Metabolic Profiling of Cisplatin and ATX-101 Induced Stress-responses in the Bladder Cancer Cell Line T24
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Proliferating cell nuclear antigen (PCNA) is a protein central in many cellular processes through its numerous protein interaction partners. Recently, it has been shown that several proteins involved in stress related processes (including DNA repair) interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM) sequence. The APIM sequence has also been discovered in cytoplasmic proteins, apparently regulating signaling transduction pathways. Targeting these interactions by a synthetic APIM-containing peptide, ATX-101, leads to improved cellular sensitivity to the DNA-damaging agent cisplatin. In this study, stress response experiments were performed to assess the effects of ATX-101, cisplatin, and cisplatin+ATX-101 on cell growth and metabolism of T24 cells. The effects were studied 24 hours after treatment addition. Assessment of cell growth was achieved by indirect DNA concentration measurements and direct cell counts. ATX-101 showed little or no growth inhibiting effects, whereas statistically significant effects were observed by the cisplatin and cisplatin+ATX-101 treatments. To study changes on the exometabolome, glucose, lactate, and glutamine concentrations in culture medium samples were measured. For all sample groups (control, ATX-101, cisplatin, and cisplatin+ATX-101), the majority of glucose consumed was found to serve the purpose of aerobic glycolysis (lactate production). Interestingly, reduced yield coefficients (lactate per glucose) were observed for samples exposed to cisplatin and cisplatin+ATX-101, due to a reduced lactate production. The endometabolome was analyzed using target mass spectrometric (MS) metabolic profiling employing RP LC-MS/MS and capIC-MS/MS. The treatment effects were compared against each other using principle component analysis (PCA), analysis of variance (Anova), and log2-comparisons. The analyses revealed minor effect of ATX-101 as a single agent treatment. However, samples exposed to cisplatin and cisplatin+ATX-101 were significantly different from untreated control samples with respect to intracellular concentration of intermediates involved in the glycolysis, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle, as well as in several amino acids. Further, samples exposed to either of these treatments were characterized by a slightly lowered energy charge (EC), due to higher amounts of mono- and diphosphates. Altogether, the observations indicate an at least additive effect by the combination ATX-101 and cisplatin. This suggests a role of ATX-101 in regulating central carbon metabolism that is yet to be fully understood, and motivates further research on the topic to reveal possible new cancer treatments in the future.