Analysis of the Stress-Induced Metabolic Response in JJN-3 Cells to Exposure to the APIM-Containing Peptide Drug ATX-101
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Cancer therapy with cytostatic drugs aims to damage the DNA and cause cell death. However, various forms of cancer are characterized by drug resistance, often resulting from increased DNA repair. Inhibiting the cells' ability for DNA repair represents a promising strategy to circumvent this resistance. The proliferating cell nuclear antigen (PCNA) is central to many of the repair processes in the cellular response to chemotherapy, through its interaction with various cellular proteins. Its key role in these processes makes it a promising therapeutic target for cancer therapy. During conditions of genotoxic stress, PCNAs interaction with its protein partners is largely dependent upon the AlkB homologue 2 PNCA-interacting motif (APIM). Inhibiting this interaction may thus improve the cellular sensitivity to cytostatic agents.The work presented in this thesis has evaluated the stress-induced metabolic response of the multiple myeloma cell line JJN-3 to exposure to the APIM-containing peptide drug ATX-101. A stress-response experiment involving 24 h of drug exposure to ATX-101 - both alone and in combination with the cytostatic cisplatin - was conducted. The results were evaluated by non-targeted metabolome analysis employing reversed-phase and amide LC-MS, and targeted analysis with GC-MS and CapIC-MS/MS. Additionally, glutamine and glucose utilization were assessed by performing enzymatic assays on samples from the growth medium. It was found that samples treated with the combinatorial treatment of ATX-101 and cisplatin had the lowest glucose uptake, particularly during the first 4 h following treatment. Yield coefficients revealed that samples treated with ATX-101 had the highest yield coefficient for lactate. For all treatments, the majority of the consumed glucose and glutamine was found to serve the purpose of energy maintenance, rather than proliferation. LC-MS analysis revealed that time 0 h samples exhibited considerable metabolic diversity, which points towards an immediate metabolic stress-response to the treatments given. ATX-101 showed potential activity as a single agent treatment, exhibiting a particularly noticeable effect on the more hydrophilic parts of the metabolome. Samples exposed to the combinatorial treatment of ATX-101 and cisplatin, or to ATX-101 alone, exhibited a high degree of metabolic similarity in the CapIC-MS analysis. Notably, samples exposed to either of these two treatment conditions exhibited low levels of UDP-N-acetylglucosamine. This possibly supports a role of ATX-101 in down-regulating the PI3K/Akt pathway, but further studies must be performed in order to confirm this finding.