3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item A novel peptide-enhanced drug delivery system for squamous cell oesophageal carcinoma(2017) Adeyemi, Samson AdebowaleCancer has been described as one of the major and leading causes of death worldwide. By the year 2030, it has been postulated that over 21.4 million new cases of cancer are anticipated, with 17 million cancer deaths annually and a total of 75 million people living with cancer within five years of diagnosis. Chemotherapy is the main therapeutic intervention for treating people living with oesophageal squamous cell carcinoma (OSCC). However, drug resistance, non-targeted delivery, sub-optimal dosage at disease sites and side effects on healthy cells have rendered it inefficient and ineffective in combating the disease even after combination chemotherapy. The paradigm shift in cancer nanomedicine employs the use of short functional peptides and ligands, conjugated to the surfaces of nanoparticles, for direct and active drug delivery systems in in vitro and in vivo assays. Smart and intelligent nanosystems remain a proactive and promising treatment alternative to circumvent the anomalies of current convectional cancer chemotherapeutics and enhance their delivery for optimal anti-tumoral effects. Based on these modalities, the conceptualization, design, optimization and characterization of a smart peptide-enhanced ligand-functionalized nano-construct – referred to herein as a PEL nanosystem – capable of encapsulating, targeting and controlling the release of endostatin (ENT), was fabricated in this study. Physicochemical parameters that characterized the design of a smart nano-construct in cancer therapy including satisfactory size, shape and surface properties, cellular uptake and internalization by tumor cells, low cellular toxicity to healthy cells and enhanced anti-tumoral activity of the encapsulated drug informed the fabrication of the PEL nanosystem. An optimized PEG-PEI-CHT nano-conjugate was developed as predicted by the Box–Behnken design model and surface-functionalized with Ly-P-1, PENT and FA as targeting moieties. Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (NMR) analysis confirmed the successful grafting of the nano-conjugates while Transmission Electron Microscopy (TEM) and Dynamic Light Scathering (DLS) analyses confirmed the synthesis of PEL nanoparticles with an average size less than 100nm. Scanning Electron Microscopy (SEM) results confirmed the morphology of the PEL nanosystem to be spherical with rough surfaces due to the attachments of the functionalized moieties. The release profile of the PEL nanosystem showed increased release of ENT at the acidic tumor micro-environment than observed at the physiological pH of healthy cells. Interestingly, the smart PEL nanosystem exhibited an enhanced targeted release of ENT for anti-tumoral effects on KYSE-30 cells relative to the unmodified nanosystem. The PEL nanosystem loaded with ENT showed a pragmatic inhibition of potent angiogenic factors including cell proliferation, nuclear apoptosis and necrosis, cell migration and invasion, as well as reduced expressions of both VEGF-C and MMP2 proteins as molecular makers for anti-angiogenesis. Athymic nude mice induced with OSSC xenografts showed a dramatic reduction in tumor volume with increased necrotic arears after treatment with the PEL ENT-loaded nanoparticles relative to the control. Overall, detailed in vitro, ex vivo cellular and in vivo experiments validated the fabricated PEL nanoparticulate systems as efficient delivery vehicles of ENT for enhancing its anti-tumoral activity by targeting the angiogenic pathway in KYSE-30 cells as presented in this study. While ENT was selected as a peptide-based anti-cancer model drug in this study due to its broad spectrum anti-angiogenic activities and limitations, the novel PEL nanosystem can be employed to incorporate alternative cancer chemotherapeutics for enhanced on-site delivery for an optimum therapeutic response in cancer therapy.Item The integrative role of Glycogen Synthase Kinase 3B (GSK3B) in adhesion-originating signals human oesophageal squamous cell carcinoma cells(2017) Mahomed, SabeehaThe serine-threonine kinase glycogen synthase kinase 3β (GSK3β), has, in recent years, become established as hub for a myriad of intracellular signalling pathways. Many of these pathways have been implicated in cell cycle progression and proliferation in a multitude of carcinomas. GSK3β is ubiquitously expressed and considered to be constitutively active, and phosphorylation at the N-terminus serine 9 residue results in the inhibition. Interestingly, another prominent phosphorylation at tyrosine 216 in the activation loop has been reported to enhance GSK3β activity 200-fold. Its’ role in human oesophageal squamous cell carcinoma (HOSCC) migration, however is not well characterised. This study established that both active and inactive GSK3β are present in high abundance in HOSCC cells at rest. In order to characterise the influence of GSK3β on the migratory phenotype of HOSCC, focal adhesion kinase (FAK), a focal adhesion-associated protein known to be increasingly activated during cell migration (FAK(Tyr397)) was used as a marker for cell migration. The abundance of active GSK3β (pGSK3β(Tyr216)) was found to fluctuate during cell migration into a wound over 24 hours. Further investigation via the abrogation of GSK3β revealed that the observed variation was not a result of migration. Instead active GSK3β was found to differentially influence the migratory response observed in HOSCC cells by either 1.) promoting laemellipod extension and cell migration or 2.) partially-abrogate these processes. These findings however, did not produce the expected biochemical alterations with respect to the abundance of pFAK(Tyr397). Moreover, the effect of GSK3β-inhibition on HOSCC was shown to be dependent on the order in which wound initiation and GSK3β occurred, as abrogation of GSK3β prior to wound initiation was seen to either 1.) simply sustain the changes in the migratory response or 2.) produce little variation in the migratory response, suggesting the existence of “rescue-signalling”. These influences thus present GSK3β as a key regulator in HOSCC migration. Additionally culturing HOSCC cells on either collagen I of fibronectin, presented general decreases in the abundance of activated FAK, suggesting that varying suggesting that no one ECM-component, but rather the cell surface receptor distribution has become more diverse. This diversity may be a contributing factor to the varied influence of active GSKβ on the migratory response observed in HOSCC.Item Investigating telomere dynamics in oesophageal squamous carcinoma cells using standard and gold nanoparticle-based assays(2017) Bernert, MartinCancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques.Item The influence of p90RSK on FAK-dependent signalling in human oesophageal squamous carcinoma cells(2017) Lachenicht, CandiceThe focal adhesion kinase or FAK plays an important role in detecting and transducing signals that are generated by cell-substrate attachment (Focal adhesions). When these pathways are activated under atypical conditions they may promote metastasis, uncontrolled proliferation and a chemo-resistant phenotype. However the mechanisms by which this protein is activated ectopically in human oesophageal squamous cell carcinomas cell lines (HOSCC) is unknown. In the current study it was hypothesised that the p90 ribosomal S6 kinase, a key member of multiple pro-survival pathways (activator of the Y-box binding protein-1), activates FAK. RSK may promote FAK activation directly, from its location at the plasma membrane, or it may modulate FAK activation indirectly via the regulation of one of its substrates. RSK inhibits the activation of the glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9. GSK3β also localises at focal adhesions and may therefore play a role in mediating FAK activity. To ascertain the role RSK plays in FAK activation, 3 inhibition studies were performed. In the first assay, RSK was specifically inhibited within HOSCC and the levels of active FAK monitored (two different environmental conditions). FAK activation was monitored by detecting the auto-phosphorylation of FAK at Tyr397. A GSK3β inhibition assay was then performed in which GSK3β was specifically inhibited and the levels of active FAK monitored. Lastly, a dual inhibition assay was performed where both RSK and GSK3β were inhibited simultaneously and the levels of active FAK monitored. The overall net changes in the phospho-protein profile indicated that all of the HOSCC cells had distinct cellular responses to the three inhibitor combinations. However RSK did not appear to activate/inhibit FAK activity directly, in most of the HOSCC cells, but rather modulated FAK activation through the inhibition of GSK3β. The effects the RSK/GSK3β pathway had on FAK activation was partially dependent on the HOSCC cells containing active levels of PTEN. Interestingly, the inhibition of both GSK3β and RSK reduced the levels of active FAK in 3 of the 5 HOSCC cell lines, indicating that this might be a good anti-cancer therapeutic. RSK appeared to play a more context specific role in FAK activation within the HOSCC cells suggesting that the grading system for moderately differentiated carcinomas needs to be improved. This paper also highlights the importance of studying the effects the microenvironment has on neoplasmic transformation as varied environmental conditions, during the RSK inhibition studies, drastically impacted the effects the RSK inhibitor had on FAK activation.