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.

Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy

“…Furthermore, increased glucose consumption serves to supply carbon to various anabolic pathways necessary for biomass duplication preceding mitosis 33 and induces resistance to classic chemotherapeutic agents. 10,34,35 Compelled by evidence that glucose transport may represent the principal ratedetermining step of glycolysis in tumor cells, 13,14 we have interrogated the glucose transporter family as a source of novel therapeutic targets. Our data clearly demonstrate myeloma dependence on GLUT4 activity, a phenomenon associated with the basal cell surface localization of the transporter.…”

“…13,14 Therefore, we established an unbiased real-time RT-PCR-based screen to determine relative expression levels of GLUTs 1 to 12 in 9 MM cell lines and NBLs ( Figure 1E). To avoid bias, myeloma lines were selected to broadly cover different genotypic disease subtypes.…”

“…12 Intriguingly, the poor efficacy of these compounds may be explained by recent observations suggesting that glucose transport may occupy the primary ratedetermining step of glycolysis in malignant cells. 13,14 Therefore, further investigation into the molecular mechanisms underlying enhanced glucose transport rates in cancer is warranted.…”

N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4

“…Both live imaging and biochemical approaches demonstrated that GLUT4 expressed in HeLa cells was able to respond to insulin in an N-glycan-dependent manner, suggesting that HeLa cells, despite their lack of GLUT4 expression (40), retain the basic machinery required for insulin-mediated cellular signaling. Similar findings have previously been reported for noninsulin-responsive fibroblasts (21), although the detailed molecular mechanism of translocation may not be exactly the same as that in insulin-responsive adipocytes (41).…”

Resveratrol Suppresses Cancer Cell Glucose Uptake by Targeting Reactive Oxygen Species–Mediated Hypoxia-Inducible Factor-1α Activation

“…Reduction of glucose uptake by resveratrol in our study was linked to diminished glycolytic metabolism as evidenced by decreased lactate production. Among the major determinants of tumor glycolytic flux (33)(34)(35), this was shown to be due to reduced Glut-1 expression rather than hexokinase activity.…”