OKSİJEN-18 TEMELLİ GC-MS İLE FARKLI KANSER HÜCRELERİNİN METABOLİK DİNAMİKLERİNİN KARŞILAŞTIRILMASI

Abstract

Cancer cells reprogram their energy metabolism to sustain rapid proliferation. Therefore, metabolomic and fluxomic approaches are important tools for investigating cancer metabolism. However, the reliability of the obtained data directly depends on sample preparation and metabolite quenching procedures. In this study, methanol, perchloric acid, and methanol–perchloric acid quenching methods were compared in A549 cells, and the selected method was subsequently applied to H218O-based fluxomic analyses in A549, Hep3B, and LoVo cell lines. Relative abundances of metabolites associated with amino acid metabolism, glycolysis, and the tricarboxylic acid (TCA) cycle were determined by GC-MS. Methanol generally provided higher and more balanced relative abundances across the investigated metabolite groups and was therefore selected for H218O labeling experiments. A549, Hep3B, and LoVo cells were incubated in medium containing 30% H218O for 5, 15, and 30 min, and relative 18O labeling profiles of selected metabolites were evaluated. TCA cycle metabolites showed higher H218O incorporation in Hep3B and LoVo cells than in A549 cells, whereas metabolites associated with glycolysis and phosphate metabolism generally exhibited higher relative labeling in A549 cells. Cell-line-specific differences were also observed in amino acid metabolism. In conclusion, the methanol-based quenching method provided a suitable sample preparation approach for GC-MS-based metabolomic and H218O-based fluxomic analyses, and the findings demonstrated distinct metabolic dynamics among the three cancer cell lines.