Interaction of antitumor α-lactalbumin—oleic acid complexes with artificial and natural membranes

Abstract

The specific complexes of human alpha-lactalbumin (alpha-LA) with oleic acid (OA), HAMLET and LA-OA-17 (OA-complexes), possess cytotoxic activity against tumor cells but the mechanism of their cell penetration remains unclear. To explore the molecular mechanisms underlying interaction of the OA-complexes with the cell membrane, their interactions with small unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles and electroexcitable plasma membrane of internodal native and perfused cells of the green alga Chara corallina have been studied. The fractionation (Sephadex G-200) of mixtures of the OA-complexes with the vesicles shows that OA-binding increases the affinity of alpha-LA to DPPC vesicles. Calcium association decreases protein affinity to the vesicles; the effect being less pronounced for LA-OA-17. The voltage clamp technique studies show that LA-OA-17, HAMLET, and their constituents produce different modifying effects on the plasmalemmal ionic channels of the Chara corallina cells. The irreversible binding of OA-complexes to the plasmalemma is accompanied by changes in the activation-inactivation kinetics of developing integral transmembrane currents, suppression of the Ca(2+) current and Ca(2+)-activated Cl(-) current, and by increase in the nonspecific K(+) leakage currents. The latter reflects development of nonselective permeability of the plasma membrane. The HAMLET-induced effects on the plasmalemmal currents are less pronounced and potentiated by LA-OA-17. The control experiments with OA and intact alpha-LA show their qualitatively different and much less pronounced effects on the transmembrane ionic currents. Thus, the modification of alpha-LA by OA results in an increase in the protein association with the model lipid bilayer and in drastic irreversible changes in permeability of several types of the plasmalemmal ionic channels.

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…Similar results were obtained regarding the bactericidal activity and membrane depolarization effect of HAMLET, bovine -lactoglobulin and pike parvalbumin in complex with OA on S. pneumococci bacteria [40,64]. Accordingly, it has been proposed that once the complex has adsorbed to the plasma membrane, it is most likely internalized by nonspecific pinocytosis, while other mechanisms of endocytosis, such as receptor-mediated endocytosis have not been ruled out [65].…”

“…Regardless of the preparation method used in the different experiments, it has been shown that the cytotoxicity of the resulting complexes was similar to that of conventionally prepared HAMLET or BAMLET [30,36,48,49,54,[61][62][63][64][65].…”

“…receptor-binding) of the complex to specific membrane regions [76]. Another study found that HAMLET and an -LA/OA complex formed at 17°C, interacts with and alters trans-membrane integral currents of artificial vesicles and natural plasma membranes of the green alga C. coralline [65]. The binding of the OA-complexes caused suppression of Ca 2+ current and Ca 2+ -activated Cl -current, as well as increased nonspecific K + leakage, indicating nonselective permeability of the depolarized plasma membrane [65].…”

“…Another study found that HAMLET and an -LA/OA complex formed at 17°C, interacts with and alters trans-membrane integral currents of artificial vesicles and natural plasma membranes of the green alga C. coralline [65]. The binding of the OA-complexes caused suppression of Ca 2+ current and Ca 2+ -activated Cl -current, as well as increased nonspecific K + leakage, indicating nonselective permeability of the depolarized plasma membrane [65]. Furthermore, all the OA-bound states of -LA had a higher affinity for interaction with the membranes compared to -LA alone, indicating a Ca 2+ -independent association.…”

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes

“…It is thus strongly suggested that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-OA complexes probably works as a delivery carrier of the cytotoxic OA to tumor cells (30,38). It is becoming increasingly evident that not only the rigid native structures but also the flexible MG structures are biologically functional, and many of the functional MG states are related to translocations of proteins or hydrophobic ligands across membrane bilayers (56 -61), and the interaction of HAMLET with artificial and natural membranes has been reported (62)(63)(64). We thus conclude that the MG-like state in HAMLET, GAMLET, and the other related complexes plays a role in transferring the hydrophobic OA molecules into tumor cells across the cell membranes.…”