calcimycin and fructose-1-6-diphosphate

We studied here, in NIH-3T3 fibroblasts, the effect of the Ca(2+)-ionophore A23187 (which is known to increase intracellular-free Ca(2+)) on the control of glycolysis and cell viability and the action of calmodulin antagonists. Time-response studies with Ca(2+)-ionophore A23187 have revealed dual effects on the distribution of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme of glycolysis, between the cytoskeletal and cytosolic (soluble) fractions of the cell. A short incubation (maximal effect after 7 min) caused an increase in cytoskeleton-bound PFK with a corresponding decrease in soluble activity. This leads to an enhancement of cytoskeletal glycolysis. A longer incubation with Ca(2+)-ionophore caused a reduction in both cytoskeletal and cytosolic PFK and cell death. Both the "physiological" and "pathological" phases of the Ca(2+)-induced changes in the distribution of PFK were prevented by treatment with three structurally different calmodulin antagonists, thioridazine, an antipsychotic phenothiazine, clotrimazole, from the group of antifungal azole derivatives that were recently recognized as calmodulin antagonists, and CGS 9343B, a more selective inhibitor of calmodulin activity. The longer incubation with Ca(2+)-ionophore also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis. All these pathological changes preceded the reduction in cell viability, and a strong correlation was found between the fall in ATP and cell death. All three calmodulin antagonists prevented the pathological reduction in the levels of the allosteric effectors, ATP and cell viability. These experiments may throw light on the mechanisms underlying the therapeutic action of calmodulin antagonists that we previously found in treatment of the proliferating melanoma cells, on the one hand, and skin injuries, on the other hand.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Benzimidazoles; Calcimycin; Calcium; Calmodulin; Cell Survival; Clotrimazole; Cytoskeleton; Fructosediphosphates; Glucose-6-Phosphate; Glycolysis; Ionophores; Mice; Phosphofructokinase-1; Solubility; Thioridazine; Time Factors

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5'-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Survival; Energy Metabolism; Fructosediphosphates; Glycolysis; Hexokinase; Ionophores; Melanoma; Mice; Phosphofructokinase-1; Tumor Cells, Cultured