title: Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET.
abstract: α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF-OA was prepared and its activity and structural changes were compared with α-LA-OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF-OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF-OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA-OA. Similar to HAMLET, LF-OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF-OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein-OA complexes.
authors.affiliation: Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; Academy of State Administration of Grain, Beijing 100037, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Fang
authors.affiliation: Beijing Technology and Business University, Beijing 100048, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; Beijing Laboratory for Food Quality and Safety, Beijing 100083, China.
authors.forename: Mai
authors.initials: M
authors.lastname: Tian
authors.affiliation: Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Beijing Higher Institution Engineering Research Center of Animal Product, Beijing 100083, China.
authors.forename: Hui Yuan
authors.initials: HY
authors.lastname: Guo
authors.affiliation: Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; Beijing Laboratory for Food Quality and Safety, Beijing 100083, China; Beijing Higher Institution Engineering Research Center of Animal Product, Beijing 100083, China. Electronic address: renfazheng@263.net.
authors.forename: Fa Zheng
authors.initials: FZ
authors.lastname: Ren
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002417:Cattle; D002942:Circular Dichroism; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D006860:Hydrogen Bonding / Q000187:drug effects; D007768:Lactalbumin / Q000737:chemistry* / Q000494:pharmacology; D007781:Lactoferrin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology; D019301:Oleic Acid / Q000737:chemistry / Q000494:pharmacology*; D009829:Oleic Acids / Q000737:chemistry* / Q000494:pharmacology; D017433:Protein Structure, Secondary
chemical_list: C506422:HAMLET complex, human; D009829:Oleic Acids; D019301:Oleic Acid; D007768:Lactalbumin; D007781:Lactoferrin
keywords: Anti-tumor; Apoptosis; HAMLET; Isothermal titration calorimetry; Lactoferrin; Oleic acid