Metabolic Stress Study of a Human Embryotic Kidney Cell Line exposed to Chemotherapeutic Drugs: Mass Spectrometry based Targeted and Non-targeted Approach
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Metabolomics, the study of metabolites in a cell, has proven as an applicable approach in cancer research to investigate the altered metabolism of cancer cells and to develop new selective chemotherapeutic drugs. ATX-101 is a novel chemotherapeutic drug that enhances the apoptotic effect of DNA-damaging agents on cancerous cells due to its APIM-sequence. APIM enables interaction with PCNA, a multifunctional protein involved in DNA repair which is overexpressed in many cancer cells. ATX-A is a non-functioning version of ATX-101 that serves as a negative control. A pilot study on cellular stress responses in human cell lines indicated that ATX-101 affected cancer cells, leaving non-cancerous cells unaffected. These findings made the foundation for this master thesis. The aim of this study is to further investigate the metabolic effects of ATX-101 and ATX-A, when used as a single agent and in combination therapy with the DNA-damaging agent cisplatin, on human embryotic kidney (HEK293) cells. The HEK293 cell line represents a model system for non-cancerous cells, and two biological replicas were used to investigate the effects of ATX-A, whereas one biological replica was used to investigate the effects of ATX-101. A time series exposure (4, 8 and 24 hours) was conducted. To study changes in the endometabolome, targeted analysis was carried out by gas chromatography coupled to a triple quadrupole mass spectrometer (GC-QqQ-MS), and non-targeted analysis was carried out by liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS). Principal component analysis (PCA) was used to uncover characteristic groupings of samples related to different treatment, hence reflecting alterations in the cells metabolism.Both targeted and non-targeted analysis revealed time-based clustering of HEK239 samples in the PCA plots, and no effects were seen due to different chemotherapeutic treatment. This suggests minor effects of both ATX-A and ATX-101 on the HEK293 cells metabolism, and holds a promise of a future selective chemotherapeutic treatment with the use of ATX-101. The results in this master thesis coincide with the pilot study and comprehensive studies carried out at NTNU.