Mass Spectrometric Metabolic Profiling of Bladder Cancer Cell Line T24 under Cisplatin and APIM-peptide Stress
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The hub protein proliferating cell nuclear antigen (PCNA) is involved in several important cellular processes. During conditions of genotoxic stress, PCNA binds to proteins containing AlkB homologue 2 PCNA-interacting motif (APIM), crucial for cellular repair systems. The novel peptide drug ATX-101, also containing the APIM-sequence, can block PCNA-interactions and thereby hinder cellular repair. This enhances DNA-damaging agents and induces apoptosis in a cancer-cell specific manner. In addition, APIM-peptides are found to affect several PCNA-interacting enzymes involved in glycolysis, TCA cycle and several signaling pathways, motivating studies on impact of ATX-101 on metabolic properties, and further on increasing understanding of the role of PCNA-APIM interactions in cells during conditions of stress. A stress response study was performed to investigate metabolic consequences of ATX-101 treatment on the bladder cancer cell line T24, both when given as a single agent and in combination with chemotherapeutic cisplatin. The endocellular metabolome was analyzed by targeted LC-MS/MS and capIC-MS/MS for quantification of amino acids, organic acids and phosphorylated metabolites, while extracellular concentrations of glucose and lactate was analyzed using enzymatic analyses of the growth medium. It was found that no entire pathways were affected by ATX-101, neither alone nor in combination with cisplatin. However, seemingly sporadic alterations of certain metabolite pools were observed in both treatments involving cisplatin, and interestingly, ATX-101 was found to enhance the cisplatin-induced alterations in some of the affected metabolite pools, such as certain amino acids and intermediates of glycolysis. Observed alterations were most likely a consequence of cellular repair caused by cisplatin damage, where a possible accumulation of nucleotides and lipid synthesis precursors suggests a downregulation of DNA and lipid synthesis due to reduced cell growth. However, the additional effects by ATX-101 confirm that metabolite production was inhibited due to disruptions of PCNA-interactions. The observed effects of ATX-101 strengthen the role of ATX-101 in cancer treatment, motivating further research on this topic.