Intracellular glucose-metabolite-profile of cancer cells using LC-MS/MS

Metabolomics studies as a novel approach in tumor molecular biology comprise the complete metabolic profiling of the living organism. Recent investigations have already indicated that a wide range of metabolites are useful in distinguishing tumors from healthy tissues and in monitoring cellular activities i.e. cell-cycle progression profile of the tumors. However, due to their high polarity, low in-vivo concentrations, rapid metabolic turnover, structural variety, instability and poor UV absorbance, these compounds are a particular challenging subclass of the metabolites to be studied. The conventional techniques for glucose metabolism ( i.e. enzyme assays, radioactive labeling) are not suitable to carry out parallel determinations of different metabolites in the same sample. To overcome this, electrospray ionization (ESI) mass spectrometry (MS) combined with on-line high performance liquid chromatographic (HPLC) separations, is a very promising technique in metabolite analysis for malignant tumors. It makes possible to identify a wide range of metabolites in one single run. Increased glycolysis is a characteristic feature of tumor cells, which is induced by the oncogene products such as serine threonine protein kinase Akt and also hypoxia inducible factor (HIF). The high glycolytic rate is necessary for keeping tumor cells alive and proliferate. It is assumed that regulatory mechanism involved in glycolysis contribute to malignant phenotype and may be considered as a rather specific target for antitumor therapy. Aims of the study: 1. Development of a suitable liquid chromatography-mass spectrometry (LC-MS) method to investigate the metabolic conversion of U-13[C]-glucose or 13C-acetate to lactic acid, pyruvate, succinate, - ketoglutarate, and ribose-5P in tumor cells. 2. Characterize the effect of various regulatory molecules (clofazamin, oxythiamine, acetoacetate) on glucose and acetate metabolism.