Mass Spectrometry Based Metabolite Profiling of Cytostatic Induced Stress Response in the Prostate Cancer Cell Line DU 145
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The discovery of AlkB homologue 2 PCNA-interacting motif (APIM), a new motif interacting with proliferating cell nuclear antigen (PCNA), has opened up a new possible therapeutic window concerning cancer treatments. This has lead to the development of a new chemotherapeutic peptide, ATX-101, that binds and blocks the APIM binding site on PCNA leading to increased apoptotic effect. APIM-proteins are involved in the DNA repair system and bind PCNA more readily when the DNA is damaged. The APIM sequence has been discovered to occur in proteins in the cytoplasm too, involved in metabolism and regulatory mechanisms. A stress response experiment was performed to assess the metabolic effect of ATX-101 alone and in combination with the cytotoxic drug Cisplatin in the prostate cancer cell line DU 145. The experimental setup consisted of 24 cell dishes with six different treatments; control, ATX-101, ATX-101 and Cisplatin, the non-functional ATX-A as a negative control, ATX-A and Cisplatin, and Cisplatin. The cells were quenched and extracted in a time series of 0, 4, 8, and 24 hours after treatment addition.Transient reduction in growth rate was observed for ATX-101 treated cells, and the combinatorial treatment of ATX-101 and Cisplatin showed periods of growth arrest. Carbon flux studies were conducted measuring the extracellular concentration of glucose, glutamine and lactate. The results showed clear indication of the Warburg effect; high production rates of lactate from a high uptake of glucose and glutamine. The Warburg effect was seen independent of treatment implying that some of the carbon going into biomass production was redirected to metabolism, production of lactate and CO2, in the samples treated with ATX-101 alone or in combination with Cisplatin. The metabolome was analyzed using target and non-target mass spectroscopy (MS) based methods; gas chromatography, ultra performance liquid chromatography (both reverse phase and hydrophilic interaction liquid chromatography), and capillary ion chromatography, all coupled to MS. The results were analyzed using principle component analysis to compare the treatment effects up against each other. General trends in the metabolite profiles showed clear effect of ATX-101 especially in combination with Cisplatin.This study showed that ATX-101 alone and in combination with Cisplatin has an effect on DU 145 regarding growth and metabolism, opening up for possible new cancer treatments in the future.