School of Molecular & Cell Biology (ETDs)

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Start date: 2015Subject: search.filters.subject.SDG-3: Good health and well-being

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    Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer
    (University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, Mandeep
    Cancer cells have an increased need for cholesterol, which is required for cell membrane integrity. Cholesterol accumulation has been described in various malignancies including breast cancer. Cholesterol has also been known to be the precursor of estrogen and vitamin D, both of which play a key role in the histology of breast cancer. Elevated cholesterol levels have been linked to breast cancer therefore depleting cholesterol levels in cancer cells can be a viable strategy for treatment. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is a cholesterol depleting compound which is a cyclic amylose oligomer composed of glucose units. It solubilizes cholesterol and is proven to be toxicologically benign in humans. This led us to hypothesise that it might deplete cholesterol from cancer cells and may prove to be a clinically useful compound. Our work provides experimental evidences to support this hypothesis. We identified the potency of HPβCD in vitro against two breast cancer cell lines: MCF7 (Estrogen positive, ER+), MDA-MB-231 [Triple negative breast cancer (TNBC)], and compared the results against two normal cell lines: MRC-5 (Normal Human Lung Fibroblasts) and HEK-293 (Human embryonic kidney) using cytotoxic, apoptosis and cholesterol based assays. HPβCD treatment reduced intracellular cholesterol resulting in significant breast cancer cell growth inhibition through apoptosis. The results hold true for both ER+ and TNBC. We have also tested HPβCD in vivo in MF-1 mice xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late, intermediate and early stage TNBC. These data suggest that HPβCD can prevent cholesterol accumulation in breast cancer cells and is a promising anti- cancer agent
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    Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer
    (University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, Mandeep
    The cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasis
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    Insights into silver(I) phosphine complexes in targeting cell death and metastatic mechanisms in malignant cell lines
    (University of the Witwatersrand, Johannesburg, 2023-09) Roberts, Kim Elli; Engelbrecht, Zelinda; Cronjé, Marianne J.
    Cancer is the leading cause of death worldwide, with 18.1 million new cases and 9.6 million deaths reported annually. Cisplatin, a popular chemotherapeutic drug, exhibits certain limitations in terms of selectivity and efficacy. This emphasizes the necessity for novel therapeutic approaches in addressing a variety of cancer types. Multiple studies have shown that silver-based compounds suppress cancer cell proliferation and induce apoptosis. Thirteen novel silver(I) mono-dentate phosphine complexes were investigated for their anticancer effects on seven different human malignant cell lines; A375 non-pigmented melanoma, A549 lung adenocarcinoma, HEP-G2 hepatocellular carcinoma, HT-29 colorectal adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarcinoma, and SNO oesophageal squamous cell carcinoma. Two non-malignant human cell lines, HEK-293 embryonic kidney cells and MRHF foreskin fibroblast cells, were used to assess the selectivity of the complexes. Cisplatin and the efficient silver(I) phosphine complexes were selected for dose-response experiments to determine IC50 concentrations for the respective cell lines. On the basis of these screening results (chapter two), five difficult-to-treat cancer cell lines, and their most efficient complexes were selected for further investigation. Various cellular characteristics were investigated in chapter three (A549, HEP-G2, HT-29); these included morphological changes, ATP levels, GAPDH levels, Ptd-L-Ser externalization, mitochondrial membrane potential, oxidative stress levels, and the activity of a metabolic enzyme, cytochrome P450 isoform CYP1B1. The antimetastatic activity of the selected complexes was assessed by evaluating their ability to impede the migration of A549 cells. The fourth chapter examines the anticancer effect of selected complexes on hormone-dependent (MCF-7) versus triple-negative (MDA-MB-231) breast cells. Changes in morphology, Ptd-L-Ser externalization, alterations in mitochondrial membrane potential, oxidative stress levels, cytochrome c release, and DNA damage were studied. Furthermore, in chapter five, molecular docking simulations were used to determine whether the most potent silver(I) phosphine complex across all cell lines bonds to estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Seven of the thirteen silver(I) phosphine complexes significantly reduced cell viability in malignant cell lines while being less toxic to non-malignant cells. Complex 4 best targeted all cancer types, with IC50 values ranging from 5.75 to 10.80 µM across malignant cell lines. In the malignant treated cells, morphological changes, reactive oxygen species production, mitochondrial membrane depolarization, and Ptd-L-Ser externalization were observed. Complexes 1 and 4 repressed cell migration in the A549 cells. The presence of damaged nuclei, metabolically inactive mitochondria and cytochrome c translocation from the mitochondria’ intermembrane to the cytosol in MCF-7 cells were observed. These findings suggest that complexes 2, 4 and 7 induced apoptotic cell death. Furthermore, in silico computational predictions suggested a promising interaction between complex 4, and ER-α and ER-β. Overall, this study demonstrates the potential of silver(I) phosphine complexes as anticancer agents, with promising effects on various cancer cell lines.
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    Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)
    (University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.
    Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.
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    HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure
    (University of the Witwatersrand, Johannesburg, 2023-09) Dlamini, Nozinhle Precious; Sayed, Yasien
    Human immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.
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    The ligandin activity of Schistosoma 26-kDa and 28-kDa glutathione transferases towards 17β-Hydroxyandrost-4-ene-3-one from a biophysical perspective
    (University of the Witwatersrand, Johannesburg, 2023) Makumbe, Hattie Hope; Achilonu, Ikechukwu Anthony
    Schistosomiasis, caused by helminth worms, ranks second amongst parasitic diseases and accounts for over 220 million fatalities globally. Statistics show that in South Africa, schistosomiasis (bilharzia) has infected approximately 4 million individuals. Currently, there are parasite resistance challenges with the sole available remedy. The World Health Organisation (WHO) acknowledges the need for new effective drugs. The 26-kDa Schistosoma bovis/haematobium (Sbh26GST) and 28-kDa Schistosoma haematobium (Sh28GST) are parasite Glutathione S-transferases (GSTs) which consist of two identical subunits that perform a vital role in mitigating the adverse effects of harmful electrophilic substances within the parasite since the parasite is devoid of the neutralizing cytochrome P-450. This automatically renders these parasite GSTs as potential therapeutic targets for schistosomiasis. Testosterone, the major hormone responsible for sexual characteristics and growth in males, can be repurposed as a drug target against schistosomiasis. In this study, we examined the structural, stability and functional interactions between the parasite GSTs and testosterone. After confirmation of inhibition, IC50 experiments were performed. The enzymes were overexpressed in Escherichia coli (E.coli) and then purified through a single-step nickel ion-immobilized metal affinity chromatography (IMAC). Extrinsic fluorescence spectroscopy was also done to provide evidence for the binding of the recombinant GSTs with testosterone. The GST activity was measured by employing 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Additionally, we investigated if the enzyme activity was influenced by the presence of testosterone. To analyse the stability of the enzymes, a SYPRO Orange-based thermal shift assay was used in the presence and absence of testosterone. In addition to empirical investigations, computational modelling, molecular docking, and molecular dynamic simulations were used to provide complementary insights to show binding affinities, prediction of binding modes and stability of the GST-testosterone complex. The secondary structural composition was found to be predominantly alpha-helical. Insights into tertiary structure analysis revealed the presence of buried solvent exposed tryptophan residues. The findings from spectroscopy with 8-anilino-1-naphthalenesulfonate (ANS) indicated that both Human GST-mu and parasite GSTs bound to ANS. Enzyme kinetic studies show that testosterone is a potent inhibitor of the parasite GSTs, with a specific activity that decreases from 16 μmol min-1mg-1 to 0.03 μmol min-1mg-1 and IC50 in the nanomolar range of 20 µM for Sh28GST. Sbh26GST exhibited a specific activity that decreased from 20 μmol min-1mg-1 to 0.14 μmol min-1mg-1, and a testosterone IC50 of 23 µM. The thermal stability assay confirmed Sh28GST to be more stable than Sbh26GST, and this stability of Sh28GST intensified when the enzyme bound to testosterone and GSH. Steady state kinetics towards glutathione (GSH) revealed a Km of 4.2mM and 6.6 mM for Sh28GST and Sbh26GST respectively. The present study has practical implications for novel application of the enzymes to serve as a basis for future studies aimed at development of inhibitors with potential therapeutic benefits through rational drug design.
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    Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines
    (University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne Jacqueline
    Breast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.
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    Establishing and characterizing organoid cultures from colon tissue of South African individuals
    (University of the Witwatersrand, Johannesburg, 2024) Du Plessis, Thea-Leonie; Kaur, Mandeep
    Colorectal cancer (CRC) has been poorly studied in South Africa, with limited studies on disease progression and development. Studies that have investigated CRC in South Africa have indicated that there is racial disparity between different racial groups that may be attributed to alternative developmental pathways, differences in genetic compositions or CRC initiators that result in these different clinical presentations. Furthermore, the lack of population-based studies substantiates the need for more intensive CRC research. A particular model used to study cancer in general is the use of two-dimensional (2D) cell cultures, which have provided novel insight into many cancers and their development processes. However, these models lack the complex biology observed in vivo. One such model that is gaining research interest is the use of three-dimensional (3D) organoid cultures. Organoids are derived from stem cells and are able to self-organize and mimic the corresponding organ from which they were derived. Research has indicated that organoids are able to maintain cell-type heterogeneity as well as gene expression levels that resemble the organ of origin. Therefore, this project aimed at standardizing a protocol to establish and characterise colorectal organoid cultures from South African patient-derived tissues. Patient samples were obtained from individual patients with informed consent and were processed to generate organoids. The morphology of the organoids was monitored across several days and across passages. Once the organoids had reached maturity and were at passage 2, characterization was performed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence which indicated that the genetic composition and spatial localization of cell types of interest in non-cancerous tissue was recapitulated in the organoids. Based on these observations, it is proposed that organoids could be a promising model to investigate CRC disease development and progression and potentially search for novel therapeutics. This project has established the protocols for growing and characterizing organoids from African samples and provides baseline data, and outlines the complexities and issues involved in growing organoid cultures for the future studies
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    A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding
    (University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, Sylvia
    During COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NC
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    Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
    (University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu Anthony
    NNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.