Doctoral Degrees (Physiological Sciences)

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Anthracycline-induced cardiotoxicity : the role of proteolytic pathways
(Stellenbosch : Stellenbosch University, 2012-03) Sishi, Balindiwe J. N. (Balindiwe Jennifer Nonkosazana); Engelbrecht, Anna-Mart; Loos, Benjamin; Van Rooyen, Jacques; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy.
Cell death in hyppxic injury : signaling mechanisms and dynamics in the decision making process
(Stellenbosch : University of Stellenbosch, 2009-12) Loos, Benjamin; Engelbrecht, Anna-Mart; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseased myocardium, type I, better known as apoptotic cell death, which is characterized by cell shrinkage and chromatin condensation, type II, or cell death with autophagy, presents a morphology with intracellular accumulation of autophagic vacuoles and type III, better known as necrosis, is characterized by cellular swelling and rapid loss in cellular membrane integrity. However, recent literature strongly argues against rigid classifications in the context of cell death mechanisms but rather suggests to adopt a view of cell death as a dynamic and integrative cellular response. Furthermore, the contribution of autophagy in cell death or cell survival is still poorly understood. Therefore the aims of this study were twofold: (i) to characterize the contribution of each cell death type in context of the severity and duration of an ischaemic insult and (ii) to determine whether manipulation of the autophagic pathway affects the contribution of cell death and translates into protection of the heart. Rodent derived cardiac myoblast cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs of simulated ischaemia (SI) under hypoxic conditions in a humidified environment containing 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusion respectively. We employed a modified ischaemic buffer containing either 2-deoxy- D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult of mild (mild SI), moderate (moderate SI) and severe (severe SI) character respectively. We evaluated the contribution of each cell death mode using a combination of viability- and ATP assays. Molecular markers for each cell death process such as LC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescence techniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MA respectively, and similar parameters were evaluated after 2 hours of mild or moderate SI. Propidium Iodide exclusion and Fluorescence Resonance Energy Transfer (FRET) in combination with mitochondrial inner membrane depolarization were employed to assess the onset of cell death dynamically. Flow cytometry was employed to evaluate the degree of protection. In addition, the ATP levels and reactive oxygen species (ROS) were evaluated. Our results strongly indicate a differential induction of cell death, which is dependent on the severity and duration of the ischaemic insult. Mild SI led to the induction of autophagy and apoptosis, whilst moderate or severe SI induced both apoptotic and necrotic cell death without an indication of autophagy. Only mild SI, but not moderate and severe SI, resulted in an ATP surge. Moreover, our data provide direct evidence that increased autophagy delays the loss of cellular membrane integrity and delays caspase-3 activation as well as mitochondrial depolarization in ischaemic cardiomyocytes. Our results show a profound effect of increased autophagy on the onset of apoptosis as well as necrosis under simulated ischaemic conditions, providing cellular protection. This ATP surge observed during mild SI was abolished with increased autophagy. Furthermore, our results indicate a profound effect of autophagy on ROS generation. Under normoxic conditions, increased autophagy induced a significant decrease in ROS while the inhibition of autophagy significantly increased ROS generation. However, when increasing or decreasing autophagy prior to the ischaemic insult, ROS increased significantly in both scenarios. The results suggest that the severity of ischaemia determines the mode of cell death differentially. An increase in autophagic responsiveness and flux, as induced through rapamycin treatment, provides a selective advantage for tissue against injury, possibly by maintaining intracellular ATP levels through the provision of metabolic substrates. Autophagy is described as an inherent cellular mechanism v which affects the onset of cell death and exhibits protective effects in the ischaemic myocardium when upregulated prior to the ischaemic insult. The protective effect of increased autophagy was mirrored in the isolated perfused rat heart model, reflected by improved functional recovery during ischaemia/reperfusion.
Chemoresistance 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.
Differential tolerance of a cancer and a non-cancer cell line to amino acid deprivation : mechanistic insight and clinical potential
(Stellenbosch : Stellenbosch University, 2012-03) Thomas, Mark Peter; Engelbrecht, Anna-Mart; Strijdom, Hans; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management.
The effect of melatonin treatment on doxorubicin-induced skeletal muscle atrophy within a cancer model
(Stellenbosch : Stellenbosch University, 2018-12) Isaacs, Ashwin Wayne; Engelbrecht, Anna-Mart; Loos, Ben; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background and Aim: Skeletal muscle atrophy is a major concern in patients suffering with malignancy. Chemotherapeutic agents, such as doxorubicin (DOX), can further exacerbate this loss of skeletal muscle. Although many cancer patients on chemotherapeutic agents suffer from this condition, there are no therapies routinely used to moderate muscle atrophy. The aim of the study was to investigate whether melatonin (MLT) can attenuate doxorubicin‐induced skeletal muscle and myotube atrophy in an in vivo rodent model of breast cancer as well as in an in vitro model of DOXinduced myotoxicity respectively. The safe and cost‐effective role of melatonin as a possible therapy to limit the burden of doxorubicin‐induced muscle toxicity in cancer patients serves as rationale for the in vivo study and the in vitro study allows for the exploration of more invasive mechanistic aspects using the cell lines, which would not be possible when viewing excised tissue. Methods: Female Sprague‐Dawley rats were inoculated with LA7 cancer cells and were randomly assigned to six groups: Control, Tumour control (TCON), Vehicle control (VEH), MLT, DOX and DOX + MLT (DM). Prophylactic treatment of MLT (6 mg/kg) was administered in drinking water daily and rats received three intraperitoneal injections of DOX (4 mg/kg, 3 times at 3‐day intervals). Following sacrifice blood samples (whole blood counts) and skeletal muscle tissue were collected for histological, immunoblot, antioxidant capacity and immunofluorescence analyses. Furthermore, C2C12 myoblasts grown to confluency and differentiated into myotubes were pretreated with MLT (50 nM) for 48h followed by DOX treatment (0.8 μM) for 24h. The effect of MLT treatment on C2C12 myotube diameter, mitochondrial reactive oxygen species (mtROS) production, sirtuin levels and autophagy activity was then assessed. Results: DOX treatment significantly reduced animal weight (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) compared to DM weight (281.5 ± 7.11 g vs. 284.0 ± 6.53 g) and gastrocnemius muscle weight (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) and cross sectional area (CSA), while increasing markers of muscle degradation compared to MLT treated groups. Serum myoglobin levels were significantly elevated in the DOX group compared to the DM group (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); while, white & red blood cell counts (WBC & RBC) were significantly decreased in the DOX group compared to the MLT treated groups respectively (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L1, p˂0.0001). Furthermore, MLT treatment significantly increased intramuscular antioxidant capacity, mitochondrial biogenesis and satellite cell number. In vitro DOX treatment resulted in increased myotube atrophy, mitochondrial ROS levels and these effects were significantly reduced with MLT pre‐treatment. Discussion: The improvement in animal weight, muscle to body weight ratio, muscle CSA as well as the reduction in myoglobin levels in the treatment groups compared to the DOX group indicate that MLT protects against DOX‐induced atrophy. Moreover, MLT pre‐treatment improved circulating levels of WBC & RBC compared to the DOX only group and attenuated skeletal muscle atrophy by reducing cell apoptosis and increasing satellite cell number suggesting that MLT assists with muscle repair. The in vitro study indicated that DOX‐induced myotube atrophy was preceded by increases in mitochondrial ROS. Conclusion: Results indicate that pre‐treatment with exogenous MLT protects against skeletal muscle wasting induced by DOX in a pre‐cachectic tumour‐bearing rat model.
An 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.
An investigation into the role of Serum amyloid A in breast cancer
(Stellenbosch : Stellenbosch University, 2021-12) Olivier, Daniel Wilhelm; Engelbrecht, Anna-Mart; Pretorius, Etheresia; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Cancer remains a global challenge that affects many lives. To this extent, much research has gone into understanding this disease and the factors that contribute to it. Over the past 40 years, an accumulation of data have shown the presence of the acute phase protein, serum amyloid a (SAA), in the blood of cancer patients, tumors and cells associated with tumors. Moreover, it seems that SAA levels also correlate with disease progression. As such, SAA was investigated as a contributing factor to cancer and its role therein. To date, only in a few publications exist where a role for SAA in triple-negative breast cancer (TNBC) have been investigated, apart from reporting its omnipresence in cancer patients. Moreover, many investigations to date have neglected, or was unable, to distinguish between the various SAA isoforms found in humans and mice. As such, no coherent role for each isoform have been established. Additionally, some studies have used recombinant SAA proteins, which have been questioned for various reasons discussed in this dissertation. Therefore, the aim of this study was to address some of the above shortfalls, in addition to establishing a role for SAA in cancer. Here, the role of the acute phase SAA isoforms, SAA1 and SAA2, were investigated, which are secreted in response to tissue injury or inflammation in models of TNBC. In vitro, a role for SAA1 was investigated through RNA interference, whereby the SAA1 gene was knocked down. Data showed that SAA1 is essential for healthy epithelial functioning, but also in the cancer cells. However, whereas SAA1 knockdown in an epithelial cell line (MCF12A) induced characteristics associated with cell death inhibition and cell repair, knockdown in the two TNBC cell lines (MDA-MB-231 and HCC70) induced characteristics of mitotic catastrophe and also caused decreased migration in these cells. Subsequently, an in vivo model was considered wherein TNBC tumors were induced in mice genetically wild-type for SAA1/2 (WT), and mice deficient in SAA1/2 (SAADKO). Here, results showed that tumor induction in SAADKO mice elicited an inflammatory response opposite to WT mice. Molecular analysis of WT and SAADKO tumors further revealed that SAADKO tumors showed signs of inhibition of apoptosis, but a high level of DNA repair, in addition to characteristic associated with lower metastatic potential, when compared to WT tumors. Histological analysis subsequently revealed that SAADKO tumors also had less necrosis. Combinedly, this data suggests that SAADKO tumors are less aggressive, leading to the conclusion that SAA1/2 contributes to cancer progression as a chronic inflammatory mediator. Therefore, SAA could potentially serve as a therapeutic target in the future.
An investigation into the role of Serum amyloid A in breast cancer
(Stellenbosch : Stellenbosch University, 2021-04) Olivier, Daniel Wilhelm; Engelbrecht, Anna-Mart; Pretorius, Etheresia; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Cancer remains a global challenge that affects many lives. To this extent, much research has gone into understanding this disease and the factors that contribute to it. Over the past 40 years, an accumulation of data have shown the presence of the acute phase protein, serum amyloid a (SAA), in the blood of cancer patients, tumors and cells associated with tumors. Moreover, it seems that SAA levels also correlate with disease progression. As such, SAA was investigated as a contributing factor to cancer and its role therein. To date, only in a few publications exist where a role for SAA in triple-negative breast cancer (TNBC) have been investigated, apart from reporting its omnipresence in cancer patients. Moreover, many investigations to date have neglected, or was unable, to distinguish between the various SAA isoforms found in humans and mice. As such, no coherent role for each isoform have been established. Additionally, some studies have used recombinant SAA proteins, which have been questioned for various reasons discussed in this dissertation. Therefore, the aim of this study was to address some of the above shortfalls, in addition to establishing a role for SAA in cancer. Here, the role of the acute phase SAA isoforms, SAA1 and SAA2, were investigated, which are secreted in response to tissue injury or inflammation in models of TNBC. In vitro, a role for SAA1 was investigated through RNA interference, whereby the SAA1 gene was knocked down. Data showed that SAA1 is essential for healthy epithelial functioning, but also in the cancer cells. However, whereas SAA1 knockdown in an epithelial cell line (MCF12A) induced characteristics associated with cell death inhibition and cell repair, knockdown in the two TNBC cell lines (MDA-MB-231 and HCC70) induced characteristics of mitotic catastrophe and also caused decreased migration in these cells. Subsequently, an in vivo model was considered wherein TNBC tumors were induced in mice genetically wild-type for SAA1/2 (WT), and mice deficient in SAA1/2 (SAADKO). Here, results showed that tumor induction in SAADKO mice elicited an inflammatory response opposite to WT mice. Molecular analysis of WT and SAADKO tumors further revealed that SAADKO tumors showed signs of inhibition of apoptosis, but a high level of DNA repair, in addition to characteristic associated with lower metastatic potential, when compared to WT tumors. Histological analysis subsequently revealed that SAADKO tumors also had less necrosis. Combinedly, this data suggests that SAADKO tumors are less aggressive, leading to the conclusion that SAA1/2 contributes to cancer progression as a chronic inflammatory mediator. Therefore, SAA could potentially serve as a therapeutic target in the future.
Manipulation of the autophagic pathway sensitises cervical cancer cells to cisplatin treatment
(Stellenbosch : Stellenbosch University, 2013-03) Leisching, Gina Renata; Engelbrecht, Anna-Mart; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction Cisplatin has been widely used to treat solid tumours and much success has come from the use of this drug in the treatment of head and neck, ovarian, testicular, cervical and small-cell lung cancers. However, the success of cisplatin treatment is limited due to its dose-limiting toxicity and its resulting side-effects, such as nephro- and ototoxicity. The devastating side-effects induced by cisplatin treatment provided the platform for this study whereby the aim was to lower the concentration of cisplatin while maintaining its cancer-specific cytotoxic action. Equally concerning is, cisplatin resistance which is becoming increasingly common, and this radically limits the clinical efficacy and utility of the drug. Adjuvant therapy has thus become necessary in an attempt to possibly curb or lessen the extent of cisplatin resistance. Due to the large body of evidence implicating the importance of autophagy in cancer, the prospect of targeting this mechanism has generally been accepted. Various chemotherapy agents induce autophagy in cancer cells; however the effect of cisplatin on autophagic induction has not been very well explored. We thus hypothesise that the manipulation of the autophagic pathway will sensitise cancer cells to a low concentration of cisplatin treatment. Furthermore, due to the functional interaction between Bcl-2 and Beclin-1 and its role in the regulation of autophagy, ratio analysis of Beclin-1 to Bcl-2 as means of detecting the role of autophagy within the cell under homeostatic and treatment/stress conditions has been conducted. Additionally, Bcl-2 has a prominent role in the malignant cell and it’s over-expression has been found to confer resistance in a variety of cancerous cell lines. We therefore hypothesise that the silencing of Bcl-2 prior to cisplatin treatment will sensitise cervical cancer cells to apoptosis and increase the Beclin-1/Bcl-2 ratio in favour of apoptosis. Materials and Methods Three human cervical cell lines were used: a non-cancerous ectocervical epithelial cell line (Ect1/E6E7) and two cancerous cervical cell lines (HeLa and CaSki). In order to determine a concentration of cisplatin that was non-toxic to the non-cancerous Ect1/E6E7 cell line, a dose-response was performed. With the use of an autophagy inhibitor (bafilomycin A1) and an autophagy inducer (rapamycin), autophagic flux capacities were assessed in each cell line through the Western blotting technique. In order to assess whether the chosen concentration of cisplatin induced autophagy, flow cytometry with the use of a Lysotracker™ dye was utilised, as well as analysis of autophagy protein levels (LC-3 II, Beclin-1 and p62). Autophagy modulation was achieved through two methods: pharmacological modulation with use of two recognised agents, namely bafilomycin A1 and rapamycin, and biological manipulation with the use of ATG5 and mTOR mRNA silencing. The effects of different treatment regimes on cell death was assessed with the use of PARP and caspase-3 cleavage through Western blotting, caspase-3/-7 activity (Caspase-Glo®), PI inclusion, LDH release and MTT reductive capacity. Additionally the effects of these treatment regimes on cell-cycle progression were also analysed. Beclin-1 and Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin in HeLa and CaSki cells. To assess the reliance of the cervical cancer cells on Bcl-2 after cisplatin treatment, Bcl-2 knock-down was achieved through RNA interference, where after the Beclin-1/Bcl-2 ratio was assessed as well as apoptosis with the use of cleaved PARP analysis (Western blotting) and Caspase-Glo©. For the ex vivo analysis, biopsies were collected from patients undergoing routine colposcopy screenings and hysterectomies at Tygerberg Hospital, Tygerberg, Western Cape. A total of 10 normal, 29 low-grade squamous intraepithelial lesions (LSIL), 33 high-grade squamous intraepithelial lesions (HSIL) and 13 carcinoma biopsies were collected for analysis, where after the expression profiles of two autophagy markers (mTOR and LC-3 II), as well as one anti-apoptotic marker (Bcl-2) were assessed. Protein levels were analysed through Western blot and confirmed through immunohistochemistry. Results Dose-response curves revealed that 15 μM of cisplatin did not induce cell death in the normal cervical epithelial cell line (Ect1/E6E7) and was therefore utilised through-out the remainder of the study. It was additionally determined that the CaSki cells were more resistant to cisplatin treatment when compared to the HeLa and Ect1/E6E7 cells. Autophagic flux analysis revealed that, although all three cell lines were cervix derived, their autophagic flux capacities differed. It was observed that the chosen concentration of cisplatin was able to induce autophagy in all three cell lines, with the HeLa cells demonstrating a particularly pronounced response. Autophagy modulation in conjunction with cisplatin treatment revealed the following: Autophagy inhibition with bafilomycin A1 lead to significant increases in caspase-3 and PARP cleavage and LDH release in both cervical cancer cell lines. The inhibition of autophagy through silencing of ATG5 induced caspase-3 cleavage and agrees with results obtained from pharmacological inhibition of autophagy with bafilomycin A1. In addition to autophagic induction, a low concentration of cisplatin induced the up-regulation of Bcl-2, which when silenced significantly improved cisplatin-induced apoptosis in both cervical cancer cell lines. Analysis of the expression profiles of mTOR and LC-3 in normal, pre-malignant (LSIL and HSIL) and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma of the cervix. Additionally, Bcl-2 expression is significantly increased in cervical carcinoma tissue, which agrees with results from other studies. Conclusion Autophagic flux capacities between the three cell lines investigated, derived from the same organ, differ significantly. This should be taken into consideration when autophagic modulation is being used as an adjuvant treatment. With regard to chemotherapy treatment in cervical cells, a low-concentration of cisplatin significantly induces autophagy in malignant and non-malignant cervix-derived cell lines where it serves a pro-survival mechanism. Inhibition of autophagy with bafilomycin A1 and ATG5 siRNA confirmed this survival effect in both cancerous cell lines where apoptosis was significantly increased. Interestingly, rapamycin pre-treatment together with cisplatin did not induce significant levels of apoptosis in HeLa cells where autophagy induction may have provided additional protection from the cytotoxic effects of cisplatin. Therefore the inhibition of autophagy through pharmacological and biological inhibition improves the cytotoxicity of a low concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer. Bcl-2 up-regulation in response to cisplatin treatment also serves as a protective mechanism by which cervical cancer cells survive. The extent of apoptotic cell death observed after biological inhibition of Bcl-2 reiterates the fact that this response may be exploited in order to favour the use of lower concentrations of cisplatin. Analysis of clinical specimens emphasised the value of the in vitro work: Cervical cancer biopsies had increased expression of both LC-3 II and Bcl-2, indicating autophagy induction and apoptosis inhibition, respectively. Thus two novel methods of improving cisplatin cytotoxicity have been demonstrated in the following study. Treatment regimens may administer more frequently and prolonged due to the minimal side-effects that accompanies low-dose cisplatin treatment.
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.
Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity : An evaluation of the protective role of melatonin.
(Stellenbosch University, 2017-03) Govender, Yogeshni (Jenelle); Engelbrecht, Anna-Mart; Loos, Ben; Marais, Erna; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Anthracyclines, such as doxorubicin (DXR), are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side-effects such as cardiotoxicity. The abundance of mitochondria in cardiomyocytes closely links mitochondrial function with myocardial function. Mitochondrial dysfunction has emerged as a critical element in the development of DXR-induced cardiotoxicity. In light of this scenario, melatonin (MLT) is a potent anti-oxidant, is non-toxic, is dually oncostatic and cardio-protective, and has been shown to influence mitochondrial homeostasis and function. Both endogenously produced and exogenously administered MLT during or prior chemotherapy shows great promise in this therapeutic avenue as demonstrated in several studies. Although research support the mitochondrial protective role of MLT, the exact mechanisms by which MLT confers mitochondrial protection in the context of DXR-induced cardiotoxicity remains to be elucidated. Aims: The aim of this study was to investigate the effect of MLT on the following mitochondrial and cellular parameters: mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential, mitochondrial fission and fusion, mitochondrial bioenergetics and biogenesis, sirtuin activity, autophagy and cell death in an in vitro model of DXR-induced cardiotoxicity. Furthermore, the effect of MLT on cardiac function and tumor growth was assessed in a tumor-bearing rat model of acute DXR-induced cardiotoxicity. Materials and Methods: H9c2 rat cardiomyoblasts were pre-treated with MLT (10 μM) for 24 hours followed by DXR treatment (3 μM) for 24 hours. Following treatment, the above mentioned mitochondrial and cellular parameters were assessed using immunoblot analysis, mitochondrial respiration analysis, flow cytometry, fluorescence microscopy and luciferase-based assays. Sprague Dawley female rats (16-18 weeks old), were inoculated with LA7 rat tumor cells. Animals received DXR (3 intraperitoneal injections of 4 mg/kg at 3-day intervals, 12 mg/kg cumulative dose) and/or received MLT (6 mg/kg) daily in their drinking water. Tumors were measured daily using digital calipers and tumor volumes calculated. Animal weights were recorded daily. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. Results: DXR treatment significantly increased cell death, mitochondrial ROS levels and mitochondrial fission and these effects were significantly reduced with MLT pre-treatment. Furthermore, MLT pre-treatment significantly increased mitochondrial membrane potential, mitochondrial biogenesis and cellular ATP levels reduced by DXR treatment. Cardiac output and total work performance of the heart was significantly increased in rats treated with DXR+MLT in comparison to rats treated with DXR alone. In addition, body and heart weights were significantly reduced in DXR-treated rats in comparison to DXR+MLT treated rats. Tumor volumes were significantly reduced in DXR+MLT-treated rats on Day 8 in comparison to DXR-treated rats. Discussion and Conclusion: The results obtained from the current study indicates that MLT treatment confers a cardio-protective effect by maintaining mitochondrial function, increasing cardiomyocyte survival and improving cardiac function during DXR-induced cardiotoxicity. Furthermore, MLT treatment alone suppresses the growth of tumors. The combination of DXR+MLT treatment rapidly reduced tumor growth, suggesting that MLT enhances the oncostatic activity of DXR. The unique ability of MLT to be both cardio-protective and oncostatic during DXR-induced cardiotoxicity is promising for the field of cardio-oncolocgy.
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.
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.
The role of the AHNAK protein in breast cancer : implications for tumour metastasis and chemoresistance
(Stellenbosch : Stellenbosch University, 2016-12) Davis, Tanja Andrea; Engelbrecht, Anna-Mart; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction – Cancer continues to have a significant impact on society. While there has been much success in characterising tumours and identifying targetable markers, two major problems are still faced today, namely therapeutic failure and advanced progression of the disease. The human AHNAK protein is a giant scaffold protein involved in multiple cellular processes and has now also been suggested to be associated with cancer, particularly with regards to tumour metastasis and chemoresponse. However, limited information and several contradicting findings have contributed to a poor understanding of the role of AHNAK in cancer. Thus, we aimed to characterise the AHNAK protein in cancer by determining the role of the protein in the chemotherapeutic response of breast cancer to doxorubicin (DXR) and also in cellular migration. Methods – For the in vitro model the non-metastatic DXR-sensitive epithelial-like MCF-7 and metastatic DXR-resistant mesenchymal-like MDA-MB-231 cell lines were used. We performed DXR treatments and assessed AHNAK’s protein expression and intracellular localisation. We also assessed these properties in a tumour-bearing mouse model. AHNAK knockdown and overexpression was achieved by means of transient plasmid transfections in both cell lines and following DXR treatments we assessed apoptotic marker expression, cell cycle modulation, epithelial-mesenchymal transition (EMT) marker expression and cellular migration. Results – DXR induced dose-independent and dose-dependent changes in AHNAK protein expression in MCF-7 and MDA-MB-231 cells, respectively, but it did not affect its intracellular localisation in these cells. In the tumour-bearing mouse model DXR also induced dose-dependent changes in AHNAK expression without affecting its localisation, similar to the MDA-MB-231 cells. In the MDA-MB-231 cells, DXR promoted apoptosis inhibition by decreasing cPARP and cCasp7 expression. Knockdown of AHNAK prevented this inhibition while overexpression induced a similar inhibitory effect. With cell cycle analyses we observed that DXR also resulted in S phase arrest in these cells. AHNAK knockdown completely prevented the DXR-induced cell cycle arrest while overexpression was sufficient to cause such an arrest on its own. No significant effects were observed with these experiments in the MCF-7 cells. DXR induced EMT in the MCF-7 cells but AHNAK knockdown or overexpression did not affect this. In MDA-MB-231 cells DXR treatment showed a trend of decreased EMT and while AHNAK knockdown had no effect on this, its overexpression showed clearer evidence of EMT reduction. AHNAK knockdown also had no major effects on cell migration in both cell lines, although its overexpression generally decreased cellular migration. Conclusions – We show that AHNAK plays a novel role in the DXR-response of breast cancer cells and this involved AHNAK’s expression, apoptosis inhibition and cell cycle modulation. Possible molecular mechanisms are proposed but require further investigation. Our results regarding the role of AHNAK in tumour cell migration is less clear and contradicting when compared to other studies. These results may have potential therapeutic implications with regards to the modulation of DXR response to improve treatment efficacy.

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