title: Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells.
abstract: BACKGROUND
  Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells.
  
  
  METHODS
  To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-(14)C]-glucose and [1-(14)C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated (13)C atoms after [U-(13)C]-glucose/[2-(13)C]-acetate labelling. Based on [1-(14)C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated.
  
  
  RESULTS
  Both [U-(13)C]-glucose and [1-(14)C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain (13)C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-(13)C]-acetate-representing a novel functional test in malignant cells-confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency.
  
  
  CONCLUSIONS
  The applied methods of energy substrate utilisation and other measurements represent simple assay system using (13)C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: A
authors: A
authors: Jeney
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: Z
authors: Z
authors: Hujber
authors: Laboratory of Environmental Chemistry and Bioanalytics, Department of Analytical Chemistry, Institute of Chemistry, Eötvös Loránd University, P.O. Box 32, Budapest, 1518 Hungary.
authors: N
authors: N
authors: Szoboszlai
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: A
authors: A
authors: Fullár
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: J
authors: J
authors: Oláh
authors: Department of Clinical Research, National Institute of Oncology, P.O. Box 21, Budapest, 1525 Hungary.
authors: É
authors: É
authors: Pap
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: Á
authors: Á
authors: Márk
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: Cs
authors: C
authors: Kriston
authors: Department of Clinical Research, National Institute of Oncology, P.O. Box 21, Budapest, 1525 Hungary.
authors: J
authors: J
authors: Kralovánszky
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.
authors: I
authors: I
authors: Kovalszky
authors: Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Pusztaszeri u. 59-67, Budapest, 1025 Hungary.
authors: K
authors: K
authors: Vékey
authors: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085 Hungary.|Tumour progression Research Group of Joint Research Organization of Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary.
authors: A
authors: A
authors: Sebestyén
keywords: Bioenergetic signature; Energy metabolism; GAPDH/β-F1-ATPase expression; Glucose/acetate utilization; Metabolic characterisation; TCA impairment; mTOR