Biochemical and Biophysical Research Communications
Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization
Introduction
Cancer is the second most common cause of death in the world after cardiovascular diseases [1]. Targeting tumor cells is one of the key problems in developing anti-cancer therapies. Generally speaking, there are two approaches to the targeting of cancerous cells. In the first variant, agents are developed which are broadly toxic, and subsequent development work is completed which aims to ensure that such agents are delivered only to cancer cells. In the second variant, the delivery of agents is rather broad, but the agent itself is known to be almost exclusively toxic to cancer cells. Here, we focus on complexes which fall into the second category. Human alpha-lactalbumin (hAl, 14.4 kDa) is a whey protein and, normally, it is only in the mammary gland during lactation. The main function of alpha-lactalbumin is alteration of galactosyltransferase substrate specificity. Alpha-lactalbumin is expressed in many breast tumors [2].
Published work indicates that multimeric complexes of lactalbumin and oleic acid (CAlOA) have antitumor activity in cellulo, yet without toxicity to normal cells [3]. The mechanism of action behind such complexes is not well understood. It was shown that CAlOA is capable of penetrating into cells [4], interacting with chromatin [5,6], and interacting with nucleotide-binding proteins [7].
In our previous work, in which we prepared CAlOA complexes [8], it was shown that they had similar activities to those described in the literature for HAMLET (Human Alpha-lactalbumin Make LEthal to Tumor cells) complexes [9]. It should be noted that CAlOA differs from HAMLET in terms of preparation and, most likely, in terms of structure. In recent years it has become obvious that hAl is not the only milk protein capable of binding oleic acid. Lactoferrin (Lf, 78 kDa), for example, also forms cytotoxic complexes with OA [10].
The aim of this research is to study the structure of OA/human alpha-lactalbumin (ChAlOA) and OA/bovine alpha-lactalbumin (CbAlOA) complexes and to determine their influences on tumor chromatin structure, including understanding potential antitumor mechanisms. The properties of ChLfOA (human lactoferrin in complex with OA) were also studied.
Section snippets
Materials and methods
Materials. Commercial oleic acid, reagents, buffers, and chromatography column sorbents were purchased from Merck (Germany).
Alpha-lactalbumin purification. Human and bovine alpha-lactalbumin (hAl and bAl, respectively) were isolated from fresh human or bovine milk by hydrophobic chromatography using phenyl-sepharose, as described [11]. Protein purities and identities were confirmed by SDS-PAGE [12], followed by Coomassie staining [13] and MALDI mass spectrometric analysis.
Human lactoferrin
Results and discussion
All isolated proteins which were used for complexes preparation were reliable identified by mass spectrometry as bAl, hAl and hLf respectively. To characterize complexes of alpha-lactalbumin with oleic acid, CbAlOA and ChAlOA were studied by SANS; similar solutions of bAl and pure OA were used as control (Fig. 1). The data are presented on the double-logarithmic scale.
Samples of CAlOA were characterized by high differential cross section of scattering in small angles that decayed away by
Conclusion
Thus, the artificially created complex of multimeric alpha-lactalbumin, formed in the presence of oleic acid, contain a stable suspension of oleic acid with particles of various sizes, up to tens, and possibly hundreds of nanometers. Alpha-lactalbumin molecules, presumably, are localized on the surface of such particles. At the same time, the lactoferrin and oleic acid complex, does not contain large micelles, and can be considered as a monodisperse system of small (∼20 nm) particles. When
Declaration of competing interest
None.
Acknowledgements
This work based on experiments provided on KWS-3 (#11603, #13835) and KWS-2 (#11589) instruments, local contacts: V. Pipich and A. Radulesku, MLZ, FRM-II Munich, Germany, and also on YuMO spectrometer (experiments #2015-10-15-17-15-09, #2015-10-15-19-57-02, #2017-04-14-14-35-32), IBR-2, local contact A.I. Kuklin, Dubna, Russia.
The work was supported by the NRC “Kurchatov Institute” (№1363).
References (25)
- et al.
HAMLET, protein folding, and tumor cell death
Biochem. Biophys. Res. Commun.
(2007) - et al.
Internalization properties of the anti-tumor α-lactalbumin-oleic acid complex
Int. J. Biol. Macromol.
(2017) - et al.
Multimeric α-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei
Exp. Cell Res.
(1999) - et al.
HAMLET interacts with histones and chromatin in tumor cell nuclei
J. Biol. Chem.
(2003) - et al.
Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET
Biochim. Biophys. Acta Mol. Cell Biol. Lipids
(2014) - et al.
The biological activities of protein/oleic acid complexes reside in the fatty acid
Biochim. Biophys. Acta
(2013) - et al.
Fractal nature of chromatin organization in interphase chicken erythrocyte nuclei: DNA structure exhibits biphasic fractal properties
FEBS Lett.
(2005) - et al.
Targeted vaccination against human α-lactalbumin for immunotherapy and primary immunoprevention of triple negative breast cancer
Cancers
(2016) - et al.
Targeting of nucleotide-binding proteins by HAMLET—a conserved tumor cell death mechanism
Oncogene
(2016)