Department of Physiological Sciences

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Browsing Department of 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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    Molecular regulation of autophagy and metastasis in breast cancer: new insights into the role of serum amyloid A
    (Stellenbosch : Stellenbosch University, 2022-04) Du Plessis, Manisha; Engelbrecht, Anna-Mart; Davis, Tanja; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Cytokines, growth factors and acute phase proteins present in the tumour microenvironment regulate inflammatory responses and alter crosstalk between various signalling pathways involved in the progression of cancer. It has recently been reported that serum amyloid A (SAA), an acute-phase protein, mainly produced by hepatocytes during an inflammatory response or infection, is also synthesized by cancer cells as well as other cells in the tumour microenvironment. SAA can activate several signalling pathways, including PI3K/Akt and MAPK signalling pathways, which are also known modulators of the intracellular degradation process, autophagy. However, no knowledge exists regarding the relationship between SAA and autophagy in breast cancer. Furthermore, it has been reported that SAA can promote the metastasis of cancer cells. Therefore, the aim of this study was to investigate the role of SAA in autophagy, metastasis and the activation of signalling pathways in in vitro and in vivo models of breast cancer. Methods: For the in vitro model the following cell lines were used, the triple-negative metastatic breast cancer cell line, MDA-MB-231, the oestrogen (ER+) and progesterone positive (PR+) epithelial-like cell line, MCF7, and the non-malignant breast epithelial cell line, MCF12A. These cell lines were transiently transfected with a control vector, pcDNA3, and an overexpression plasmid, pcDNA3-hSAA1, to overexpress SAA1. This study also investigated whether SAA1/2 is required for tumourigenesis in an in vivo tumour-bearing mouse model with double knockout of Saa1 and Saa2. We assessed autophagy, metastasis, proliferation, apoptosis and signalling pathway marker activation in these two models. Results: The overexpression of SAA1 in the MCF12A, MDA-MB-231 and MCF7 cell lines resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF12A and MCF7 cell lines. Furthermore, the overexpression of SAA in these cell lines resulted in the inhibition of autophagy, while the expression of the cargo recruiter, p62, was increased in the MCF7 cell line. SAA also promoted the migration of the MDA-MB- 231 and MCF7 cell lines, while no significant changes in the expression of the EMT markers were detected. The overexpression of SAA1 decreased the activation of the MAPK and PI3K signalling pathways in the MCF12A cell line, activated these pathways in the MDA-MB-231 cell line and inhibits Akt signalling in the MCF7 cell line. The overexpression of SAA1 resulted in a decrease in the colocalization of pERK and LC3-II in the MCF12A cell line and increased the colocalization of pERK and LC3-II in the MDA-MB-231 cell line. Furthermore, SAA1/2 knockout in vivo resulted in the induction of autophagy, while increasing the expression of p62. The knockout of SAA1/2 also promoted the resistance of these cancer cells to apoptosis, possibly through the regulation of autophagy. The knockout of SAA also inhibited the mesenchymal phenotype by downregulating the expression of vimentin. Lastly, the knockout of SAA1/2 resulted in the inhibition of the PI3K pathway protein, PKB/Akt, while increasing the activation of the MAPK, p38. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1β, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Conclusions: We have determined for the first time a novel role for SAA in autophagy in breast cancer cells. SAA overexpression inhibited autophagy in breast cancer cells. Additionally, SAA promotes migration and proliferation through the cell-type specific regulation of the PI3K/Akt and MAPK, ERK1/2 and p38, signalling pathways. Furthermore, the double knockout of SAA1/2 induced autophagy, promoted tumour cell survival, inhibited metastasis and regulates the activation of the PI3K/Akt and p38 signalling pathways. Double knockdown of SAA1/2 also resulted in an altered inflammatory profile in vivo. Our results therefore suggest that SAA plays an important role in breast cancer tumourigenesis.
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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.
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    The role of Serum Amyloid A in NLRP3 inflammasome signalling in breast and colon cancer
    (Stellenbosch : Stellenbosch University, 2022-04) Fourie, Carla; Engelbrecht, Anna-Mart; Davis, Tanja; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Cancer is a complex disease with multiple interactions targeting the organism on cellular, tissue and systemic levels. The main research focus for the past decades has been on the genome and on the molecular level where signaling pathways were dissected for the development of targeted therapies. However, in order to develop more efficient therapeutic regimes, a better understanding on systemic level is required. Over the past few years, the role of serum amyloid A (SAA) has gathered significant evidence which highlights its role in the pathogenesis of several cancers, including breast and colorectum carcinomas. To date, SAA has been shown to bind to several pattern recognition receptors, which might suggest that inflammasomes play a role in the tumour-promoting properties of SAA. Inflammasomes are cytoplasmic multiprotein complexes characterized by a sensor protein, an adaptor protein, and inflammatory caspases. However, the role of inflammasomes in cancer remain controversial. The aim of this study was therefore to investigate the role of SAA in inflammasome signaling in breast and colon cancer. Methods: In this 3-part animal study, tissues were subjected to immunoblotting, real-time PCR, haematoxylin and eosin staining and immunohistochemistry. For the first inflammatory model, wild-type and SAA double knockout C57BL/6 mice received 2.5% dextran sulfate sodium, which was administered for a total of 5 days. To assess tumourigenesis, colitis-associated cancer and triple negative breast cancer models were used, respectively. For colitis- associated cancer, wild-type and SAA double knockout C57BL/6 mice received an intraperitoneal injection of 12.5 mg/kg azoxymethane. After one week, dextran sulfate sodium treatment was administered at a concentration of 2.5% for a total of 5 days, followed by a recovery period of 16 days. Dextran sulfate sodium treatment was administered for a total of 3 cycles. Triple negative breast tumours were established in wild-type and SAA double knockout C57BL/6 mice by injecting EO771 cells subcutaneously at the fourth mammary fat pad. The experimental endpoint was reached when tumour volumes reached 300-400 mm3. Results: In this study we have showed that in an in vivo model of dextran sulfate sodium induced colitis, SAA ablation exerted pro-inflammatory properties independent of the NLRP3 inflammasome. The ablation of serum amyloid A1/2 was associated with the increased expression of pro- inflammatory cytokines. In contrast, in an in vivo colitis-associated cancer and in a triple negative breast cancer model, the ablation of SAA suppressed canonical NLRP3 inflammasome activation, which was associated with anti-inflammatory properties. These findings suggest that during tumourigenesis, SAA functions as an endogenous damage associated molecular pattern in the tumour microenvironment. Conclusion: Here we show for the first time, in models of CAC and TNBC, the novel role of SAA in the activation of the NLRP3 inflammasome and the generation of pro-inflammatory cytokines, two mechanisms known to promote tumour development and metastasis. This study emphasizes the notion that the tumour-induced systemic environment acts as a critical regulator of cancer progression and metastasis. In conclusion, simultaneously targeting SAA and NLRP3 components could be beneficial for cancer treatments.