Self-assembled β-lactoglobulin–oleic acid and β-lactoglobulin–linoleic acid complexes with antitumor activities

Abstract

β-Lactoglobulin (β-LG) can bind to fatty acids such as oleic acid (OA) and linoleic acid (LA). Another whey protein, α-lactalbumin (α-LA), can also bind to OA to give the complex α-LA-OA, which has antitumor properties. Based on reports that the activity of α-LA-OA is highly dependent on OA, as well as the acquisition of similar complexes using other proteins, such as lysozyme and lactoferrin, we speculated whether β-LG could also kill tumor cells after binding to other fatty acids. Therefore, we prepared complexes of β-LG with OA (β-LG-OA) and LA (β-LG-LA) in the current study and evaluated them in terms of antitumor activity and thermostability using the methylene blue method and differential scanning calorimetry, respectively. The structural features of these complexes were also evaluated using fluorescence spectroscopy and circular dichroism. The binding dynamics of OA and LA to β-LG were studied using isothermal titration calorimetry. Cell viability results revealed that β-LG-LA and β-LG-OA exhibited similar antitumor activities. Interestingly, the binding of β-LG to LA led to an increase in its thermostability, whereas its binding to OA had very little effect. The environments of the tryptophan residues in the β-LG-OA and β-LG-LA complexes were very different, with the residues being blue- and red-shifted, respectively. Furthermore, the hydrophobic regions in β-LG were buried after binding of OA, which was slightly changed in β-LG-LA. Circular dichroism results showed that β-LG-OA enhanced the tertiary structure, which was partially lost in β-LG-LA. There were more binding sites for OA than for LA on β-LG, although the binding constants of the 2 fatty acids were similar, with both acids interacting with the protein though van der Waals and hydrogen bonding interactions. This study could help provide a deeper understanding of the structural basis for formation of antitumor protein-fatty acid complexes.

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

“…Alpha-lactalbumin protein triggers cell death in normal rat intestinal epithelial cells (63) and virus-transformed mouse leukaemia cells (64). These results and HAMLET results showing HAMLET cytotoxicity superior to that of the oleic acid alone (3)(4)(5)15,17,38,44,50,54), indicate that α-lactalbumin protein is also playing role in HAMLET-induced cell death.…”

“…Other studies found that the protein-lipid complex is more cytotoxic than similar amounts of oleic acid or sodium oleate alone (3)(4)(5)15,17,38,44,50,54). It is not yet clear whether these are true differences in cytotoxicity for the different protein carriers of oleate, or whether these different results reflect difficulties in solubilising oleic acid alone for delivery in water-based cellular systems, or are a result of accuracy and precision in measuring the oleate amounts present in the HAMLET-like compounds.…”

“…Other proteins from which cytotoxic HAMLET-like compounds can be made include α-lactalbumin of goat (referred to as GAMLET), horse, pig, and camel (19,46,47), and other proteins altogether, such as bovine β-lactoglobulin (3,4,17), bovine lactoferrin (48), bovine serum albumin (5), pike fish parvalbumin (17), horse lysozyme (referred to as ELOA) (49), recombinant dog milk lysozyme mutant (47), horse heart apo-myoglobin (47,50), chicken ovalbumin (5), recombinant β2-microglobulin (47), and human immunoglobulin G (5). Structural changes have been made to α-lactalbumin, and it continues to be able to make cytotoxic compounds when complexed with oleic acid.…”

“…The fatty acids that produce the most cytotoxic HAMLETs are C18:1:9 cis (oleic acid) and C18:1:11 cis (vaccenic acid), and others that produced HAMLETs of lesser cytotoxicity are C18:3 cis (α-linolenic acid and γ-linolenic acid), C18:1:6 cis (petroselinic acid), C18:2 cis (linoleic acid), and C16:1:9 cis (palmitoleic acid) (55). When complexed with bovine β-lactoglobulin, oleic acid and linoleic acid have similar cytotoxicities (3).…”

“…In vitro cell death assays of HAMLET and BAMLET frequently use the α-lactalbumin protein without oleic acid as the control, and find that the apo-protein is not cytotoxic (3,(13)(14)(15)(16)21,24,38,44). Calcium depleted α-lactalbumin takes on the same folded structure as the native calcium-bound form (66) and the apo-protein used in cell death assays can be assumed to be folded.…”