Rodríguez-Enríquez Sara, Marín-Hernández Alvaro, Gallardo-Pérez Juan Carlos, Moreno-Sánchez Rafael
Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez, Tlalpan, México City, Mexico.
J Cell Physiol. 2009 Dec;221(3):552-9. doi: 10.1002/jcp.21885.
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.
肿瘤糖酵解的代谢控制分析表明，己糖激酶（HK）和葡萄糖转运蛋白（GLUT）发挥主要的通量控制作用（71%）。为了解释它们为何是主要的控制步骤，我们分析了大鼠肝癌AS - 30D细胞和人宫颈癌HeLa细胞中的GLUT和HK动力学以及GLUT1、GLUT2、GLUT3、GLUT4、HKI和HKII的含量。我们开发了一种改进的方案，以D - [2 - (3)H - 葡萄糖]作为生理底物来测定GLUT的动力学参数。动力学分析显示，在两种肿瘤细胞中，低葡萄糖浓度和高葡萄糖浓度下均存在两个组分。在低葡萄糖浓度和37℃条件下，肝癌细胞和HeLa细胞的V(max)分别为55±20和17.2±6 nmol（min×mg蛋白质）⁻¹，而K(m)分别为0.52±0.7和9.3±3 mM。在AS - 30D肝癌细胞中，细胞松弛素B（IC(50)=0.44±0.1；K(i)=0.3±0.1 μM）和根皮素（IC(50)=8.7 μM）可部分抑制GLUT活性。在生理葡萄糖浓度下，GLUT1和GLUT3分别是HeLa细胞和AS - 30D细胞中的主要活性亚型。HeLa细胞中的HK活性（60 mU/mg蛋白质）远低于AS - 30D细胞中的HK活性（700 mU/mg蛋白质），但两种HK均受到6 - 磷酸葡萄糖（G6P）的强烈抑制。HKII是AS - 30D癌细胞和HeLa细胞中的主要亚型。与对G6P敏感的HK相比，GLUT的V(max)和催化效率（V(max)/K(m)）值低得多，这表明在癌细胞中，转运蛋白对糖酵解通量的控制作用高于HK。因此，GLUT似乎是一个更合适的治疗靶点。
