Browsing by Author "Mitchell, Megan Irvette"

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    Circadian rhythms as novel chemotherapeutic strategies for breast cancer
    (Stellenbosch : Stellenbosch University, 2014-12) Mitchell, Megan Irvette; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition.
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    Metabolic reprograming and cancer resistance : an investigation into the metabolic control of cancer-associated fibroblasts on breast cancer cell survival and metastasis
    (Stellenbosch : Stellenbosch University, 2018-03) Mitchell, Megan Irvette; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Cancer-associated fibroblasts (CAFs) constitute the most abundant mesenchymal cell type present within the tumour microenvironment. Recent evidence suggests that nutrient deprived cancer cells survive as a result of their ability to undergo extensive metabolic reprogramming exploiting the metabolic capacities of surrounding CAFs. Additionally, it has been proposed that CAFs also play a role in enhancing tumourigenicity and the metastatic capability of cancer cells. However, the mechanisms underlying the interactions between epithelial cancer cells and surrounding stromal fibroblasts remain to be elucidated. We therefore hypothesize, that nutrient deprived breast cancer epithelial cells could influence cancer-associated fibroblasts (CAF’s), to produce metabolites which may be utilized by cancer cells for survival, chemo-resistance and enhanced migration. Methods: E0771 cancer cells were subjected to glucose starvation after which cell viability, oxidative stress analysis and cell death was assessed. E0771 conditioned media was then generated and proteomic analysis on conditioned media was performed. This media was also used to treat mouse embryonic fibroblast (MEF) cells. The activation of a CAF phenotype was assessed by means of Western blotting and confocal microscopy. Furthermore, cell viability assays, oxidative stress, glucose uptake and GLUT4 translocation were assessed. MEF conditioned media was then generated and again proteomic analyses were performed. MEF conditioned media was then used to treat glucose deprived E0771 cells. Where after cell viability, cell death and migration were assessed. The effects of CAFs on chemotherapy resistance and metastasis was assessed by treating E0771 cells with doxorubicin and MEF conditioned media, following which, cell viability, apoptosis and migration assays were performed. An in vivo tumour bearing mouse model was established using female C57/BL6 mice treated with doxorubicin. Primary epithelial organoids were isolated from tumours and a 3D branching morphogenesis assay was performed. Results: 12 hours of glucose deprivation resulted in no significant changes in mitochondrial reductive capacity or markers of apoptosis, however, a significant increase in mitochondrial oxidative stress was observed. Proteomic analysis of glucose deprived E0771 conditioned media revealed an increase in proteins associated with exosome-like vesicles and an increased clustering of proteins involved in epithelial-to-mesenchymal transition and glucose metabolism. 2-NBDG glucose uptake was significantly increased in conjunction with an increase in the fluorescent intensity of the HA-GLUT4-GFP construct following exposure to E0771 conditioned media, indicating the increase in glucose uptake is in part mediated by GLUT4 translocation. Furthermore the treatment of E0771s with MEF conditioned media lead to a significant increase the speed of migration and EMT. Furthermore, increased invasiveness of epithelial organoids was observed following exposure to MEF-CM in Dox treated animals, with an increase in a more epithelial-like phenotype. Conclusion: Our data suggest that glucose deprivation induces a state of oxidative stress in the E0771 cells which is transferred to MEFs leading to the “activation” of a CAF-like phenotype, and that this “activated” phenotype contributes significantly to the pro-survival and pro-metastatic abilities of breast cancer cells. Furthermore, our results contribute significantly to the understanding of the molecular mechanisms underlying the interaction between epithelial cancer cells and fibroblasts within the tumour microenvironment.