each of the compounds alone with a less pronounced effect on the MCF- 12A cell line in comparison to the MCF-7 cells. The experimental compounds initiated apoptosis with ESE-16 eliciting a greater effect than ESE-15-ol. The combination of the oestrone analogues with 2-DG resulted in increased apoptosis in contrast to the compounds alone. ESE-16 alone and in combination with 2-DG lead to the most prominent morphological changes, with ESE-15-ol decreasing cell density slightly. The combination of ESE-15-ol with 2-DG decreased cell density with membrane blebbing apparent. The MCF-12A cell line was less susceptible to morphological changes after treatment of ESE-15-ol with 2-DG however ESE-16 and the combination with 2- DG resulted in similar attributes seen in MCF-7 treated cells. ESE-15-ol resulted in accumulation of cells in the G2 cell cycle phase which was further amplified after the combination of 2-DG. A sub-G1 accumulation was observed after treatment with ESE-16 with a shift to a G2 accumulation after the combined treatment of ESE-16 with 2-DG. After 48 hours, ESE-15-ol alone and in combination with 2-DG on MCF-7 cells resulted in depolarisation of the mitochondrial membrane. A slight decrease in the membrane potential was observed after treatment with ESE-16 and this was further increased after the combined treatment of ESE-16 with 2-DG. The MCF-12A were less susceptible after 24 hour treatment than 48 hour exposure of the experimental compounds. The presence of autophagic-like vacuoles were apparent in all treatment groups as well as the increased expression of LC3-II. Conclusion: The combined treatment of synthetic oestrone analogues with 2-DG displayed greater therapeutic efficacy than each of the compounds alone. As a result, the apoptotic and autophagic pathways were induced and a shift in cell cycle progression was observed. Mitochondrial involvement was apparent and the compounds significantly affected cell viability. This suggests that the combinations between the antimitotic oestrone analogues and glycolysis inhibitor 2-DG act synergistically to induce apoptosis and autophagy in MCF-7 breast cancer cells. en_ZA
dc.description.availability Unrestricted en_ZA
dc.description.degree MSc en_ZA
dc.description.department Pharmacology en_ZA
dc.identifier.citation Anderson, RD 2017, Improving breast cancer therapy through oestrone analogue and glycolysis inhibitor synergism, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/63050> en_ZA
dc.identifier.uri http://hdl.handle.net/2263/63050
dc.language.iso en en_ZA
dc.publisher University of Pretoria
dc.rights © 2017 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.
dc.subject Breast cancer en_ZA
dc.subject Combination therapy en_ZA
dc.subject Glycolysis inhibitors en_ZA
dc.subject Oestrone analogues en_ZA
dc.subject Mechanisms en_ZA
dc.subject Synergism en_ZA
dc.subject Cell death en_ZA
dc.subject In vitro en_ZA
dc.subject MCF-7 en_ZA
dc.subject MCF-12A en_ZA
dc.title Improving breast cancer therapy through oestrone analogue and glycolysis inhibitor synergism en_ZA
dc.type Dissertation en_ZA


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