Rivka Beitner

Clotrimazole and bifonazole detach hexokinase from mitochondria of melanoma cells.

Cancer cells are characterized by a high rate of glycolysis. Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the only glycolytic enzyme which binds to mitochondria, is exceptionally high in cancer cells, and believed to play a key role in regulating cell energy metabolism and cancer cell growth rate. We have previously found that clotrimazole (1-(alpha-2-chlorotrityl)imidazole) and bifonazole (1-(alpha-biphenyl-4-ylbenzyl)imidazole), the antifungal azole derivatives, which were recently recognized as calmodulin antagonists, are calmodulin antagonists which most effectively reduce glycolysis and ATP level in B16 melanoma cells. They act through allosteric regulation and detachment of glycolytic enzymes from cytoskeleton. Here we report of a novel, additional, mechanism of action of these drugs. We show that they induce a dose-dependent detachment of hexokinase from mitochondria of B16 melanoma cells. This effect preceded the decrease in cell viability. These results suggest that clotrimazole and bifonazole may be promising drugs in treatment of melanoma.

The role of glucose 1,6-bisphosphate in the regulation of carbohydrate metabolism in muscle muscle

Abstract Glucose 1,6-bisphosphate is a regulator of the key enzymes of carbohydrate metabolism, and its concentration in muscle changes under different physiological, hormonal and pathological conditions. It is suggested that this compound regulates the different pathways of glucose metabolism.

Detachment of glycolytic enzymes from cytoskeleton of melanoma cells induced by calmodulin antagonists.

Glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We have previously found that different calmodulin antagonists decrease the levels of allosteric activators of glycolysis, and reduce ATP content and cell viability in B16 melanoma cells. Here we report of a novel, additional, mechanism of action of calmodulin antagonists in melanoma cells. We show that these drugs cause a detachment of the glycolytic enzymes, phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of B16 melanoma cells. This effect was dose- and time-dependent, and preceded the decrease in cell viability. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton-membrane and would affect cytoskeleton structure. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by calmodulin antagonists, as well as their reported inhibitory action on cell proliferation, make these drugs most promising agents in treatment of cancer.

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.

The levels of cyclic gmp and glucose 1,6-diphosphate, and the activity of phosphofructokinase, in muscle from normal and dystrophic mice.

A striking reduction in the levels of glucose 1,6-diphosphate and an increase in cyclic GMP were found in muscle from dystrophic mice. Concomitant to these changes, the allosteric activity of phosphofructokinase was found to be markedly reduced. These findings could offer an explanation for the observed reduction in glycolysis in the dystrophic muscle.

Detachment of glycolytic enzymes from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma cells induced by clotrimazole and its correlation to cell viability and morphology.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We report here that clotrimazole (l-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, induced a dose-dependent detachment of the glycolytic enzymes, phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase (D-fructose-l,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton membrane, and would also affect cytoskeleton structure and cell shape. We show here that clotrimazole decreased the viability of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. After 3h of incubation with clotrimazole, complete cell destruction was detected. Ultrastructural cell damage was manifested by disintegration of the outer membrane by scanning electron microscopy (SEM). The detachment of glycolytic enzymes from cytoskeleton, induced by clotrimazole, preceded the decrease in cell viability, which indicates that this is an early effect and not a result of cell death. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation, and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by clotrimazole, as well as its reported inhibitory action on cell proliferation, makes this drug the most promising agent in the treatment of cancer.

Taxol (paclitaxel) induces a detachment of phosphofructokinase from cytoskeleton of melanoma cells and decreases the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP

Glucose utilization through glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Here we report of a novel mechanism of action of taxol (paclitaxel; Baccatin III N-benzyl-beta-phenylisoserine ester), the anti-microtubule agent with remarkable anticancer activity. We show that taxol affects both levels of regulation of glycolysis in melanoma cells; it decreases the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis, and also causes a detachment of phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis, from the cytoskeleton of B16 melanoma cells. These effects of taxol were dose-dependent, and preceded the decrease in ATP levels and cell viability. Thus, taxol not only inhibits the essential dynamic processes of microtubule network, but also reduces glycolysis, through the novel mechanisms described here.

Clotrimazole decreases glycolysis and the viability of lung carcinoma and colon adenocarcinoma cells

Glycolysis is known to be the primary energy source in most cancer cells. We investigated here the effect of clotrimazole (1-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, 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 LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. We found that clotrimazole induced a significant, dose- and time-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP, and cell viability. These findings suggest that clotrimazole causes a reduction in glycolysis and ATP levels, which eventually leads to cell destruction after 3 h of treatment. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, this substance is most promising agent in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cancer cell growth.

The effect of cyclic GMP on phosphofructokinase from rat tissues

In view of the recently proposed hypothesis of biologic regulation through opposing influences of cyclic AMP and cyclic GMP, and since cyclic AMP is a well-known allosteric activator of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the effect of cyclic GMP on the activity of this enzyme from several rat tissues was investigated. It was found that cyclic GMP exerted an inhibitory effect on the activity of rat heart and skeletal muscle phosphofructokinase. This effect was most pronounced under conditions in which the enzyme was partially inhibited by ATP or by citrate. Cyclic GMP also antagonized the deinhibitory action of cyclic AMP and other allosteric activators, such as glucose 1,6-bisphosphate or AMP, on the ATP or citrate-inhibited heart or muscle phosphofructokinase. In contrast to the heart and skeletal muscle phosphofructokinase, the adipose-tissue enzyme was not affected by cyclic GMP to any significant degree. The antagonistic action of cyclic GMP to the activation of heart-phosphofructokinase, may suggest a mechanism by which the activity of phosphofructokinase is synchronized with the activity of glycogen phosphorylase, as a result of acetylcholine action in heart, to achieve a decrease in total glycogenolysis and glycolysis.

Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells

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.