Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by browse.metadata.advisor "Loos, Ben"

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    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.
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    Investigating the role of rilmenidine and spermidine on autophagic flux and cell death in a model of Alzheimer's disease
    (Stellenbosch : Stellenbosch University, 2020-04) Lumkwana, Dumisile; Loos, Ben; Kinnear, Craig; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction - Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by multiple cognitive deficits. The neuropathology of AD is underpinned by two molecular hallmarks; intracellular protein aggregates known as neurofibrillary tangles (NFTs), composed of hyper-phosphorylated Tau and extracellular amyloid beta (Ab) plaques, composed of Ab peptides derived from the amyloid precursor protein (APP). Both of these occur as a result of an imbalance in proteostasis, leading to neuronal toxicity. Although, we have advanced our understanding of the molecular machinery that regulates the rate of protein degradation through autophagy at basal levels and increasingly so the many aspects of its dysfunction in AD, the deviation of autophagic activity from basal levels and its change during disease pathogenesis in neuronal tissue remains largely unclear. Over the recent years, substantial progress has been made in modulating autophagy using pharmacological agents in vitro and in vivo and mounting evidence points towards autophagy modulation using pharmacological agents as one of the major therapeutic strategies for neurodegenerative diseases. Although spermidine and rilmenidine both enhance autophagy, the relationship between autophagy activity, the extent of protein clearance and cell death onset remains unclear. Moreover, the impact of their concentration on autophagic flux and subsequent protein clearance as well as neuronal toxicity is unclear. Therefore, this study aimed to unravel the impact of using a high and low concentration of spermidine and rilmenidine on autophagic flux, neuronal toxicity and protein clearance using distinct neuronal injury model systems. Methods - Murine hypothalamus-derived GT1-7 neuronal cells and the mouse neuroblastoma (N2a) cell line stably expressing Swedish double mutation APP695 (Swe) associated with AD pathology were used. GT1-7 cells transfected with mRFP-GFP-LC3 and GFP-LC3-RFP-LC3DG were treated with a low and high concentration of spermidine and rilmenidine in the absence and presence of saturating concentrations of bafilomycin, after which the autophagic flux profile was characterized, assessing cellular viability, autophagosome pool, autolysosome pool, autophagosome flux, transition time, p62 puncta and autophagic vacuoles. Cellular viability assays, western blotting, fluorescence microscopy, transmission electron microscopy and correlative light and electron microscopy linked to quantitative morphometric analysis was performed. In addition, potential protective effects of a low and high concentration of spermidine and rilmenidine were assessed in a paraquat (PQ)-induced neuronal toxicity model and in an APP over-expression model. Cellular viability, ROS damage, cell death onset and autophagic activity were assessed in the PQ-induced toxicity model, while in the APP model, cellular viability and protein clusters were assessed and quantified using cellular viability assays and single molecule imaging, i.e. direct Stochastic Optical Reconstruction Microscopy (d-STORM). Moreover, a Correlative Light and Electron Microscopy (CLEM) method was developed and implemented to morphometrically assess the effects of a low and high concentration of spermidine on the localization of autophagosomes in 3 dimensions. Finally, potential protective effects of spermidine at two distinct concentrations were assessed using transgenic mice expressing GFP-LC3, treated with PQ to induce neuronal toxicity associated with neurodegeneration. Results - Our results indicate a concentration-dependent effect of spermidine and rilmenidine on autophagic flux with the detected change in flux depending on the specificity and sensitivity of the method employed. In addition, in the in vitro model of PQ-induced toxicity, our results revealed that spermidine at a low concentration and not a high concentration protected against cell toxicity, ROS damage, cell death as well as microtubule destabilization. To our surprise, both concentrations of rilmenidine failed to protect against ROS damage and cell death and also failed to robustly upregulate autophagy in the same model. Moreover, in the in vitro model of neuronal toxicity induced by APP over-expression, our results showed that both concentrations of spermidine and rilmenidine protected against cytotoxicity. Here, spermidine at a low concentration effectively cleared APP clusters and reduced their size, especially after 48 h of APP over-expression, while rilmenidine, reduced the number and size of APP clusters in a concentration-dependent manner at the same time point. Taken together, the in vitro results reveal a concentration-dependent effect, that is cell type and injury specific, impacting autophagy and cell death control. Moreover, we have successfully implemented a 3D CLEM protocol and revealed that spermidine, in combination with BafA1 decreases autophagosome volume while increasing their surface area in a concentration-dependent manner. Lastly, in vivo, our results reveal that PQ-induced toxicity impacts the brain regions differentially, with the hippocampus being highly susceptible to PQ-induced injury followed by the cortex. Moreover, our results show that both dosages of spermidine robustly protected against oxidative stress, neuronal damage, microtubule destabilization, and upregulated autophagy, in the hippocampus and cortex. However, the low dose of spermidine resulted in more enhanced protection, although, autophagy was here regionspecifically upregulated in a manner dependent on the dose utilized, with the higher dose of spermidine increasing LC3-II in the hippocampus, and the lower dose increasing LC3-II in the cortex. Conclusion - Our results indicate the critical importance of using multiple tools to assess autophagy and show that a concentration-dependent effect of the two selected drugs on autophagic flux exists. In addition, we provide evidence of the distinct, context dependent protective roles of spermidine and rilmenidine in an in vitro model of APP over-expression as well as the protective roles of spermidine using in vitro and in vivo models of PQ-induced neuronal toxicity. These results suggest that firstly administration of spermidine may represent a favourable therapeutic strategy for the treatment of AD and secondly, concentration / flux screening may be more critical for optimal autophagy control than previously thought. Future studies, using an in vivo model over-expressing APP are warranted to further verify the protective effects of spermidine, to foster clinical translation and therapeutic intervention in neurodegenerative disorders.
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    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.