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Metabolomic analysis of Human Hepatocellular Carcinoma by proton NMR spectroscopy

Metabolomic analysis of Human Hepatocellular Carcinoma by proton NMR spectroscopy. Aicha Demidem INRA, Theix. Colloquium LIX, Ecole Polytechnique. Palaiseau november 2010. Context. Hepatocarcinoma – HCC Deadly disease : 3rd position (WHO) Diagnostic Relatively late.

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Metabolomic analysis of Human Hepatocellular Carcinoma by proton NMR spectroscopy

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  1. Metabolomic analysis of Human Hepatocellular Carcinoma by proton NMR spectroscopy Aicha Demidem INRA, Theix Colloquium LIX, Ecole Polytechnique. Palaiseau november 2010

  2. Context • Hepatocarcinoma – HCC • Deadly disease: 3rd position (WHO) • Diagnostic • Relatively late

  3. Diagnostic of Hepatocarcinoma(HCC) • MarkerSerique • AFP (α-fœto protein) weak sensibility • Anatomo-pathologie, mutation of β-catenin after hepatic resection

  4. AIMS OF STUDY • Identify new biomarkers from tissues and sera (from patients having hepatocarcinoma) to improve the early detection of this deadly disease • Identify metabolic pathway of cellular transformation in the aim to determine new therapeutic targets

  5. Method • Metabolomic : approach without a priori * Nuclear Magnetic Resonance (NMR) * Mass Spectroscopy (SM) • Identification and quantification of metabolites from tissues and biological fluids • Literature data Metabonomic studies of human hepatocellular carcinoma using 1H NMR spectroscopy Yang et al., J of Proteome Res, 2007

  6. Material & Method: NMR • Spectral acquisition • NMR 400 MHz • 1H Spectra • 31P Spectra

  7. Spectral Treatments • Software : MestReNova • Reference : creatine 3,035 ppm

  8. Materials and methods: Experimental protocol • Collection of liver tissues (+ 4°C) • Collection of blood (serum sample) • Preparation of tissues for extract and acquisition of NMR spectra • Histology report Prelevement of pathological and healthy tissues

  9. Ten patients underwent liver resection for tumor Comparative metabolic profile from tumoral tissue versus adjacent tissue from the same patient by 1H-NMR Biopsies of both tumor and non-tumoral tissues were obtained from these patients

  10. Clinical Material

  11. Results: Identification of Metabolites Publications HMDB

  12. Results: Identification of Metabolites

  13. Results: 2D spectral to identify metabolites

  14. Results : Spectrum difference Healthy Tissue Pathological Tissue Difference PT / HT PT/HT lactate alanine glutamate glutathione

  15. ascorbic acid Results : Spectrum difference PT/HT Healthy Tissue Pathological Tissu Difference PT / HT glucose glucose / glycogen

  16. Analysis of Relative Concentrations( Univariate data analysis) Mann-Whitney test a = 5%

  17. Analysis of relative concentrations

  18. Results from Principal Component Analysis (PCA) Glycolysis is predominant: Warburg effect Ros increased Ros increased Ros increased Differentiation Differentiation Differentiation Anaerobic creatine system / phosphocreatine is predominant • Allow to describe the information from a set of data (linear combinations of variables) The 2 dimension analysis represents 51% of totalvariance Correlation circle

  19. Correlation between the grade of HCC and the increase of glycolysis?

  20. Interpretation 1- Increase of lactate and alanine content(final products of glycolysis) Biomarkers of Warburg effect : correlation with progression/stage of disease 2- Increase of glutamate (aggressive form) and glutamine level (less aggressive form) : Promoters of the tumoral progression Actual target: Glutaminase Inhibitors Convert an aggressive form to an non aggressive form 3 -Decrease of glucose and glycogen level : Result from the conversion of glucose to lactate: hyperactive glycoysis Phenomena common to tumor cells

  21. Conclusion • Comparative metabolomic analysis of healthy hepatic tissue and HCC allowed generating assumptions on the redox status and bioenergetics of HCC

  22. Conclusion • Metabolomic analysis of HCC allows to propose : • - Metabolic tumoral biomarker (s ) as candidate HCC • Hypothesis (ses) on the metabolic pathways implicated in HCC pathological evolution (aerobic glycolysis, glutamine and creatine system) Metabolomic study represents an good way of generating experimental data for metabolic modeling in integrative biology Transfer to in vivo studies using Magnetic Resonance Imaging (MRI)

  23. INRA Anne Fages Daniel Morvan Pascale Rio Georges Stepien MNR/MS Team Guy Bielicki Jean Pierre Renou Estelle Pujos Hospital Team Armand Abergel Emmanuel Buc Denis Pezet

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