title: 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.
authors.affiliation: Department of Molecular Biology, University of Bergen, Thormøhlensgt. 55, 5008 Bergen, Norway.
authors.forename: Hanzhen
authors.initials: H
authors.lastname: Wen
authors.forename: Wilhelm R
authors.initials: WR
authors.lastname: Glomm
authors.forename: Oyvind
authors.initials: O
authors.lastname: Halskau
mesh_terms: D000818:Animals; D002417:Cattle; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008563:Membrane Lipids / Q000378:metabolism*; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D019301:Oleic Acid; D007768:Lactalbumin
keywords: 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; 6-Dodecanoyl-2-Dimethylaminonaphthalene; 8-aminonaphtalene-1,3,6-trisulfonic acid; ANTS; BLA; BLAOA; Bilayer packing; Cell death; DHPE; DPX; ELOA; EYPC; GP; GUV; HAMLET; HLA; LUV; Laurdan; Membrane; OA; Oleic acid; PBPS; PMV; bovine α-lactalbumin; bovine α-lactalbumin complexed with oleic acid; egg yolk phosphatidylcholine; equine lysozyme complexed with oleic acid; general polarization function; giant unilamellar vesicle; human α-lactalbumin; human α-lactalbumin made lethal to tumors; large unilammelar vesicle; oleic acid; p-xyelene-bis-N-pyrimidium bromide; plasma membrane vesicle; porcine brain phosphatidylserine; α-Lactalbumin