Misfolded α-lactalbumin Induces Apoptosis In Tumor Cells

Proteins are the executives of different biological functions in vivo. The functions of proteins are based on their structure. Therefore, to research the relationship between structure and function will help us better understand various complex phenomena in lives,α-lactalbumin is a member of lysozyme family and has similar structure to lysozymes. It can bind to fatty acids and novel functions were found. It was reported that complex ofα-lactalbumin and oleic acid named after HAMLET (humanα-lactalbumin made lethal to tumor cells) induces apoptosis in tumor cells but spares healthy cell. In our research, we take both bovineα-lactalbumin (apo-BLA) and humanα-lactalbumin (holo-HLA) as examples to investigate the effects of oleic acid onα-lactalbumin in various pH. The results from "phase diagram" method indicate that there are two intermediates in the conformational transition of apo-BLA induced by acid. One intermediate populated at pH 3.0 while the other accumulates at pH 4.0-4.5. The interaction ofα-lactalbumin and oleic acid was measured by ITC and a binding constant of about 1×10-5 M-1 was observed. The binding of oleic acid to apo-BLA is driven entirely by large favorable entropy increases but with unfavorable enthalpy increases for the first and the third sequential binding sites of the protein. Cell viability experiments indicate the aggregates of either BLA or HLA induced by oleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 and human promyelocytic leukemia cells of HL60.The effects of three fatty acids on cytotoxic aggregate formation of Ca2+-depleted bovineα-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37℃and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovineα-lactalbumin made lethal to tumor cells), a complex of the partially unfoldedα-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity ofα-lactalbumin, and will be helpful to the understanding of the molecular mechanism of HAMLET/BAMLET formation.