1,721,187 research outputs found
Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer
No full textThesis Submitted in fulfillment of the requirements for the degree Philosophiae Doctor (PhD.) in Molecular and Cell Biology in the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa 2019Cancer 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 agentNational Research Foundation (NRF) tMM202
Investigation of serum elements and biochemical variables as a risk factor for female infertility
No full textCharacterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer
No full textA research report submitted in partial fulfillment of the requirements for the degree of Master of Science in Molecular and Cell Biology in the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2022The 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 metastasisMM202
Investigating the Effect of Cholesterol Depletion on IL-6 Cytokine Induced Epithelial-to-Mesenchymal Transition in Colorectal Cancer
No full textA research report submitted in fulfillment of the requirements for the Master of Science in Molecular and Cell Biology, In the Faculty of Commerce, Law and Management, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022Colorectal cancer (CRC) is the third leading cause of cancer globally. Poor patient prognosis is attributed to late diagnosis and high metastatic potential of the disease. The process of metastasis usually favours the conversion between epithelial and mesenchymal cellular states, termed epithelial-to-mesenchymal transition (EMT). EMT has been reported to contribute to drug resistance, following treatments with chemotherapy, which allows for cancer recurrence and metastasis. Additionally, increasing evidence indicates the role of the cholesterol biosynthesis pathway in EMT progression. The lipid composition of epithelial cells compared to mesenchymal cells is distinct and cells that undergo EMT show an elevation in cholesterol levels to facilitate increased proliferation and oncogenic signalling. This study aimed to investigate the effect of cholesterol depletion in EMT induced CRC and explored how cholesterol depletion influences the susceptibility of EMT induced cells to the chemotherapy cocktail referred to as FOLFOX (5-fluorouracil and oxaliplatin). Results showed that following EMT induction by proinflammatory cytokine interleukin-6 (IL-6), in HT-29 cells, cellular cholesterol content increased by ~30%. Cholesterol depletion with compounds methyl-β-cyclodextrin (MβCD) and KS-01, in post-EMT cells, showed to reverse EMT phenotypic changes as noted by increased epithelial marker E-cadherin (by 46.9% and 55.3%, respectively), decreased mesenchymal marker vimentin (by 41.0% and 52.8%, respectively) and reduced invasive potential (by 43.0% and 47.8%, respectively). Interestingly, EMT induced cells displayed increased sensitivity to the FOLFOX chemotherapy, following cholesterol depletion, as observed by enhanced susceptibility to apoptosis (by 46.7% and 60.3%, respectively) and attenuation of multidrug resistance (by 80.6% and 71.8%, respectively). Ultimately, this study highlights cholesterol depletion to be a promising strategy for potential treatment of metastatic CRC.National Research Foundation (NRF)MM202
A Phenotype Prediction Framework for Classifying Colorectal Cancer Patients’ Response to FOLFOX Treatment: An Integrated Approach
No full textA research report submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2024Colorectal cancer (CRC), characterised by its prevalence and heterogeneity, poses a significant challenge in understanding drug resistance, especially in the context of FOLFOX treatment. This study presents an innovative methodology that integrates diverse data analysis approaches to address the challenge of predicting the phenotype of CRC patients resistant or sensitive to FOLFOX. The initial analysis involved dierential and co-expression analyses, identifying pivotal hub genes crucial to drug resistance in CRC, regulating intricate molecular networks. Subsequent enrichment analysis revealed their significant roles in biological functions, particularly influencing DNA repair and nuclear division. To capture inherent topological characteristics within genetic expression data, a novel technique utilising topological data analysis (TDA) was employed. By applying persistence homology to generate persistence images, the Vietoris-Rips complex was constructed using the signed-topological overlap matrix, comprehensively capturing numerous topological features, including high-dimensional Betti-1 and Betti-2. This provided valuable insights into the structural patterns of gene expression between the hub genes. Furthermore, the integration of whole-slide images enhanced understanding of tissue anatomy, which is crucial for predicting cancer stages. Using a MobileNet architecture, a deep learning model classified cancer stages, contributing to a holistic understanding of colorectal tumor microenvironments. For predictive modelling of drug resistance, a multilayer perceptron applied topological summaries generated by TDA. The developed framework, GeTopology, exhibited remarkable performance metrics, achieving an overall 83% accuracy in predicting the FOLFOX response, demonstrating a 3% improvement over a previously published phenotype prediction framework (NSCLC ) that utilised similar data modes. Robust accuracies were consistently observed in independent datasets, classifying both cancer patients and healthy individuals. The results indicated an approximate 10% increase in model prediction accuracy compared to NSCLC, emphasising the potential clinical impact of this integrative approach. In conclusion, this study advances the understanding of drug resistance in CRC by proposing a novel approach that integrates topology with histopathological images, oering transformative insights into predictive modelling and precision medicineMM202
Establishing and characterizing organoid cultures from colon tissue of South African individuals
No full textA Dissertation Submitted in fulfillment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa, 2024Colorectal 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 studiesNRFMM202
The effect of cholesterol depletion on TGF-ß-induced epithelial-mesenchymal transition in pancreatic cancer cells
No full textA dissertation submitted in fulfilment of the requirements for the degree Master of Science, to the Faculty of Science, Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2024.Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic cancer that relies on the epithelial to mesenchymal transition (EMT) program for its spread. EMT is a cell plasticity program that involves the reorganization of cell structure as cells transition from an epithelial to a mesenchymal phenotype. The dysregulated cholesterol metabolism resulting from metabolic reprogramming in PDAC is thought to play a role in EMT by affecting EMT-related signalling pathways. However, no publication has yet investigated the impact of EMT on cholesterol content in PDAC. To shed light on these dynamics, EMT was induced in PANC-1 cells using TGF-β1, thereafter the effect of cholesterol-depleting agents (KS-01 and methyl-β-cyclodextrin) alone or in combination with chemotherapeutic agents (Gemcitabine (GEM) and 5-Fluorouracil (5-FU)) on cholesterol content, EMT state, drug resistance, and invasion were investigated. Our results showed that mesenchymal cells rely on reduced membrane cholesterol levels, synthesis, and uptake, while storing more cholesterol and promoting efflux. EMT also promoted drug resistance via upregulation of ABCB1 expression and reduced hENT1 expression. Targeting cholesterol using cyclodextrins promoted a cholesterol compensatory mechanism, leading to a hybrid EMT state, drug resistance, and metastatic potential. Treating mesenchymal PANC-1 cells with GEM or 5-FU monotherapies were seen to promote EMT-transcription factors, as well as promote cholesterol efflux, synthesis, and import, an unexpected result as these chemotherapeutic agents are not known to affect cholesterol. When GEM was combined with KS-01, drug resistance, invasion, EMT-transcription factors, vimentin, and E-cadherin was promoted indicating the promotion of a hybrid EMT state. Interestingly however, combining KS-01 with 5-FU resulted in an interplay that was seen to mitigate the EMT-promoting effects typically associated with cholesterol depletion alone. The exact mechanism linking the cholesterol compensatory mechanism to EMT remains complex and unknown. Based on work presented in this dissertation, it is proposed that targeting cellular cholesterol should be continued to be investigated, particularly in understanding the repercussions of the use of cholesterol depleting agents for the treatment of other disorders in patients with PDAC.University of the Witwatersrand, Johannesburg (Kirsch Foundation)Self-Initiated Research Grant from South African Medical Research Council [SAMRC]MMM202
Analysing RNA-sequence data for pancreatic ductal adenocarcinoma tissue samples to identify potential biomarkers
No full textA dissertation submitted in fulfilment of the requirements for the degree Master of Science, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancer and is the fourth leading cause of death with a five-year survival rate of less than 10%. Patients are asymptomatic until detection is observed at a metastatic stage, hence contributing massively towards the high mortality rate. This study was conducted to explore PDAC and its two main subtypes, the classical and basal-like subtype, in an in-depth level via bioinformatic analysis. Bioinformatics is a computational approach to evaluate biological data by analysing omics data including genomic expression and proteomic sequences. A workflow consisting of programmes and web-tools was used to analyse PDAC RNA-sequence data. The sample sets were grouped according to tumour, stage, and subtype. The workflow began with quality control using FastQC and Trimmomatic. Alignment of sequencing files and counts were done through HISAT2 and HTSeq. The main component of this workflow was differential gene expression analysis to identify differentially expressed genes (DEGs), statistically significant genes, per compared conditions. WGCNA was used for co-expression analysis to identify the hub genes involved in regulating the biological network. Lastly, in-silico validation was done by using available web tools to support the findings of this workflow. The identified tumour genes included S100A11, PKM, GPRC5A, LAMC2 and ITGA2, which may represent as universal biomarkers as sample extraction was performed from data generated from individuals belonging to 8 different countries. KRT13 and IL6 were identified in the advanced stage and their role in cancer progression have been explored in this current study. The basal-like subtype had CAV1, DCVLD2 and TGFB2 genes that contribute to treatment resistance. The common dysregulated genes in the basal-like subtype and advanced stage were analysed to evaluate the link between subtype and stage which included WNT3A, TP63, KRT13 and IGF2BP. Coexpression analysis revealed hub genes for tumour (KIF4A, SPAG5, RRM2 and AURKA), basal-like subtype (BUB1, DEPDC1 and KIF14) and classical-subtype (PTPRN and CAMK2B). Through a machine learning model, recall, precision and accuracy scores per sample conditions for the DEGs were all above 94%. These potential biomarkers all have significant roles in promoting cancer progression, aggression and resistance. Hence, these may serve as a less invasive screening method for PDAC as DEGs were classified based on tissue or blood (extracellular vesicle) biomarkers. However, further wet laboratory validation is required for these biomarkers.South African Medical Research Council ( (SAMRC), Cape Town- Self-Initiated Research (SIR) Grant.MM202
Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling
Get PDFT lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells
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