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Development of a breast cancer spheroid model for use in drug discovery and cancer biology researchJurgens, Tamarin January 2020 (has links)
The screening of lead compounds during cancer drug discovery still results in many hits that never reach the clinic. This is in part due to the distance between 2D cell culture, which is mostly used for screening assays and the complexity of an in vivo tumour setting. It is thus essential that screening setups are developed that better bridge the gap between in vitro and in vivo studies. Culturing cells in 3D has been shown to provide an architecture and gene expression profile that better resembles that found in tumours. A number of methods have been developed but many are laborious, require specialised equipment, or are prohibitively costly. In Southern Africa, we do not always have access to expensive machinery and reagents. Therefore, in this thesis, we aimed to set up a simple, easy to use, cost-effective 3D spheroid methodology for triple negative breast cancer that could be used in any laboratory with minimal reagents.
The non-metastatic triple negative breast cancer cell line, BT-20, was identified as the cell line most amenable to spheroid formation and measurement, compared to the metastatic cell lines MDA-MB-231 and MCF-7. Both these cell lines formed inconsistent, difficult to measure spheroids. A low attachment methodology was settled on to induce cell aggregation and spheroid formation. This proved to be an easy, low-cost, and reproducible method. Using the developed methodology, the efficacy of several novel compounds with anti-proliferative activity in 2D cancer cell line cultures were tested for their ability to affect cell survival in the spheroid model.
Our studies show that one non-sulfamoylated 2-methoxyestradiol derivative, EE-15-one, caused a loss of cells in the 2D cell survival assay while it had no effect on BT-20 spheroids. In contrast, two other 2-methoxyestradiol derivatives had similar effects in 2D and 3D. Another novel anticancer compound, STX1972, also had anti-proliferative capabilities in 2D which were lost in cells grown in 3D. As a result, our screening approach on a small number of samples was able to identify 2 of 5 compounds that had no effect on cancer cell growth in 3D. Further analysis of EE-15-one suggests that this compound does not inhibit cell cycle progression like the other derivatives tested but instead inhibits cell adhesion. Our data shows that integrin-based adhesion is replaced by cadherin-dependent cell-cell adhesion in spheroids. Changing the mode of adhesion correlates strongly with the efficacy of this compound suggesting that it is an inhibitor of integrin dependent cell adhesion. Further analysis show that the efficacy of this compound was not dependent on hypoxia, further strengthening the suggestion that it directly acts on cell adhesion.
It is commonly accepted that a tumour consists of a heterogeneous mix of subpopulations of cancer cells, each with altered genetic backgrounds. Furthermore, such subpopulations can affect each other changing behaviours of neighbouring cells. To replicate this scenario in vitro, we initiated the development of a co-culture spheroid system of different triple negative breast cancer cell lines to investigate how they behave within a spheroid. Our data shows that when a co-culture spheroid system of BT-20 and MDA-MB-231 cells is generated, the cells with migratory ability were able to migrate away from the spheroid onto rigid surfaces. In contrast, the non-metastatic BT-20 cells remained within the confines of the spheroid. This suggests that indeed different cell populations will continue to behave differently within a 3D cell culture setting.
In conclusion, we have developed a robust, cost-effective 3D culture system that has shown great potential for used in high throughput screening of novel anti-proliferative compounds. We have shown that these spheroids use different adhesive strategies than their counterparts kept in 2D. This suggests that these cells also change their cellular behaviour which is essential for better mimicking the in vivo tumour setting. / Thesis (PhD)--University of Pretoria, 2020. / NRF-DAAD / MRC / CANSA / Physiology / PhD / Restricted
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Deciphering the Role of Adrenergic Hormones in Embryonic Cardiac Calcium Signaling and MetabolismPeoples, Jessica 01 January 2018 (has links) (PDF)
The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are critical regulators of mammalian cardiovascular physiology. NE and EPI mediate stress responses to enhance cardiovascular function, however dysregulation of adrenergic signaling leads to heart failure, congenital heart malformations, and sudden cardiac death. Adrenergic hormone-expressing cells were found in the early embryonic heart, and NE has been determined essential for embryonic cardiac development. Despite extensive work in adults, the regulatory roles and adrenergic targets of these hormones during embryonic cardiac development have not yet been fully determined. Prior transcriptomic studies from our lab showed that expression of signal transduction and metabolic genes in embryos lacking adrenergic hormones were by far the most affected categories of genes. Thus, we hypothesized that adrenergic hormones stimulate early calcium signaling, and are required for sufficient supply of energy substrates for the metabolic shift from anaerobic glycolysis to aerobic respiration during heart development. We utilized the dopamine ?-hydroxylase knock-out (Dbh-/-) mouse model to examine effects of adrenergic-deficiency on calcium signaling and metabolism during heart development. Using calcium-imaging and patch-clamp techniques, we found that calcium transients, voltage-gated calcium channels, and L-type calcium currents in adrenergic-deficient embryonic hearts were not affected relative to controls indicating adrenergic stimulation did not influence early calcium signaling. Metabolomics analyses of adrenergic-deficient hearts revealed disruption in glycolytic and pentose-phosphate pathways as well as reduced activity of respective regulatory enzymes, glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase indicating compromised glucose metabolism. Addition of pyruvate to embryonic hearts led to significant recovery of ATP concentrations and oxygen consumption rates, thereby supporting the hypothesis that adrenergic-deficient hearts are "starved" of metabolic substrates required for transitions from anaerobic glycolysis to aerobic metabolism. Overall, we showed that adrenergic hormones are not necessary for calcium signaling in the embryonic heart, but are essential regulators ensuring sufficient metabolic substrate and boosting enzymatic activities to fuel aerobic metabolism.
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Delineating the Cytotoxic Mechanism of Purine Metabolite AICAR in Lung Cancer CellsAftab, Fareesa 01 January 2022 (has links) (PDF)
Lung cancer is the leading cause of cancer death and the second most common cancer in the United States. Targeted therapies have led to an increase in survival gains; however, their use is dependent on presence of druggable mutations and hindered by emergence of drug resistance. 5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR or Acadesine) is a natural metabolite of de novo purine synthesis pathway, catalyzed by adenylosuccinate lyase (ADSL), and it is a precursor to 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP). ZMP mimics adenosine monophosphate (AMP) and activates adenosine monophosphate-activated protein kinase (AMPK) and has been shown to specifically induce apoptosis in aneuploid cells. Cell viability assay was used to establish that AICAR can induce cytotoxicity in EGFR and KRAS mutated lung cancer cells irrespective of the mutational status.; thereby, making AICAR a potential anti-cancer drug candidate. However, AICAR's mechanism of cytotoxicity is not understood in lung cancer. FINDSITEcomb2.0 analysis, western blot and thermal shift binding assay identified the cytoplasmic tail of mucin 1 (MUC1-CT), a membrane glycoprotein overexpressed and aberrantly glycosylated in adenocarcinomas, is directly bound and degraded by AICAR. We also found that AICAR can disrupt Janus kinase 1 (JAK1) and MUC1-CT interaction and downregulate JAK1 and epidermal growth factor receptor (EGFR) signaling pathways. Moreover, EGFR mutant patient-derived xenograft tumor and mouse lung tumor tissue-derived organoids showed an enhanced reduction in size with co-treatment of AICAR, EGFR and JAK inhibitors. Moreover, AICAR significantly reduced xenograft tumor weight and size in nude mice without body weight loss Furthermore, MUC1 overexpression rescued AICAR-induced cytotoxicity in H1975 organoids. Therefore, this study is the first to discover MUC1 as the novel binding partner of AICAR and modulator of AICAR's toxicity and the first to show that AICAR can disrupt JAK1-MUC1-CT interaction.
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Bacterial Association Networks from Healthy and Cancer-associated Gut MicrobiomesLoftus, Mark 01 January 2021 (has links) (PDF)
The human gastrointestinal tract is colonized by a diverse community of symbiotic microorganisms, mainly bacteria, that are known to play essential roles in maintaining the health of their human host. Disruption of this bacterial community has been associated with numerous diseases, including Colorectal Cancer (CRC). CRC is one of the leading causes of cancer-related deaths worldwide. As such, focus has been placed on the modulation of the bacterial community within the cancer-associated gut microbiome as the next step in possible CRC treatment and prevention strategies. To use the bacterial community for these purposes, a better understanding of the associations that exist between bacteria within the healthy human gut microbiome and how these associations have changed within the CRC-associated gut microbiome is direly needed. In this dissertation, we first utilized whole-genome shotgun sequence data from four previously published healthy human cohorts to explore the composition and community structure of the healthy gut microbiome across populations. We show that despite species carriage differences, bacterial communities across healthy human populations are similar in both their structure and functional capacities. In addition, we found that positive associations occur between taxonomically and functionally related species in the gut microbiome. In follow-up work, we employed a similar approach to study the bacterial community composition and structure in late-stage CRC patient gut microbiomes. We found key differences between CRC and healthy gut bacterial communities, suggesting an overgrowth of potentially pathogenic species classified as oral microbes. Additionally, a striking difference in the bacterial community structure was found which we believe to be a bacterial response to probable ecosystem changes associated with tumor formation in the CRC-associated gut microbiome. Overall, our findings shed new light on how the bacterial community is structured within the healthy gut microbiome and how this structure has changed in the late-stage CRC-associated gut microbiome.
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Chaperonin Containing TCP1 (CCT) as a Target for Cancer TherapyCarr, Ana 01 January 2017 (has links)
Treatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the CCT2 subunit is highly expressed in breast cancer and could be therapeutically targeted. To determine whether CCT is a marker of disease progression in other cancers, we analyzed CCT2 gene expression in liver, prostate and lung cancer, using publicly available genetic databases, and confirmed findings by assessing CCT2 and client proteins, like STAT3, in tumor tissues by immunohistochemistry. We found that CCT2 was high in all cancers, especially SCLC, and correlated with decreased patient survival. We tested CT20p, the peptide therapeutic developed by our laboratory to inhibit CCT, on SCLC and primary lung cells, finding that CT20p was only cytotoxic to SCLC cells. Since SCLC currently lacks targeted therapeutics, our work yielded a new targeted agent that could improve lung cancer mortality. To establish a mechanism of action for CT20p, we partially knocked out CCT2 in TNBC cells, which decreased tumorigenicity in mice and reduced levels of essential proteins like STAT3. To confirm, we overexpressed CCT2 in non-tumorigenic cells and conferred tumor-like characteristics such as increased migration and elevated STAT3. These studies positioned us to develop and validate a strategy for discovery of new small molecule inhibitors of CCT. We thus advanced the field of cancer research by demonstrating that CCT could have diagnostic potential for cancers, such as SCLC and TNBC, that are a significant cause of human death and showed that targeting CCT is a promising therapeutic approach.
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Chaperonin-containing TCP-1 Modulates Breast Cancer Growth and MalignancyShowalter, Anne 01 January 2020 (has links)
Among women worldwide, breast cancer is the second leading cause of death. Despite improved screening techniques and new treatments such as immunotherapies and targeted hormone therapy, survival rates of advanced or metastatic disease are at 27%. Current treatments focus on the primary tumor and are generally ineffective against metastases due to the location and heterogeneity of the metastatic tumor cells. However, all cells rely on the proteostasis network (PN), a collective term for protein synthesis, folding, and degradation, consisting of both chaperones and chaperonins. Chaperonin-containing TCP-1 (CCT) consists of 16 heterologous subunits (CCT1-8) and folds ~10% of the known proteome. However, breast cancer cells rely heavily on CCT clients including oncogenes and mutated tumor suppressors as well as essential cytoskeletal proteins. The objective of my research is to investigate the role of CCT in cancer by overexpressing or depleting a single CCT subunit, CCT2 (2nd subunit of CCT) in both breast cancer and normal breast epithelial cells. We found increasing CCT2 in a 2D setting leads to a more invasive phenotype in breast cancer cells characterized by increased proliferation and migration. Additionally we observed increased tubulin, one of CCT's obligate clients as well as increasing other CCT subunits. Loss of CCT2 leads to cell death, reduction of other CCT subunits, and in vivo prevents tumors from forming. To further understand the role of CCT, we grew CCT2 overexpressing breast cancer lines as spheroids, an in vitro tumor analogue. The CCT2 overexpressing spheroids grew faster and larger than control cells. Additionally, they express increased actin, another CCT obligate client, as well as show significantly increased attachment with both cell-to-cell and cell-to-substrate connections, which could be suggestive of a metastatic phenotype. Finally, we used a CCT inhibitor, CT20p, to treat breast cancer cells and found it induces immunogenic cell death (ICD). ICD stimulates the immune system to attack the tumor cells and mice vaccinated with dying CT20p-treated cells did develop protective immunity against challenge with live cancer cells. Investigating the role of CCT in breast cancer advances both a novel marker and potential target for new cancer treatments, meeting an urgent need to reduce the mortality of advanced and metastatic breast cancer.
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The Identification and Segmentation of Astrocytoma Prior to Critical Mass, by means of a Volumetric/Subregion Regression Analysis of Normal and Neoplastic Brain TissueHiggins, Lyn 01 January 2018 (has links)
As the underlying cause of Glioblastoma Multiforme (GBM) is presently unclear, this research implements a new approach to identifying and segmenting plausible instances of GBM prior to critical mass. Grade-IV Astrocytoma, or GBM, is an aggressive and malignant cancer arising from star-shaped glial cells, or astrocytes, where the astrocytes, functionally, assist in the support and protection of neurons within the central nervous system and spinal cord. Subsequently, our motivation for researching the ability to recognize GBM is that the underlying cause of the mutation is presently unclear, leading to the operative that GBM is only detectable through a combination of MRI and CT brain scans, cooperatively, along with a resection biopsy. Since astrocytoma only becomes evident at critical mass, when the cellular structure of the neoplasm becomes visible within the image, this research seeks to achieve earlier identification and segmentation of the neoplasm by evaluating the malignant area via a volumetric voxel approach to removing noise artifacts and analyzing voxel differentials. In order to investigate neoplasm continuity, a differential approach has been implemented utilizing a multi-polynomial/multi-domain regression algorithm, thus, ultimately, providing a graphical and mathematical analysis of the differentials within critical mass and non-critical mass images. Given these augmentations to MRI and CT image rectifications, we theorize that our approach will improve on astrocytoma recognition and segmentation, along with achieving greater accuracy in diagnostic evaluations of the malignant area.
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The interaction between Rad9 and Tousled-like kinase 1 in the cell cycle and the DNA damage responseKelly, Ryan 24 December 2013 (has links)
Genomic integrity is preserved by checkpoints, which are signal transduction pathways that serve to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a proliferating cell nuclear antigen (PCNA)-like clamp that is loaded onto DNA at structures resulting from damage, and is important for initiating and maintaining checkpoint signaling. Rad9 possesses a C-terminal tail unrelated to PCNA that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. This thesis establishes that Rad9 is indeed phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. In addition, TLK1 and Rad9 were shown to interact constitutively, and this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, this thesis demonstrates that TLK1 is required for progression through S-phase in normally cycling cells, and that depletion of TLK1 results in a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 is transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, this work proposes that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn plays a role in regulating the DNA damage response. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-12-24 10:31:57.987
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The effects of parthenolide on cervical cancer prone cells /Megha, Rishi. January 2009 (has links)
Thesis (B.S.) Magna Cum Laude --Butler University, 2009. / Includes bibliographical references (leaf 26).
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The Biology of the Receptor for Advanced Glycation End Products (RAGE) in CancerKadasah, Sultan Ftayes Saeed January 2020 (has links)
Overexpression of the Receptor for Advanced Glycation End Products (RAGE) has been implicated in multiple diseases, including several types of cancer. In different types of cancer, RAGE has been shown to promote cell survival by either autophagy or activation of the transcription factor NF-κB. Based on what is known about RAGE, we hypothesized that the RAGE/ligand interaction at the cell surface promotes pancreatic cancer and melanoma cell survival by both pathways, autophagy and NF-κB activation.
To study the role of RAGE in pancreatic cancer resistance to chemotherapy, BxPC-3, MIA PaCa-2, PANC-1, and RAGE overexpressing PANC-1 FLR2 cell-lines were used. A significant decrease in cell viability was observed upon gemcitabine treatment with further significant reduction in cell viability upon combination of gemcitabine with the RAGE inhibitor IgG 2A11. In our studies we showed that RAGE plays a central role in pancreatic cancer cell resistance to gemcitabine by increasing autophagy.
To test the importance of RAGE localization in mediating drug resistance, three melanoma cell-lines (WM115, WM266, and SK-MEL2) with their daughters, RAGE overexpressing cells (WM115-RAGE, WM266-RAGE, and SK-MEL2-RAGE) were used. Wild type cell-lines only expressed RAGE intracellularly while RAGE overexpressing cells expressed RAGE both at the cell surface and inside cells. We show in this study that only the cell surface RAGE is involved in melanoma resistance to dacarbazine.
We next tested the effects of RAGE/RAGE ligand interaction at the cell surface in pancreatic tumor growth. We used two carcinoma cell-lines, PANC-1 and MIA PaCa-2, for this purpose. Both cell-lines were transiently transfected with a NF-κB/Luciferase reporter plasmid to test the effects of the interaction between RAGE and its ligands on the activation of the NF-κB signaling pathway. We observed higher NF-κB activity upon treatment with RAGE ligands (AGE, S100P, and S100A8/A9) compared to non-treated cells. Higher activity of NF-κB was coupled with a higher expression of cyclin D1 and lower expression of p53, NF-κB target genes. / Cobre grant "P20GM109024"
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