Cytotoxicity of bovine α-lactalbumin: Oleic acid complexes correlates with the disruption of lipid membranes

Abstract

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.

α-Lactalbumin:Oleic Acid Complex Spontaneously Delivers Oleic Acid to Artificial and Erythrocyte Membranes

“…Our earlier results indicate that BLAOA may incorporate into the lipids upon membrane interaction and cause a tighter lipid packing in the membrane 21 . The membranes become denser after being treated with BLAOA ( Fig.…”

“…Recently, we published a paper focusing on the effects that one such protein-fatty acid complex has on the lipid membrane 21 . We prepared BLAOA, a complex similar to BAMLET, using a simple incubation of BLA in the presence of OA under mild conditions favoring partial unfolding (Ca 2+ -depletion and 37 ºC) 22 .…”

“…That is, the more tension there is between inner and outer leaflet due to BLAOA action, the stronger this effect would be. BLAOA adversely affects the integrity of the membrane in a manner that is proportional to the amount of OA in the complex, and a concomitant tightening packing of the leaflet structure is observed21 . Thus, BLAOA seem to provide both the necessary tension and the necessary defects for this mechanism to be effective.…”

Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

“…A great number of experiments were performed on α-La interaction with lipid bilayers, using both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) as model membranes [13,24,26,41,[43][44][45][47][48][49]. Besides being an excellent model system for studying protein-membrane interactions and describing specific mechanisms for amphitrophic protein behavior [11], the specific interest in the analysis of α-La-membrane interaction is mainly based on the fact that the bovine and human variant of this protein can alter its biological function and gain a tumoricidal property if partially unfolded and bound to oleic acid [32,[50][51][52]. These complexes are named HAMLET (human α-La made lethal to tumor cells) and BAMLET (bovine α-La made lethal to tumor cells) [50,51] and their interaction with membrane has been shown to be crucial to trigger tumor cell death [32,53,54].…”

The Use of Liprotides To Stabilize and Transport Hydrophobic Molecules

“…These complexes are cytotoxic to certain cells (2,3) and bacteria (4) and appear to work by transferring the poorly soluble fatty acid, oleic acid (OA), to cell membranes (5,6) . It is generally believed that the cytotoxicity of these complexes is due to the fatty acid and that the protein component only works as a transporter of the fatty acid (7,8) . Recently a number of protein-fatty acid complexes have been found to be similar to HAMLET in terms of cellular toxicity and structure.…”