Doctoral Degrees (Physiological Sciences)
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Browsing Doctoral Degrees (Physiological Sciences) by browse.metadata.advisor "Nell, Theo A."
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- ItemChemoresistance in a breast cancer animal model: the role of obesity and inflammation(Stellenbosch : Stellenbosch University, 2019-12) Mentoor, Ilze; Nell, Theo A.; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: Globally an increase in obesity incidence represent a major health concern amongst a rising incidence of impaired treatment outcomes of breast cancer patients. Adipose tissue and/or adipocytes, in the tumour microenvironment serve as an exogenous energy source for the survival of breast cancer cells, especially since adipose tissue is abundant in breast tissue. Breast cancer cells modulate lipid metabolism (de novo fatty acid synthesis and lipolysis), by altering the secretion of adipokines through adipocytes, resulting in the release of free fatty acids to provide energy substrates for breast cancer cells to sustain its high proliferation demand for survival. Evidence on the role of obesity and lipid metabolism especially fatty acids in breast cancer treatment resistance is lacking. This motivates investigation to identify and understand the underlying physiological and molecular mechanisms by which chemotherapeutic treatment resistance is achieved. We therefore hypothesise that obesity-induced inflammation alters lipid metabolism in adipose tissue/adipocytes and contribute to the development of doxorubicin chemotherapeutic treatment resistance in breast cancer cells. Methods: A diet induced obesity animal model was established by feeding female C57BL6 mice a high fat diet for 12 weeks. After developing the diet induced obesity phenotype, breast tumour xenographs were induced by subcutaneous inoculation in the fourth mammary gland with E0771 triple negative breast cancer cells.Once tumours became palpable, mice received either vehicle treatment (Hanks balance salt solution) or doxorubicin treatment (cumulative dose of 12 mg/kg). Plasma inflammatory markers, fatty acid profiles and protein expression of lipid metabolism markers (de novo fatty acid synthesis and lipolysis) was determined in mammary adipose and tumour tissue. To validate the in vivo model findings, we developed an in vitro model using a conditioned media approach. A human adipose tissue derived stem cell line was used for the differentiation of mature adipocytes after which conditioned media was collected to assess the paracrine effect between adipocytes and doxorubicin treated MDA-MB-231 triple negative breast cancer cells. Cell viability was assessed with WST-1 assays. Western blots were used to determined alterations in protein expression of apoptotic and lipid metabolism markers (de novo fatty acid synthesis and lipolysis). An Inflammatory marker as well as free fatty acid profile was also analysed in treatment conditioned media. Results: Diet induced obesity significantly increased tumour growth and decreased doxorubicin treatment efficacy in E0771 triple negative breast tumours (p<0.0001), resulting in treatment resistance. Our findings also showed that diet induced obesity supressed de novo fatty acid synthesis (decreased SCD-1) and lipolysis (decreased HSL) in mammary adipose tissue of doxorubicin treated mice. Conversely an increase in de novo fatty acid synthesis (increased SCD-1) and lipolysis (increased ATGL) was found in tumour tissue, leading to significant changes in FAs composition of both tissues. Diet induced obesity also significantly increased plasma leptin (p=0.025) and resistin levels (p=0.046) and increased NFĸB protein expression in mammary fat of doxorubicin treated mice, thereby inducing systemic and local inflammation. Furthermore, we also report that adipocytes promoted acquired breast cancer treatment resistance by significantly increasing the cell viability of doxorubicin treated MDA-MB-231 triple negative breast cancer cells (Dox+CM vs Dox, p=<0.0001). This was achieved by attenuating doxorubicin’s efficacy to induce apoptosis (decreased cleaved-caspase-3, p<0.05), in a paracrine manner. Adipocytes also induced inflammation (increased leptin and MCP-1) as well as lipolysis (increased HSL) in doxorubicin treated breast cancer cells (Dox vs Dox+CM p=0.03), thereby altering the free fatty acid profile of breast cancer cells. Conclusion: Our data suggest that adipose tissue/adipocytes significantly contribute to treatment resistance in triple negative breast cancer cells. We have demonstrated in both in vivo and in vitro models that adipose tissue/adipocytes secretory factors induce inflammation in the breast tumour microenvironment, which leads to the induction of lipolysis in triple negative breast cancer cells. This resulted in altered metabolic behaviour i.e. increased free fatty acid utilization, which can be utilized as energy substrates or induce lipid saturation in order confer to acquired treatment resistance by evading apoptosis We propose that this could be a novel mechanism by which adipose tissue/adipocytes within the tumour microenvironment can contribute to the development of breast cancer treatment resistance under obesogenic conditions. This study also significantly contributed to the identification and understanding of molecular mechanisms underlying breast cancer treatment resistance in obese patients.
- ItemAn evaluation of the hepatic proteomic signature in identifying cancer tolerance and resistance mechanisms in a mouse allograft system(Stellenbosch : Stellenbosch University, 2017-03) Van Niekerk, Gustav; Engelbrecht, Anna-Mart; Loos, Benjamin; Nell, Theo A.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background The unfavourable therapeutic index of most treatment modalities has greatly impeded progress in the development of effective cancer therapy. Therefore a need exists for treatment modalities that are less taxing on a patient’s health status (i.e. maintain a patients reserve capacity and thus prolong survival) while additionally not invoking counter evolutionary strategies from tumour cells. Plant biologist have long distinguished between the host’s ability to accommodate pathogen burden, as oppose to its ability to antagonise pathogen load. Hence, the disease trajectory is not only dependent on the hosts’ ability to resist an infection, but also on the capacity to tolerate pathogen burden. This distinction have only recently been applied to animals. A number of observations suggest that this distinction may be of great immunological relevance, including the prevalence of asymptomatic carriers and natural variation in the population with regards to disease progression. Thus, the tolerance/resistance (T/R) paradigm represents a novel approach for understanding disease progression. We hypothesise that similar mechanisms might underlie host-tumour dynamics. Study aims and experimental design The current study aimed to evaluate the application of the T/R framework within an oncological context. A syngeneic mice model system was used to compare tolerance and resistance between two cancer cell lines. C57BL/6 mice were inoculated with either mammary carcinoma cells (EO771) or melanoma cells (B16). In a clinical setting, health status would not only be influenced by tumour load, but also by therapeutic interventions such as cytotoxic therapies, which must also be tolerated. Thus, a second study was performed using chemotherapeutic regimes as a variable to explore the effect of high (5 mg/kg) and low (2 mg/kg) dose doxorubicin (DXR) treatment on tolerance and resistance in mice. In addition, attempts were made to identify mechanisms underlying differences between groups with regards to variation in tolerance and resistance. To this end, a combination of immunoblotting and proteomic analyses were performed. Methodology: quantifying tolerance and resistance Resistance was quantified as the slope of a regression line, with tumour volume as response variable, and time as independent variable. Tolerance was measured similarly, but with body weight as response variable and tumour load as independent variable. Differences in regression slopes was used to compare tolerance and resistance. To confirm tolerance, differences in gastrocnemius muscle cross-sectional area (MCA) were compared between groups. Results Mice inoculated with melanoma (B16) cells showed a significantly lower resistance compared to mice inoculated with breast cancer EO771 cells. With regards to tolerance, B16 cells also exhibited lower tolerance, though tests for homogeneity of regression slopes demonstrated that these differences did not reach significance (p = 0.0856). Similarly, B16 and EO771 groups did not exhibit any difference in MCA. Comparing the effect of high and low dose DXR on mice bearing EO771 revealed that DXR decreases resistance: both low dose and higher dose DXR increased tumour growth as demonstrated by significantly steeper slopes in DXR groups compared to the tumour control group. In order to explain the increase in EO771 tumour growth in mice receiving DXR, the activation of a panel of signalling proteins associated with cell growth and survival (cRaf, ERK, p38 MAPK, JNK, PTEN, PI3Kp85, PDK1, Akt, mTOR, Bcl-2) as well as apoptotic markers (Caspase 3, 8 and 9) in tumour samples were evaluated by western blot analyses. However, the only significant finding include elevated ERK activation in mice receiving DXR, suggesting that extracellular signalling molecules might drive tumour growth. Since the liver plays a critical role in energy homeostasis, as well as in the production and clearance of circulating factors, western blot analyses were performed on liver samples. Markers of autophagy (p62 and LC3B-II) as well as growth signalling proteins (Akt and mTOR) and apoptosis (Caspase 3) were evaluated by western blot analyses. Mice inoculated with B16 demonstrated a marked increase in both p62 and LC3B-II, signifying an increase in autophagosome pool size, most likely due to dysfunctional lysosomal fusion. Surprisingly, other makers in both EO771 and B16 did not significantly differ from control liver samples. Subsequently, liver proteomics were performed making use of a Gene Ontology approach in order to describe biological, functional, structural and other processes that are uniquely altered between groups. Interestingly, a comparison between livers of mice inoculated with B16 melanoma cells and EO771 breast cancer cells also suggested that autophagic activity was not upregulated compared to the control group. DXR groups also did not exhibit differences in autophagic processes, though proteins involved in the proteasomal pathway were upregulated in mice receiving high doses of DXR. An increase expression of enzymes associated with retinoic acid metabolism was observed in the B16 group, which might explain decrease tolerance and resistance in this group. An increase in steroid metabolism was also observed in mice receiving DXR. Since cholesterol form a key component of cell membranes, it is possible that cholesterol synthesis might enable rapidly growing tumours of mice receiving DXR. Finally, concurrent up- and downregulation of certain proteins involved in radical scavenging in DXR mice might suggest a differential free radical scavenging response, thus explaining why anti-oxidant therapies have not proven successful in clinical settings in response to DXR. Collectively, these observations highlight alteration in hepatic activities through which tolerance and resistance mechanism might manifest. In summary, this study have demonstrated the implementation of the T/R framework within an oncological setting. Evidence suggest that defects in hepatic autophagy might contribute to lower tolerance, and possibly also resistance. Autophagy was not significantly upregulated in response to DXR which was associated with lower tolerance. Similar, mice inoculated with B16 tumours exhibited lower tolerance as well as evidence for suppressed lysosomal fusion with autophagosome. These observations suggest that a compromised autophagic apparatus might contribute towards the lower tolerance. Proteomic results are also suggestive of a potential role played by altered liver metabolism, including retinoic acid and steroid metabolism. Future studies evaluating the role of this pathways might identify novel tolerance-promoting pathways.