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

Abstract

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.

Introduction

Glycolysis is the primary energy source in cancer cells, exceeding the capacity of mitochondrial oxidative energy metabolism (Eigenbrodt et al., 1985; Fiechter and Gmünder, 1989; Beckner et al., 1990; Greiner et al., 1994). Glycolysis is known to be controlled by allosteric regulators (for reviews, see Beitner, 1979, Beitner, 1984, Beitner, 1985, Beitner, 1990), as well as by reversible binding of the glycolytic enzymes to cytoskeleton (Arnold and Pette, 1968; for reviews see Clarke et al., 1985; Beitner, 1993; Pagliaro, 1993). The latter mechanism has recently attracted much attention. It has been shown by many laboratories that glycolytic enzymes bind reversibly to the cytoskeletal elements, particularly to the actin filaments and also to tubulin/microtubules (Arnold and Pette, 1968; Clarke and Masters, 1975; Clarke et al., 1985; Pagliaro and Taylor, 1988, Pagliaro and Taylor, 1992; Walsh et al., 1989; Lilling and Beitner, 1990, Lilling and Beitner, 1991; Lilling et al., 1991; Beitner, 1993; Lehotzky et al., 1993). The binding of glycolytic enzymes to cytoskeleton was demonstrated both in vitro, using purified components, and in vivo, in whole tissues, as well as in different cultured cells (Minaschek et al., 1992).

All glycolytic enzymes bind to cytoskeleton (Clarke et al., 1985) except hexokinase, which binds reversibly to mitochondria, where it is linked to oxidative phosphorylation (Gots et al., 1972; Viitanen et al., 1984; Mohan et al., 1989; Adams et al., 1991). Cytoskeletal glycolysis provides local ATP in the vicinity of the cytoskeleton (Beitner, 1993), which is known to interact dynamically with plasma membrane upon membrane-induced events (Geiger, 1983). Binding of glycolytic enzymes to cytoskeleton also affects cell structure, as glycolytic enzymes were found to cross-link actin-containing filaments into ordered supramolecular structures (Clarke et al., 1985). Many factors and conditions control the binding of glycolytic enzymes to cytoskeleton (Beitner, 1993; Parra and Pette, 1995), which is being recognized as an important modulator of metabolic functions in the cell. The actin cytoskeletal network is involved in events regulating cell proliferation and differentiation, and alterations in actin state were reported during malignant transformation of cells in culture, and in naturally occurring tumors (for review, see Rao and Cohen, 1991). Cell cycle-related changes in F-actin distribution were shown to correlate with glycolytic activity (Bereiter-Hahn et al., 1995).

Our previous experiments have revealed that growth-promoting hormones, insulin and growth factors, stimulate glycolysis by increasing the binding of glycolytic enzymes to cytoskeleton and by raising the level of glucose 1,6-bisphosphate, the signal molecule, which is a potent allosteric activator of glycolysis. We have also shown that all these effects of insulin and growth factors are prevented by treatment with calmodulin antagonists, which strongly suggest that Ca²⁺/calmodulin is involved in their stimulatory action on glycolysis, which supplies energy for cell growth (Chen-Zion et al., 1992a, Chen-Zion et al., 1992b, Chen-Zion et al., 1993; Beitner, 1993; Livnat et al., 1993, Livnat et al., 1994, Livnat et al., 1995). Calmodulin is a multifunctional Ca²⁺ binding protein that has been implicated in the regulation of numerous cellular events, including that of normal and abnormal cell proliferation (Veigl et al., 1984; Hait and Lazo, 1986; Rasmussen and Means, 1987; Reddy, 1994). Calmodulin antagonists were reported to inhibit cellular proliferation of various cells (Hait and Lee, 1985; Susuki et al., 1986; Ford et al., 1989; Mac Neil et al., 1993a; Hait et al., 1994), including melanoma (Ito and Hidaka, 1983; Mac Neil et al., 1984; Al-Ani et al., 1988).

Recent experiments from our laboratory have revealed that calmodulin antagonists decrease the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis, and reduce ATP content and cell viability in B16 melanoma cells (Glass-Marmor et al., 1996). In the present research, we investigated whether calmodulin antagonists also exert an effect on the cytoskeleton-bound glycolytic enzymes, phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis, and aldolase (d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), in B16 melanoma cells. We used the same calmodulin antagonists as in our previous research (Glass-Marmor et al., 1996), namely: thioridazine (10-[2-(1-methyl-2-piperidyl)ethyl]-2-methylthiophenothiazine), an antipsychotic phenothiazine, clotrimazole (1-(α-2-chlorotrityl)imidazole) and bifonazole (1-(α-biphenyl-4-ylbenzyl)imidazole), the antifungal azole derivatives, that were recently recognized as calmodulin antagonists (Hegemann et al., 1993; Mac Neil et al., 1993a), and CGS 9343B (1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate), a more selective inhibitor of calmodulin activity (Norman et al., 1987).