zy-17617b 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

Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of four different calmodulin antagonists: thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), CGS 9343B (1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a] [4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2 H-benzimidazol-2-one (1:1) maleate), clotrimazole (1-(alpha-2-chlorotrityl)imidazole) and bifonazole (1-(alpha-biphenyl-4-ylbenzyl)imidazole), on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in B16 melanoma cells. We found that all four substances significantly reduced the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP, in a dose- and time-dependent manner. Cell viability was reduced in a close correlation with the fall in ATP. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate did not result from the cytotoxic effects of the calmodulin antagonists, since their content was already reduced before any cytotoxic effect was observed. These findings suggest that the fall in the levels of the two signal molecules of glycolysis, induced by the calmodulin antagonists, causes a reduction in glycolysis and ATP levels, which eventually leads to cell death. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, these substances are most promising agents in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cell growth.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Calmodulin; Cell Survival; Clotrimazole; Fructosediphosphates; Glucose; Glucose-6-Phosphate; Imidazoles; Melanoma, Experimental; Mice; Thioridazine; Tumor Cells, Cultured