Control of cellular proliferation by modulation of oxidative phosphorylation in human and rodent fast-growing tumor cells.

The relationship between cell proliferation and the rates of glycolysis and oxidative phosphorylation in HeLa (human) and AS-30D (rodent) tumor cells was evaluated. In glutamine plus glucose medium, both tumor lines grew optimally. Mitochondria were the predominant source of ATP in both cell types (66-75%), despite an active glycolysis. In glucose-free medium with glutamine, proliferation of both lines diminished by 30% but oxidative phosphorylation and the cytosolic ATP level increased by 50%. In glutamine-free medium with glucose, proliferation, oxidative phosphorylation and ATP concentration diminished drastically, although the cells were viable. Oligomycin, in medium with glutamine plus glucose, abolished growth of both tumor lines, indicating an essential role of mitochondrial ATP for tumor progression. The presumed mitochondrial inhibitors rhodamines 123 and 6G, and casiopeina II-gly, inhibited tumor cell proliferation and oxidative phosphorylation, but also glycolysis. In contrast, gossypol, iodoacetate and arsenite strongly blocked glycolysis; however, they did not affect tumor proliferation or mitochondrial metabolism. Growth of both tumor lines was highly sensitive to rhodamines and casiopeina II-gly, with IC(50) values for HeLa cells lower than 0.5 microM, whereas viability and proliferation of human lymphocytes were not affected by these drugs (IC(50) > 30 microM). Moreover, rhodamine 6G and casiopeina II-gly, at micromolar doses, prolonged the survival of animals bearing i.p. implanted AS-30D hepatoma. It is concluded that fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target.

Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase.

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.