title: Kinetics of transport and phosphorylation of glucose in cancer cells.
abstract: Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez, Tlalpan, México City, Mexico. sara.rodriguez@cardiologia.org.mx
authors.forename: Sara
authors.initials: S
authors.lastname: Rodríguez-Enríquez
authors.forename: Alvaro
authors.initials: A
authors.lastname: Marín-Hernández
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Gallardo-Pérez
authors.forename: Rafael
authors.initials: R
authors.lastname: Moreno-Sánchez
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003080:Cold Temperature; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000037:antagonists & inhibitors / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000494:pharmacology; D006019:Glycolysis / Q000502:physiology; D006367:HeLa Cells; D006593:Hexokinase / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism*; D010693:Phloretin / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
chemical_list: D051246:Glucose Transport Proteins, Facilitative; D007328:Insulin; D007527:Isoenzymes; D003571:Cytochalasin B; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D005947:Glucose; D010693:Phloretin