18 H 2 O 2 H 2 O 2 25 11 H 2 O 2 in vitro H 2 O 2 H 2 O 2 GSH
1. H 2 O 2 PPP TCA Ralser, et al., 29; Rui, et al., 21 PPP TCA GSH spermidine ROS H 2 O 2 H 2 O 2 H 2 O 2 18 H 2 O 2 H 2 O 2 H 2 O 2 25 25 in vitro 1 H 2 O 2 H 2 O 2 ROS 2. 2.1. American Type Culture Collection ATCC Manassas VA USAJCRB Dulbecco s modified Eagle s medium DMEM Sigma-Aldrich
Co. LLC MO USA 1 U/mL G.1 mg/ml 1% 5%CO 2 37 C 2.2. MTT 3 P. 1 H 2 O 2 24 5% H 2 O 2 EC 5 5 % Effective Concentration 96 1 1.5 1 4 5%CO 2 37 C 2.3. GPx GPx Gluthathione Peroxidase Activity Assay Kit BioVision Inc. Milpitas CA USA GPx GSSG GSH NADPH GPx 6 8 2.4. H 2 O 2 H 2 O 2 in vitro H 2 O 2 7 µm 9.5 mm 37 C 3 H 2 O 2 H 2 O 2 H 2 O 2 BIOXYTECH H 2 O 2 Assay Kit 2.5. H 2 O 2 H 2 O 2 CM-H 2 DCFDA Life Technologies Corp. Carlsbad CA USA CM-H 2 DCFDA H 2 O 2 96 PBS 2 7µM CM-H 2 DCFDA-hanks 15µL 5 H 2 O 2 492 nm 528 nm
2.6. CE-TOFMS CE-TOFMS 3 P. 1 3. 3.1. H 2 O 2 18 H 2 O 2 H 2 O 2 GPx 1 H 2 O 2 EC 5 KP3 MKN7 H 2 O 2 AsPC-1 HeLa PC14 H 2 O 2 H 2 O 2 GPx H 2 O 2 H 2 O 2 Marklund, et al., 1982 1. H 2 O 2 GPx Cancer Cell Tissue EC5 [mm] GPx [mu/mg protein] MKN7 stomach 1.38 28.7 (± 2.8) KP3 pancreas 1.21 28.2 (± 2.8) A431 unknown.6 35.2 (± 1.3) BxPC-3 pancreas.54 MKN74 stomach.54 25.5 (± 1.6) DLD-1 colon.53 A549 lung.51 17.8 (± 1.2) HT29 colon.5 56.7 (±.4) MKN28 stomach.48 2.4 (± 2.6) HepG2 liver.47 5.9 (±.2) Panc-1 pancreas.44 ) KMRC-1 kidney.43 MCF-7 breast.42 11. (±.6) ACHN kidney.41 58.2 (± 4.2) PSN-1 pancreas.38 22.9 (± 1.) PC14 lung.34 HeLa cervix.33 33.3 (± 2.) AsPC-1 pancreas.32 12. (±.5) EC 5 H 2 O 2 24 5% GPx 4
18 9 94 H 2 O 2 KP3 MKN7 2 1A H 2 O 2 H 2 O 2 -EC 5 H 2 O 2 25 H 2 O 2 -EC 5 r >.5 P <.5 1B H 2 O 2 KP3 MKN7 1C H 2 O 2 -EC 5 25 H 2 O 2
A) 12 KP3 6 MKN7 PC3-12 -6 6 12 KMRC MCF-7-6 AcHN -12 PC2 B) C) 1.5 GSH Correlation coefficient [r] 1. H 2 O 2 A 18 9 94 3 74.4B H 2 O 2 -EC 5 H 2 O 2 -EC 5.5 25 C EC 5 z r >.5 (p <.5) -.2.2.4.6.8 1 3PG DHAP F1,6P Carnosine Creatine Citrate NADP+ FAD NAD+ PRPP camp F6P G6P Ru5P 2-Oxoglutarate GMP Ornithine S-Lactoylglutathione cis-aconitate Glu Thiamine Pyruvate Asp G1P PEP metabolites KP3 MKN7 KMRC-1 BxPC-3 AcHN MCF-7 HepG2 Panc-1 A431 PC13 A549 DLD1 AsPC1 HeLa MKN28 MKN74 HT29 PSN1 cancer cell lines -3. 3.
3.2. 25 H 2 O 2 -EC 5 25 H 2 O 2 in vitro H 2 O 2 H 2 O 2 25 PPP glucose 6-phosphate G6P fructose 6-phosphate F6P fructose 1,6-diphosphate F1,6P dihydroxyacetone phosphate DHAP3-phosphoglycerate 3PGpyruvate ribulose 5-phosphate Ru5Pphosphoribosyl pyrophosphate PRPP TCA 2-oxoglutarate cis-aconitate FAD thiamine camp NAD 13 2A PPP TCA Ru5P 1 H 2 O 2 5 EC 5 pyruvate pyruvate PRPP Ru5P 2-oxoglutarate GSH spermidine H 2 O 2.1~1 mm EC 5 H 2 O 2 nm µm spermidine
A) B) 1 1 8 6 4 2 Ctrl NAD FAD Citrate Creatine Carnosine F1,6P DHAP 3PG PEP G1P Asp Pyruvate Thiamine Gln cis-aconitate GMP 2-Oxoglutarate Ru5P G6P F6P camp PRPP GSH Spermidine H2O2 concentration [%] 5 Spermidine GSH Pyruvate PRPP Ru5P 2-Oxoglutarate R5P G6P DHAP F6P F1,6P 3PG.1.1 1 1 Metabolites Concentration [mm] 2. H 2 O 2 A In vitro H 2 O 2 H 2 O 2 Ctrl 1 GSH spermidine H 2 O 2 9.5 mm 7µM B 2. H 2 O 2 Metabolite EC5 [mm] Spermidine 51.7 GSH 29.3 Pyruvate.9 PRPP 1. Ru5P 2.3 2-Oxoglutarate 4. R5P 2.1 G6P 21.5 DHAP 25.1 F6P 3.9 F1,6P 42.6 3PG 51.5 EC 5 H 2 O 2 5%
3.3. H 2 O 2 In vitro H 2 O 2 acetate 2-oxoglutarate succinate Andrae, et al., 1985; Bunton, 1949 in vitro H 2 O 2 in vitro in vitro H 2 O 2 in vitro G6P F6P Ru5P R5P PRPP 3PG DHAP F1,6P 2-oxoglutarate pyruvate H 2 O 2 1 2 1 H 2 O 2 H 2 O 2 3 H 2 O 2 KP3 MKN H 2 O 2 HeLa HeLa H 2 O 2 1 mm H 2 O 2.5 mm
Ru5P m/z 168.99, MT 1.9 Ru5P [mm].1.1 1 1 H2O2 H2O2 + - + - Relative Area R5P m/z 199., MT 11.3 H2O2 R5P [mm].1.1 1 1 H2O2 + - + - Relative Area PRPP m/z 38.98, MT 1.6 Relative Area H2O2 PRPP [mm].1.1 1 1 H2O2 + - + - DHAP [mm].1.1 1 1 H2O2 + - + - DHAP1 m/z 266.97, MT 11.7 Relative Area Relative Area Relative Area F1,6P m/z 436.96, MT 1.9 F1,6P [mm].1.1 1 1 H2O2 H2O2 + - + - H2O2 DHAP2 m/z 357., MT 11.7 DHAP [mm].1.1 1 1 H2O2 H2O2 + - + - 3. H 2 O 2 in vitro.1 1 mm H 2 O 2 24 HeLa KP3 MKN7 H 2 O 2 6 in vitoro H 2 O 2 3
3.4. H 2 O 2 H 2 O 2 H 2 O 2 KP3 HeLa 3 12 KP3 4 GSH ATP H 2 O 2 H 2 O 2 48 H 2 O 2 5A H 2 O 2 48 5B H 2 O 2
.7 G6P.6.5.4.3.2.1 Glucose Low High Low High Amount [fmol/cell] HeLa KP3 HeLa KP3 HeLa KP3 Amount [fmol/cell].2 F6P.15.1.5 Glucose Low High Low High 14 ATP 12 1 8 6 4 2 Glucose Low High Low High Amount [fmol/cell].7 6-phosphogluconate.6.5.4.3.2.1 Glucose Low High Low High Amount [fmol/cell].6 Ru5P.5.4.3.2.1 Glucose Low High Low High Amount [fmol/cell] 5 GSH 4 3 2 1 Glucose Low High Low High Amount [fmol/cell] 2.5 Pyruvate 2.5 2-oxoglutarate Amount [fmol/cell] 2 1.5 1.5 Amount [fmol/cell] 2 1.5 1.5 Glucose Low High Low High Glucose Low High Low High 4. 1 g/l4.5 g/l 12 HeLa KP3
A) Low Glc. H2O2 + High Glc. H2O2 + High Glc. H2O2 - Low Glc. H2O2 - B) HeLa KP3 1 1 Cell viability [%] 8 6 4 Cell viability [%] 8 6 4 2 2 1 4.5 Glucose [g/l] 1 4.5 Glucose [g/l] 5. H 2 O 2 A H 2 O 2 H 2 O 2 1 g/l Low. Glc. 4.5 g/l High Glc. 6 7 µm CM-H 2 DCFDA Hanks 1 mm H 2 O 2 B H 2 O 2 1 g/l 4.5 g/l 6.1 mm H 2 O 2 48 MTT 3 ** P <.1
4. 18 H 2 O 2 KP3 MKN7 H 2 O 2 GPx 1 H 2 O 2 KP3 MKN7 1C 2 2 1 3 H 2 O 2 5 H 2 O 2 G6P F6P G1P in vitro 2A G6P F6P CE-TOFMS in vitro 6 pyruvate 2-oxoglutarate pyruvate acetate 2-oxoglutarate succinate Andrae, et al., 1985; Bunton, 1949; Liu, et al., 211 pyruvate Desagher, et al., 1997 acetate LC-MS H 2 O 2 HeLa H 2 O 2 KP3 4 3 pyruvate 2-oxogrutarate
R5P H 2 O 2 Newsholme, et al., 1985 5. H 2 O 2 H 2 O 2 Andrae, U., Singh, J. and Ziegler-Skylakakis, K. (1985) Pyruvate and related alpha-ketoacids protect mammalian cells in culture against hydrogen peroxide-induced cytotoxicity, Toxicol Lett, 28, 93-98. Bunton, C.A. (1949) Oxidation of Alpha-Diketones and Alpha-Keto-Acids by Hydrogen Peroxide, Nature, 163, 444-444. Desagher, S., Glowinski, J., and Premont, J. (1997) Pyruvate protects neurons against hydrogen peroxide-induced toxicity, The Journal of Neuroscience, 17, 96-967. Liu, J., et al. (211) Metabolomics of oxidative stress in recent studies of endogenous and exogenously administered intermediate metabolites, Int J Mol Sci, 12, 6469-651. Marklund, S.L., et al. (1982) Copper- and zinc-containing superoxide dismutase,
manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues, Cancer Res, 42, 1955-1961. Newsholme, E.A., Crabtree, B. and Ardawi, M.S. (1985) The role of high rates of glycolysis and glutamine utilization in rapidly dividing cells, Biosci Rep, 5, 393-4. Ralser, M., et al. (29) Metabolic reconfiguration precedes transcriptional regulation in the antioxidant response, Nat Biotechnol, 27, 64-65. Rui, B., et al. (21) A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress, BMC Syst Biol, 4, 122. Iino, K., Kitagawa, M., Soga, T., Tomita, M., Metabolomic responses of cancer cells against H 2 O 2 -induced oxidative stress, 8 th International Conference of the Metabolomics Society, WA USA. 212/6/25-28,,,,, 7,. 212/1/1-12 Iino, K., Kitagawa, M., Soga, T., Tomita, M., Remarkable antioxidative properties of central carbon intermediates in cancer cells identified by CE-TOFMS-based metabolomics, 35,. 212/12/11-14