|dc.description.abstract||Hypoxia is a condition of mild oxygen (O2) deficiency and is observed in most solid tumors. It is believed to trigger malignant transformation, alter metabolism, and it contributes to, for example, metastasis, invasiveness, vasculogenesis, angiogenesis, and resistance to cell death. Hypoxia is a great contributor to the aerobic glycolysis seen in cancer cells due to effects of hypoxia-inducible factor 1 (HIF-1). HIF-1, a dimer of HIF-1, accumulates under hypoxic conditions and is a transcriptional activator with target genes encoding proteins that mediate adaptive responses to the altered environment. Enzymes and metabolites involved in several metabolic pathways, for instance the glycolysis and the TCA cycle, are affected by hypoxia, motivating further studies on the hypoxic influence on cell metabolism.
Metabolic adaptations of hypoxia were investigated in the prostate cancer cell line DU-145 and the glioblastoma astrocytoma cell line U-87 MG by using a hypoxic O2 concentration around 1 % and a control concentration of 21 % (normoxia). Metabolic state was analyzed 48, 72, and 96 hours after hypoxic stress initiation (96 hour results are not utterly presented due to lack of carbon sources). HIF-1 concentrations at the different sampling times were determined by ELISA but unfortunately no conclusive results were obtained. The exometabolomes were analyzed by NMR to examine whether hypoxic conditions had any effects on the glucose and glutamine consumption and on the production of lactate. The DU-145 cells consumed less glutamine under hypoxia than under normoxia, while the opposite trend was observed for the U-87 MG cells. Both cell lines produced less lactate per glucose consumed under hypoxia relative to normoxia the first 48 hours, whereas the hypoxic cell cultures produced more lactate per glucose consumed between 48 and 72 hours of incubation.
The endometabolomes were analyzed by targeted RP LC-MS/MS and capIC-MS/MS. Metabolic state adaptations of the endometabolomes under hypoxia were compared against normoxia using principle component analysis (PCA), log2-comparisons and visualization in metabolic network charts, and analysis of variance (ANOVA). The hypoxic DU-145 cell cultures showed higher levels of glycolytic intermediates and generally lower concentrations of TCA cycle intermediates compared to the normoxic cell cultures. The hypoxic U-87 MG cells showed mostly lower concentrations of TCA cycle intermediates compared to the normoxic cells. The glycolytic intermediates were in both higher and lower concentrations under hypoxia after 48 hours, whereas all glycolytic intermediates had decreased levels after 72 hours. Both cell lines mostly had reduced levels of amino acids under hypoxia relative to normoxia the first 48 hours, and increased concentrations after 72 hours of incubation.
Overall, no drastic adaptations in the central metabolism caused by hypoxia was observed, and few statistically significant changes were found. However, this was a pilot project and more experiments must be performed to further examine the observed trends and to expose the effects of hypoxia on the two cell lines.||en