2-Methoxyestradiol, an endogenous metabolite of 17-β-estradiol exerts antipropliferative, antiangiogenic and antitumor effects in vitro and in vivo and is currently in clinical trials phase II for various types of cancers including breast cancer. Due to low oral bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-Methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE), a novel bissulphamoylated derivative of 2-methoxyestradiol exerts in vitro antipropliferative effects. Although 2-MeOE2bisMATE holds therapeutic potential as an anticancer agent, several questions remain regarding the signal transduction and exact mechanism of action used by 2-MeOE2bisMATE. In vitro effects of 2-MeOE2bisMATE were investigated in a breast adenocarcinoma cell line (MCF-7) and a non-tumorigenic epithelial breast cell line (MCF-12A) by analysing its influence on cell growth, cytotoxicity, morphology, cell cycle progression, mitochondrial membrane potential, reactive oxygen species production and induction of apoptosis and autophagy. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 47% in MCF-7 cells and to 79% in MCF-12A cells after 48h of exposure. Haematoxylin and eosin staining revealed several 2-MeOE2bisMATE-treated cells with the presence of apoptotic bodies. Transmission electron microscopy demonstrated membrane blebbing, nuclear fragmentation and chromatin condensation indicating the occurrence of apoptosis. Increased lysosomal staining was revealed by fluorescent microscopy using propidium iodide, Hoechst 33342 and acridine orange; suggesting cell death via autophagy. Data obtained employing flow cytometry using rabbit polyclonal anti-LC3B conjugated to DyLight 488 verified the induction of autophagy in 2-MeOE2bisMATE-treated cells. In addition, cell cycle progression revealed an apoptotic sub-G1 peak, confirming the induction of apoptosis by 2-MeOE2bisMATE. Reactive oxygen species generation increased when cells were exposed to 2-MeOE2bisMATE. Annexin V-FITC and the investigation of a possible reduction in the mitochondrial membrane potential verified induction of apoptosis by 2-MeOE2bisMATE. All of the above-mentioned results were observed more prominently in the tumorigenic MCF-7 cell line when compared to the non-tumorigenic MCF-12A cell line. Data obtained from this in vitro study contributes to the embedded scientific knowledge regarding the signaling transduction mechanism exerted by 2-MeOE2bisMATE. / Dissertation (MSc)--University of Pretoria, 2011. / Physiology / unrestricted

Mastitis is often bacterial in origin. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), endotoxins from gram-positive and gram-negative bacteria, respectively, are potent inducers of mammary gland inflammation. Inflammation can alter expression of transporters responsible for transport of substrates important in synthesis of milk constituents and cellular metabolic energy. Since, gram-positive and gram-negative bacterial infections cause a different clinical course of mastitis, I investigated whether LTA and LPS differentially alter proton-coupled (MCT1) and sodium-coupled monocarboxylate transporter (SMCT1, SMCT2) expression and functional outcomes of altered expression. Human mammary epithelial cells (MCF-12A) were incubated with 1 microgram/mL LPS or LTA for 6, 12 and 24 hours and mRNA expression of TNF-alpha, IL-1β, IL-6, MCT1, SMCT1, and SMCT2 were measured using Quantitative RT-PCR. LPS decreased SMCT1, but increased SMCT2 expression after 6 h, while LTA increased MCT1 expression at 6 h, followed by gradual decrease in expression until 24 h. To know whether such differential changes in transporter expression by LPS and LTA could cause changes in cellular energy production, I quantified creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) and oxygen consumption rates using HPLC and Hansatech oxygen electrode, respectively. At 12 h, LPS increased concentrations of Cr, CrP, ATP and ADP, whereas LTA caused changes in CrP and ADP concentrations relative to control. Both LPS and LTA decreased oxygen consumption rates after 12 h. Furthermore, to know whether changes in transporter expression lead to differences in substrate availability, I performed uptake studies for carnitine using radiolabelled tritium L-carnitine. LPS and LTA challenge did not affect the affinity, but caused a 2-3-fold increase in maximal activity (Vmax) of carnitine transport. Although increases in Vmax were not significant, the increase in Vmax after 12 h exposure by LPS and LTA corresponds to changes in mRNA expression of the OCTN2 transporter (previously reported in the laboratory). In conclusion, LPS and LTA differentially alter mRNA expression of transporters, which leads to changes in cellular energy levels and oxygen consumption rates and possibly to changes in the functional activity of transporters. Whether such differences contribute to the different clinical course of mastitis warrants further investigation.

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. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Purpose: Breast cancer is one of the most frequent and fatal diseases women all around the globe are challenged with today. In women, breast cancer has the highest mortality rate of all cancers and the incidence rate is on the increase. It is estimated that by the year 2025, 19.3 million women will become a victim of this grave health problem. This disease is a result of the formation of malignant tumours caused by genetic alterations that are involved in the proliferation of cells, cellular differentiation and the disturbance in homeostasis which subsequently leads to the abnormal multiplication and growth of cells. Breast cancer is considered a multifactorial disease with various risk factors such as age, radiation exposure, hormone therapy, oral contraceptives, dietary factors, environmental exposure and genetic predispositions. Breast cancers can be subdivided and classified based on their cellular surface receptors such as Estrogen Receptors, Progesterone Receptors and Human Epidermal Growth Factor Receptor 2. Of the various subtypes, the triple-negative breast cancer subtype which is negative for all 3 surface receptors and presents as the most aggressive form of breast cancer with a poor prognosis. Between 10-20% of all breast cancer cases are classified as triple-negative breast cancer. Due to the hormonal status of triple-negative breast cancer, treatment options are limited and thus of great concern. Chemotherapy remains the most common treatment modality, but prognosis is poor with relapse within years ultimately leading to poor survival outcome. Due to this lack of effective treatment plans, an alternative treatment with minimal side effects and better survival remains an imperative area to explore. A wide scope of literature highlights red palm oil and its health benefits, with its growth inhibitory potential drawing great attention. Red palm oil, extracted from the Elaeis guineensis palm tree is red in colour due to the abundance of carotenoids, tocotrienols and tocopherols found in the oil. Various compounds make up the oil such as lycopene, carotenes, vitamin E and coenzyme Q10. Most studies have researched the effects of vitamin E extracted from the oil as a contributor to its growth inhibitory activity. This study focuses on the effects of the commercial red palm oil as a whole with all its compounds on the proliferation of breast cancer cells as well as the effect it has on various genes associated with breast cancer. Method: This study investigated the effect of red palm oil concentrations (1, 10, 100, 500 and 1000 μg/ml) on breast cancer cells—MCF-7 and MDA-MB-231 with comparison to a non-cancerous cell line—MCF-12A for 24-, 48- and 72-hour treatment periods. The parameter investigated was cell proliferation through the CCK-8 cell proliferation assay and the morphology following red palm oil treatment was observed and captured. Additionally, this study also investigated the effect of red palm oil on the expression of Human Mammaglobin (hMAM) and Maspin genes through the PCR assay and results visualised through agarose gel electrophoresis. Data was statistically analysed using GraphPad version 6.0 software. Results: Following treatment of red palm oil, no apparent changes in the cell morphology was observed despite using variable treatment concentrations over variable times for MCF-7, MDA-MB-231 and MCF-12A cells relative to their respective controls. Immortalised MCF-12A cells showed a significant increase in proliferation with the varying treatment concentrations, but more prominently with the highest concentration at 24, 48 and 72 hours. MCF-7 cells showed significant decreases at 24 and 72 hours. Decreased proliferation was observed at all dosages used, particularly at 10, 100, and 500 μg/ml. Furthermore, MDA-MB-231 cells demonstrated a gradual increase in cell proliferation for the 3 selected time periods in the varying concentrations. Additionally, red palm oil did not alter the gene expression of Maspin at any of the varying treatments for MDA-MB-231 nor MCF-7 cells. However, changes in hMAM gene expression were observed at treatment concentration of 100 μg/ml in MDA-MB-231 cells that were incubated for 24 and 48 hours. However, the hMAM expression was not affected in treated MCF-7 cells. Conclusion: Red palm oil, as an alternative dietary oil, seems to have potential growth inhibitory properties as demonstrated by the change in the cell proliferation of the MCF-7 cells. Literature show that various individual compounds extracted from red palm oil have anti-proliferative and inhibitory effects on breast cancer cells making them good candidates for therapy. However, this study concludes that red palm oil as a whole component would not be a suitable therapeutic agent for highly aggressive triple-negative breast cancer.