Masters Degrees (Physiological Sciences)

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Browsing Masters Degrees (Physiological Sciences) by browse.metadata.advisor "Davis, Tanja"

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    The cellular response of triple-negative breast cancer to short-term starvation: implications for chemosensitivity
    (Stellenbosch : Stellenbosch University, 2021-03) Prangley, Charne; Engelbrecht, Anna-Mart; Davis, Tanja; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Breast cancer is currently the most common cancer among women globally. Triplenegative breast cancer (TNBC) is an aggressive and often drug-resistant sub-type of breast cancer that is correlated with poor patient outcomes. As a result, adjuvant therapies that may improve drug sensitivity are currently being sought. Due to the unique metabolic hallmarks of cancer, metabolic adjuvant therapies have become an area of increasing interest. We therefore set out to investigate the effect of short-term starvation (STS) on the growth, viability, and metabolism of TNBC cells and a benign breast epithelial cell line. We also investigated the effect of STS on chemotherapyinduced cytotoxicity in these cells to determine whether STS may enhance the effect of doxorubicin in TNBC. Methods: Three cell lines were utilised for this study: a benign breast epithelial cell line (MCF- 12A), and two triple-negative breast cancer cell lines (BT-549 and MDA-MB-231). Western blotting was employed to determine the effect of starvation over time on growth and proliferation signalling pathways (PI3K/Akt) and markers of autophagy (Atg5, p62 and LC3-II). Immunocytochemistry was utilised to quantify autophagic puncta. Cell cycle progression and viability were assessed using flow cytometry and a WST1 assay, respectively. The effect of STS on chemosensitivity was then established by incubating cells in standard or starvation-mimicking media for 24 hours, whereafter they received doxorubicin at a concentration of 2.5 μM. Chemosensitivity was then established in terms of live cell number, cell death and viability, and cell cycle progression. Results and Discussion: In response to STS, the MCF-12A cells downregulated pro-growth signalling pathways, while the MDA-MB-231 cells showed significant upregulation. A 24-hour starvation period had no significant effects on these pathways or on autophagic flux in the BT-549 cells. Both the MCF-12A and MDA-MB-231 cell lines significantly upregulated autophagic flux in response to STS, with the latter achieving the most significant effect at 24-hours. This may have offered protection to these cells, as a period of starvation prior to drug administration reduced doxorubicin-induced G2/M arrest. Additionally, STS had no other significant effects on chemosensitivity in these cells. In the BT-549 cells, however, starvation was able to significantly increase the percentage of dead cells in the group that received STS prior to doxorubicin treatment. As autophagy was not significantly increased in this cell line during starvation, this suggests that autophagy may indeed play a role in drug resistance. Conclusion: In summary, the cell lines which displayed an upregulation of autophagy at 24 hours of starvation were not sensitised to doxorubicin in terms of cell death, and also experienced amelioration of doxorubicin-induced G2/M arrest. This supports the notion that autophagic upregulation may protect cancer cells from doxorubicin-induced cytotoxicity and contribute to drug resistance. However, to gain a more thorough understanding of this phenomenon, future studies investigating the mechanisms by which autophagy promotes chemoprotection are recommended.
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    Dynamic interactions between skeletal muscle and breast cancer cells following chemotherapeutic treatment
    (Stellenbosch : Stellenbosch University, 2019-04) Conradie, Daleen; Engelbrecht, Anna-Mart; Isaacs, Ashwin; Davis, Tanja; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Background: Breast cancer is the most common cancer found among women of South Africa with the prominent effective form of treatment being chemotherapy. Many cancer patients receiving chemotherapeutic treatment experience skeletal muscle wasting, however, the contribution of muscle wasting to the metastatic properties of breast cancer and response to current treatment strategies has not yet been fully investigated. The aims of this study were to investigate the reciprocal interactions between mouse breast cancer cells (E0771) and mouse myotubes (C2C12) as well as the effects of doxorubicin (DXR) on these interactions. Methods: Conditioned media was collected from two separate cycles. The initial cycle of conditioned media was collected from E0771 breast cancer cells after treatment with/without 1.6 μM of DXR. Myotubes were then treated with/without DXR as well as conditioned media collected during the initial cycle from the E0771 cells. A new series of E0771 cells were then treated with/without DXR as well as with the second cycle of conditioned media collected from the myotubes. Mitochondrial integrity of myotubes was investigated using MitoSOX™ stain analysis while myotube cell viability and integrity was assessed using a Cell Tracker™ stain analysis. Cell viability of E0771 cells was assessed with an MTT assay and the migratory properties (wound closure) using a migration scratch assay. Western blot analyses were used to determined alterations in proliferation, apoptotic, and epithelial-mesenchymal transition (EMT) signaling pathways. Results: Treatment of myotubes with 1.6 μM of DXR significantly induced mitochondrial ROS production (5.580 ± 0.4, p<0.001) when compared to Control but myotube integrity was maintained. Treatment of E0771 cells with 1.6 μM of DXR compared to Control significantly decreased cell viability (60.354% ± 1.237, p<0.001), significantly increased the phosphor/total ERK expression ratio (3.946 ± 0.520, p<0.001), and significantly decreased the cleaved/total PARP expression ratio (0.651 ± 0.027, p<0.001). Additionally, a significant increase in the percentage of wound closure was also observed in the DXR group (16.049% ± 1.11, p<0.01) compared to Control after 24-hours. E0771 cells treated with myotube conditioned media after treatment of DXR (C.DXR), induced a significant decrease in expression of the cleaved/total PARP ratio (0.662 ± 0.097, p<0.01) as well as a significant difference in percentage of wound closure (17.19 ± 0.758, p<0.001) compared to C.Control. Following treatment of the E0771 cells with myotube conditioned media, harvested after the treatment of conditioned media from DXR treated E0771 cells (C.C.DXR), a significant increase in cell viability (121.743% ± 3.442, p<0.05) when compared to C.C.Control. Additionally, comparison of C.C.DXR to C.C.Control observed a significant decrease in expression of total Akt (65.554% ± 17.55, p<0.05), MCM2 (55.167% ± 14.64, p<0.05), and the cleaved/total PARP ratio (0.456 ± 0.111, p<0.001) was observed. Conclusion: Investigation of the dynamic interactions between myotubes and breast cancer cells revealed novel evidence of the influence of the myotube environments on cancer progression. Our study also revealed novel evidence that this myotube environment significantly affected the response of breast cancer cells to the chemotherapeutic treatment of DXR. These findings identified new mechanisms that may promote breast cancer metastasis, which can be utilized to improve chemotherapy in cancer patients.
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    The paracrine effects of fibroblasts on Doxorubicin-treated breast cancer cells
    (Stellenbosch : Stellenbosch University, 2019-04) Fourie, Carla; Engelbrecht, Anna-Mart; Davis, Tanja; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Breast cancer is frequently diagnosed in women in both developed and developing countries and poses a major health problem throughout the world. The current standard treatment for breast cancer patients is radiation, surgery and chemotherapy or a combination of surgery with chemotherapy. The unresponsiveness of cancer cells to chemotherapeutics, however, is still a main concern. During chemotherapeutic treatment with Doxorubicin, normal and healthy neighbouring cells are also damaged. Apoptotic or senescent fibroblasts in the tumour microenvironment can then secrete a variety of bioactive molecules which promote tumour growth, metastasis and drug resistance. Methods: Mouse embryonic fibroblasts (MEFs) were cultured and treated with Doxorubicin to induce apoptosis and senescence respectively. An SA-ß-gal stain was used to determine the number of senescent cells in the cell population and expression of apoptotic and senescent markers were determined through western blotting. Conditioned media was collected from the MEFs after apoptosis and senescence induction and used to assess the paracrine effects between fibroblasts and E0771 cells. Results and discussion: Doxorubicin (1 μM) was able to significantly induce apoptosis in MEFs after 24 hours. During senescence induction, 2 μM of Doxorubicin treatment for 4 hours was unable to induce 80% of senescence in the MEF population. The western blot analyses show that the expression of many apoptosis and senescence markers significantly increased or decreased after Doxorubicin treatment. Furthermore, the results indicate that senescent fibroblasts (56%) were able to significantly increase cell viability in E0771 cells following treatment with Doxorubicin. Conclusion: Our results highlight the fact that the tumour microenvironment is extremely complex and how important it is that chemotherapeutic agents such as Doxorubicin should specifically target cancer cells. Once healthy, neighbouring stromal cells such as fibroblasts are affected by chemotherapeutic agents, they have the ability to secrete paracrine factors that enhance breast cancer growth and induce therapeutic resistance by evading cell death.