Author: Saumya Arora
Arora, Saumya, 2024 Identifying the membrane signalling pathways regulated by newly discovered natural products that block Glioblastoma invasiveness, Flinders University, College of Medicine and Public Health
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Glioblastoma (GBM) is the most fatal, invasive and recurrent form of central nervous system cancer. Despite the standardized treatment, median survival rate is constrained at 15 months post treatment and unimproved prognosis. Prognosis impacted by inevitable recurrence is primarily due to highly invasive nature of tumor cells. Currently, the research focus is on developing cytocidal and antiproliferative drugs, leaving a research gap of limited testing of agents to prevent GBM recurrence. Overexpression of Aquaporins (water channels) and Calcium permeable Glutamate receptors with intricately regulated intracellular Calcium levels are known to enhance the invasive ability of GBM cells. Additionally, Glutamate, the most significant excitatory neurotransmitter, is oversecreted by GBM cells to cause excitotoxic death of neural cells and to promote invasion of surrounding tumor cells. The novel, naturally sourced compounds RAD060 (SN096), RAD062 (SN098) and RAD203 (Xanthurenic Acid) have been tested for 4.5 to 5-hour period and proven to exert invasion inhibitory effect without cytotoxicity. The novel objective of this study was investigation of impact of longer duration of application of test compounds on GBM cell viability. 1:1 combinations of these compounds, to discover a plausibly differential impact on cellular viability, were also examined. Additionally, optimization of protocol was aimed to explore a potential impact on intracellular calcium flux in the presence and absence of Glutamate.
A 24-hour application period to adult (U251) and pediatric (KNS42) GBM cells was followed by Alamar Blue cell viability assay. 1 µM concentration of the compounds was further explored for their effect on Calcium oscillations across GBM cell membranes using a fluorescent Calcium indicator, Fluo-3 AM. The protocol was optimized with microplate reader, followed by troubleshooting impediments. To obtain the instant change in intracellular Calcium levels, the stained cells were visualized with microscopic live cell chamber when exposed to Calcium with or without Glutamate.
Conclusively, 1 µM Xanthurenic Acid (XA) was found to be toxic towards U251 (adult glioma cell line), while 1:1 combinations of lower concentrations were proved to be non-cytocidal. Inhibition of Glutamate receptors by RAD203 was verified, on comparison with control of known Glutamate receptor inhibitor (CNQX); however, inefficiency at optimized concentration was noted in KNS42. RAD060 had similar effect as RAD203 (XA), while RAD062 treated cells allowed Calcium influx. Mechanism of the compounds could not be ascertained due to lack of statistical significance for obtained results.
Keywords: Glioblastoma, Invasion, Brain cancer, Glioblastoma treatment, Anti-invasion, Aquaporin, Calcium oscillations
Subject: Medical Biotechnology thesis
Thesis type: Masters
Completed: 2024
School: College of Medicine and Public Health
Supervisor: Dr Sunita Ramesh