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Differential toxicity of two murine endothelial cells to ROS duress: understanding oxidative stress-induced blood-brain barrier dysfunction
(University of the Western Cape, 2020) Alamu, Olufemi Akinyinka
The blood-brain barrier (BBB) is a critical interface between the blood circulation and brain tissue which performs critical selection of circulating molecules that gain access to the brain tissue. Its unique ability to adjust to changes in the constituents of the blood circulation confer in the BBB a dynamic nature enabling changes in its properties to suit the homeostatic needs of the brain. Dysfunction of the BBB has been established to be pivotal to the initiation and/or maintenance of an array of neurological disorders, most of which involve the production of excess reactive oxygen species (ROS) and oxidative stress in their pathophysiology. Thus, clinical trials of exogenous antioxidant agents have been proposed and initiated, with most results being inconclusive. Extensive studies of the impact, capacity and plasticity of endogenous antioxidants in the cells that constitute the blood-brain barrier, especially the brain endothelial cells, therefore, became necessary for the rational choice, timing, and the mode of application of antioxidants in the management of oxidative stress-mediated neurological diseases. In the present study, incremental hydrogen peroxide (H2O2) concentrations were dosed in cell culture medium to simulate OS and gauge the capacity of the brain endothelial cell (BECs) models of the BBB to resist ROS toxicity, the contribution of endogenous antioxidants to this resistance and to study the morphological changes in ROS-mediated BBB dysfunction. Two types of mammalian endothelial cells, b.End5 and bEnd.3, commonly used for in vitro studies of the blood-brain barrier, were selected for use in this study. A combination of spectrophotometry, fluorescent microscopy, trypan blue exclusion method and flow cytometry were used to assess cell viability, cellular glutathione (GSH) content, cell cycle changes, cellular death by apoptosis and necrosis in BECs exposed to incremental H2O2 concentrations for 24hr. Exogenous antioxidants were variably administered to study the effects of externally incident antioxidants when the cells were under H2O2 exposure. Results showed that b.End5 cell line significantly tolerated higher concentrations of H2O2 than the bEnd.3 cell line. GSH contents for both cell lines were fairly similar under physiological conditions but after exposure to H2O2, b.End5 cells demonstrated higher resistance to GSH depletion than the bEnd.3 cell line, although the two cell lines were obtained from the same animal species. Along incremental concentrations of H2O2, increased cell proliferation, cell necrosis and apoptosis and cell cycle arrest were observed concurrently. At H2O2 concentrations that defined OS, live cells were depleted in b.End5 cells used while there was significant increases in apoptotic and necrotic cells with apoptotic cells as the significant majority comparatively. Cell cycle studies showed arrest of cell division at the G2/M phase of the cell cycle at higher concentrations of H2O2. Application of exogenous antioxidants ameliorated the H2O2-induced cellular depletion as well as improved recovery in cellular viability following withdrawal of H2O2 after 24hr exposure. It was conclusive that apoptotic pathway of cellular death is a major pathway of BECs response to OS. Also, there was differential H2O2 toxicity and GSH de novo synthesis capacity between the b.End5 and bEnd.3 cell lines despite their common origin from the same animal species and their possession of similar contents of endogenous antioxidant GSH under physiological conditions. This finding calls for more caution for the choice of cellular models for specific studies of the BBB to ensure that results obtained are reproducible, reliable and sufficiently conclusive. Furthermore, our results tend to suggest that the processes responsible for the endothelial component of BBB dysfunction under conditions of oxidative stress occur concurrently and include increased proliferation, necrotic and apoptotic cell death as well as cell cycle arrest. Additionally, study suggests that the clinical administration of antioxidants could be an appropriate intervention for the alleviation of neurological diseases.
Authentic leadership and its effects on organizational citizenship behaviour in a provincial government department in the Western Cape.
(University of the Western Cape, 2015) George, Lee-Ann Melissa
Leaders are often thought of as being the top management team of the organisation, illuminating the way forward for individuals by directing organisational activities towards a shared vision (Fernald, Solomon & Tarabishy, 2005). As organisations are constantly facing challenges in establishing a profitable presence in a competitive marketplace, effective leadership is one difference between organisations that successfully meet the challenges and those that do not (Wherry, 2012). In order for any organisation to cope with the demands of a dynamic and ever changing environment, it is necessary for management to move towards a leadership style that allows for the empowerment of employees (Carson & King, 2005). Scholars have identified a form of leadership termed “authentic leadership” where authentic leaders display traits such as honesty, sincerity, high moral standards, ethics and trustworthiness (Avolio et al., 2004; May 2004). According to George (2003), authentic leaders are self-aware and transparent therefore this behaviour sends a strong message to their followers influencing what they, the follower, attend to, how they view themselves and ultimately how they behave. Within organisations where authentic leaders are present, the importance of employee initiative and cooperation become very important (Le Pine, Erez & Johnson, 2002). The individual or employee initiative and cooperation can be viewed as in role (within formal job descriptions) or extra role (outside of formal job description) behaviour. Extra-role behaviour is also defined as organisational citizenship behaviour. This research study investigated if the dimensions of authentic leadership (self-awareness, moral perspective, balanced processing and relational transparency) had an effect on the dimensions of organisational citizenship behaviour (altruism, conscientiousness, sportsmanship, courtesy and civic virtue). The population for this study was a provincial government department within the Western Cape. A non-probability sample based on the method of convenience was utilised of which 131 respondents completed three sets of questionnaires namely; a Biographical questionnaire, Authentic Leadership Questionnaire (Avolio, Gardner & Walumbwa, 2007) and the Organisational Citizenship Questionnaire (Podsakoff, Mackenzie & Fetter, 1990). Statistical analyses involved both descriptive (measures of central tendency and dispersion) and inferential statistics (correlation and multiple regression). The findings indicated that a moderate to weak relationship exists between the dimensions of authentic leadership (self-awareness, moral perspective, balanced processing and relational transparency) and the dimensions of organisational citizenship behaviour (altruism, conscientiousness, sportsmanship, courtesy and civic virtue). Organisational citizenship behaviour of the employees within the organisation is not largely influenced by their leader’s authentic leadership style. Therefore, other factors such as work ethic, organisational commitment, work motivation or personality may have greater influence on organisational citizenship behaviour than authentic leadership. However, a few limitations associated with the study were identified and it is suggested that a qualitative approach be implored as well as other provincial, local or national government departments in the Western Cape be used to contribute to greater representativeness and generalisability. Variables identified in this study are embodied in the human resource functions of the organisation and managers should utilise the findings of this study to better understand human behaviour within the workplace.
Protein expression and antifungal effect of fluconazole-resistant Candida species following effective in vitro treatment with K21, a novel antifungal agent
(University of the Western Cape, 2019) John, Cathy Nisha
Background: Oropharyngeal candidiasis, caused by the fungus Candida, is the most common opportunistic infection affecting the quality of life of immunocompromised patients. Fluconazole is widely used as the first line of treatment for fungal infections. However, the inappropriate and misguided use of the drug has led to the evolvement of fluconazole-resistant Candida organisms. This arising resistance resulted in the urgent need for the development of new antimicrobial drugs. The aim of the present study was to investigate the antifungal action of K21, a novel antimicrobial quarternary ammonium compound, on fluconazole-resistant Candida species. Materials and Methods: An in vitro study was conducted using a total of 143 Candida isolates obtained from HIV-positive patients. The ethical aspects of the study complied with the declaration of Helsinki (2013). The time-kill assay was used to evaluate the rate of action of K21 over time on Candida while the fungicidal effect of K21 against C. albicans (ATCC 90028) and C. glabrata (ATCC 26512) was observed at 2 hours. The minimum inhibitory concentration (MIC) of K21 was compared with the MICs of fluconazole. Synergy between K21 and fluconazole was evaluated by both the checkerboard microdilution method and time-kill assay. The modes of action of K21 and drug delivery were determined by performing postembedding immunogold labelling and for the sight-specific target and protein expression of Sap 1-3 and Sap 4-6 within the Candida cell. Results: Of the 143 isolates, 108 were fluconazole-resistant, 15 were fluconazole-intermediate and 20 were fluconazole-susceptible using the broth microdilution assay and breakpoint values recommended by the Clinical and Laboratory Standards Institute (CLSI). The MIC of K21 was 31.24 µg/mL for C. albicans when determined by the broth microdilution assay. About 103 Candida species were resistant and 13 were categorised as intermediate to FCZ with a MIC range between 64 µg/mL - 256 µg/mL and 16 µg/mL - 32 µg/mL respectively. However, the majority of the Candida species (n = 86) showed intermediate susceptibility to K21 with a MIC range between 62.48 µg/mL - 124.95 µg/mL and only 9 of the Candida species were resistant to K21 with a MIC value of ≥249.89 µg/mL. A statistically significant (p value = 0.000) was observed when the MIC values of K21 and FCZ were compared. No antagonism was observed in the study among the Candida strains. The time-kill and synergism assays showed significant differences over time with synergy between K21 and FCZ demonstrated for C. albicans (ATCC 90028 and NCPF 3281) C. dubliniensis (NCPF 3949a), C. tropicalis (ATCC 950) and C. lusitaniae (ATCC 34449). Scanning electron microscopy displayed major alterations in the morphology of Candida species between 2 hours and 24 hours, exhibiting cell lysis and cell death. Transmission micrographs of C. albicans (ATCC 90028) treated with K21 showed shrunken nuclei with disruption of cell walls and cell membranes. Immunogold labelling of C. albicans (ATCC 90028) and C. dubliniensis (NCPF 3949a) with Sap 1-3 antibodies exhibited the presence of gold particles confined to the cell wall and cell membrane, but, when exposed to Sap 4-6 antibodies there were a few non-specific gold particles found in C. albicans (ATCC 90028) and an absence of gold particles in C. dubliniensis (NCPF 3949a). C. tropicalis (ATCC 950) showed few gold particles along the cell membrane when treated with Sap 1-3 antibodies and no gold particels were found when treated with Sap 4-6 antibodies. Conclusion: The present study suggests that K21 acts as a potent antifungal agent and can be considered for development as an alternative treatment for fluconazole-resistant Candida species especially in immunocompromised patients.
The effects of various combinations of different classes of anticancer drugs and tyrosine kinase inhibitors on the human MCF-7 and triple-negative MDA-MB 231 breast carcinoma cell lines
(University of the Western Cape, 2020) Abrahams, Beynon
Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy. The epidermal growth factor receptor (EGFR) is abnormally activated in many cancers and its signal transduction pathways play an important role in regulating cell proliferation and survival. It is often overexpressed in TNBC. P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for active transport of substances across the cell membrane out of a cell. It functions as an efflux pump and is overexpressed in many cancer cells. The altered expression of both EGFR and P-gp parallels an aggressive clinical course and the development of resistance to anticancer and adjuvant endocrine therapies. Anti-EGFR compounds that specifically target the intracellular and extracellular domains of the EGFR include tyrosine kinase inhibitors (TKIs), e.g., gefitinib and erlotinib and monoclonal antibodies, e.g., cetuximab and trastuzamab, that are amongst the most effective agents that are currently used together with chemotherapeutic agents in clinical practice. However, the emergence of multidrug resistance (MDR), a mechanism that is not well understood, has, over time, eclipsed the progress that has been made in drug therapeutics. In this study we examined the effects of doxorubicin (DOX), cisplatin (CDDP) and one investigational TKI (EGFR inhibitor I, hereinafter referred to as EGFRi), individually and in combination, on human MCF-7 breast carcinoma cells and a triple negative breast cancer (TNBC) cell line (MDA-MB-231), with specific focus on P-gp and EGFR inhibition. Analyses of MCF-7 and MDA-MB-231 TNB carcinoma cells exposure to DOX, CDDP and EGFRi included growth and dose-response curves (cytotoxicity assays), caspase3/7 assays for apoptosis detection, P-glycoprotein function (intracellular Calcein-AM accumulation) and EGFR and P-glycoprotein (ABCB1 gene) mRNA expression, using quantitative Real-Time PCR. When used as an individual treatment, EGFRi demonstrated a higher growth inhibition in both MCF-7 and MDA-MB 231 cell lines, compared to DOX and CDDP. Combination treatment of DOX with CDDP at equimolar log dose concentrations, synergistically inhibited cell growth that was significantly different to the compounds used individually. The pairwise combinations of EGFRi with both DOX and CDDP also demonstrated synergistic and antagonistic drug interactions consistent with the Bliss independence and Loewe additivity synergy models. Apoptosis was confirmed in both MCF-7 and MDA-MB-231 TNBC cells after 24-hour drug treatments. Intracellular calcein accumulation that is indicative of P-glycoprotein inhibition, was determined using the Calcein-AM assay. Drug treatments significantly increased the intracellular accumulation of calcein inside the cells, but only at very high concentrations in MCF-7 cells. No significant levels of calcein accumulation were detected in MDA-MB-231 cells, following individual treatment with EGFRi, DOX and CDDP. The pairwise combination drug treatments of EGFRi + DOX demonstrated significant accumulation of calcein only in MCF-7 cells. RT-qPCR was utilized to quantify the gene expression levels of EGFR and ABCB1 in both MCF-7 and MDA-MB-231 TNBC cell lines following drug treatment. The expression levels of EGFR gene were significantly reduced in both cells following drug treatment, which correlates with the cellular growth inhibition results. The expression levels of ABCB1 gene could not be quantitated following optimization of expression due to undesirable CT values that was outside the normal acceptable ranges. This is a due to the undetectably low expression levels of ABCB1 gene in our samples. Our scientific findings confirmed our hypothesis of the EGFRi’s ability to successfully reduce the efflux function of P-glycoprotein as well as demonstrating its combinatorial potential with DOX and CDDP, as compounds from different classes are more effective in enhancing growth inhibition efficacy in the two human breast carcinoma cell lines.
“Green Synthesis of Silver Nanoparticles using Cowpea Pods and the Evaluation of anticancer effects in vitro
(University of the Western Cape, 2025) Nkomana, Wendy
Cancer remains a major global health burden, and the number of cases is expected to double by 2040. Current treatments such as surgery, chemotherapy and radiotherapy have shown improved health and survival rates but are limited by several drawbacks. Chemotherapy and radiotherapy may result in adverse effects and drug resistance, while surgery is primarily effective on solid and tissue confined tumours. Consequently, there is a pressing demand for novel cancer therapies that are effective and have less adverse side effects. Nanotechnology has shown potential to develop novel anticancer strategies that incorporate materials on the nanoscale range. Metallic nanoparticles, particularly silver nanoparticles (AgNPs) have demonstrated impressive anticancer properties, and a potential to be effective in both localized and metastatic cancers. Their effectiveness is due to their physicochemical properties such as large surface area, tuneable size and shape, making them valuable for various biological applications such as biotechnology, medicine, microbiology and pharmaceuticals. The physical and chemical synthesis methods of AgNPs are costly, energy-intensive, and use environmentally harmful chemicals. Green synthesis using agricultural waste like cowpea pods (CP) provides sustainable, more affordable, and environmentally friendly alternatives. In this study, CP aqueous extract was obtained through maceration in distilled water and used as a reducing and stabilizing agents in the biosynthesis of CP-AgNPs. The CP-AgNPs were synthesized by optimizing reaction parameters such as pH, temperature, CP-Extract concentration, AgNO3 concentration and reaction time. CP-AgNPs were characterized using Ultraviolet-visible (UV-Vis) spectroscopy, Dynamic Light Scattering (DLS), High Resolution-Transmission Electron Microscopy (HR-TEM) and Attenuated Total Reflectance Fourier Transform Infrared (ATR- FTIR). The stability of CP-AgNPs was evaluated in biological media such as, Phosphate Buffered Saline (PBS), Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco’s Modified Eagle’s Medium (DMEM) with supplements (complete) and without supplements (incomplete). The total phenolic content (TPC) of CP-Extract and CP-AgNPs was determined by Folin-Ciocalteu (FC) assay using Gallic acid as a reference standard. The anticancer effects of CP-Extract, CP-AgNPs, free doxorubicin (Dox), and co-treatment of CP-AgNPs with Dox were evaluated on normal skin fibroblast (KMST-6), pancreatic cancer (Panc-1) and prostate cancer (PC-3) cell lines using the WaterSoluble Tetrazolium Salts-1 (WST-1) assay. The CP-AgNPs had a surface plasmon resonance peak at 420 nm. DLS revealed a hydrodynamic diameter of 64.94 ± 4.293 nm (PDI 0.2596 ± 0.0152), while HR-TEM displayed an average core size of 10.99 ± 2.6 nm confirming synthesis of small CP-AgNPs. The ATR- FTIR identified the functional groups involved in the reduction of Ag+ to CP-AgNPs. CP-Extract was non-toxic to all the cell lines. CP-AgNPs reduced the cell viability of Panc-1 cells to ~58%, with minimal toxicity against KMST-6 and PC-3 cells. The co-treatment of CP-AgNPs and Dox did not show synergistic effects as the cell viability after co-treatment was similar to when the treatments were used on their own. In conclusion, the CP-AgNPs showed favourable cytotoxic effects against Panc-1 cells thus indicating that agricultural waste can be used as a sustainable source of reducing and stabilizing agents for green synthesis of bioactive AgNPs.