ABOUT LACTOFERRIN

Lactoferrin (LF) is an endogenous iron-containing glycoprotein from the transferrin family. It is found in almost all mammals in the postnatal period in most secretions of the exocrine glands of the digestive, respiratory and reproductive tracts: in milk, saliva, bile, pancreatic juice, bronchial secretion, seminal fluid, tears, synovial fluid and others [Janssen RT, 1983, Masson PL, 1966, 1971, Saito K, 1992, Nikolaev AA, 1985], as well as in blood plasma, where it enters from neutrophils, in which it is the main component of secondary secretory granules [RadoTA, 1984].

Lactoferrin is a species-specific glycoprotein, which is due to the structure of its molecule.

Chemical analysis [Metz-Boutigue MH, 1984] and c-DNA cloning [Powell MJ, 1990, Rey MW, 1990] have shown that the human Lf molecule consists of a single polypeptide chain 692 amino acid residues long with a molecular weight of about 80 kDa. The protein contains intramolecular disulfide bonds and does not contain free sulfhydryl groups. According to X-ray structural analysis, the protein chain forms two globules - N- and C-terminal, linked by a helix [Baker EN, 1998]. These globular structures, in turn, each consist of two domains: N1, N2 and CьCр. In each globule of the human Lf molecule, at the border between domains N1 and N2 and C1 and C2, respectively, there are two similar centers for binding iron ions. The binding of iron ions is not the same: one ion is split off during treatment with a pH 4.0 buffer (“acid-labile”), and the other only at pH 2.0 (“acid-stable”). The iron content in the “acid-stable” site is 1020 [Mazurier J, 1980, Iyer S, 1993].

In the physiological state, the lactoferrin molecule is only 10-20% saturated with iron ions, which largely determines the functions of LF as an antioxidant.The N2 and C2 domains contain one asparagine residue each, to which a polysaccharide chain of the N-acetyl-lactosamine type is attached [Spik G, 1982].

HUMAN LACTOFERRIN

The lactoferrin of various mammals is highly homologous in structure to human lactoferrin

The human LF gene is located on the short arm of the 3rd human chromosome, region Зр21.3 [Iijima H, 2005]. The enormous interest in LF among researchers of various profiles is caused by its multifaceted physiological functions [Vorland LH., 1999, Tsuda H, 2002, Valenti P, 2004, Ward PP, 2005, Shimazaki K, 2005]

the LF molecule of cow (LFc) consists of 689 amino acid residues and has 69% homology with the primary structure of the LF molecule of human. The spatial structure of human and cow LF also has much in common, but is not identical [Goodman RE, 1991, Pierce A, 1991, Moore SA, 1991].

The molecular mechanisms underlying these physiological functions and the unique biological role of LF are not actually clear. However, it is currently known that LF plays an important role in the metabolism and transport of iron ions and other variable-valence metal ions in the body [Harrington JP, 1992]; has the ability to bind to the surface of bacteria, viruses, fungi and protozoa [Yu RH, 2000], as well as to specific receptors on blood cells, liver, vessels, etc. [Suzuki YA, 2002]. In recent years, the RNAse activity of LF [Kanyshkova TG, 2003] has been discussed, as well as its role as a transcription activator [Choi S-Y, 2005]. The listed properties of LF determine its most important functions: antioxidant, antibacterial and immunomodulatory. The implementation of these properties of LF in the body determines its action as a detoxifying, anti-inflammatory and anti-carcinogenic agent.

Antioxidant properties

The antioxidant properties of LF have been confirmed in various model systems [Bemun S., 1997, Huang Sh.-W., 1999, Steijns J., 2000].

They are explained by the ability of LF to bind iron ions Fe3+ and thereby reduce the amount of Fe2+ formed as a result of the Fenton reaction (Fe3++02 - Fe2+ +O2 reaction 11). Since Fe2+ ions are a catalyst in the formation of hydroxyl radical by the Haber-Weiss reaction (O2,+H2O2 – OH+0H+02 reaction 6) (Zenkov N.K. et al., 1993), depletion of the medium in iron ions leads to a decrease in the intensity of oxidation processes.

The greater activity of apo-LF than LF saturated with Fe3+ by 45% and 100% confirms that it is precisely the strong coordination binding of iron ions to the LF molecule that underlies one of the mechanisms for the implementation of the antioxidant properties of LF [Bemun S, 1997].

There is another mechanism that explains the antioxidant properties of LF. Fe3+ ions, bound to the LF molecule, are capable of being reduced to Fe2+ in the presence of free radicals and, thus, inactivate free radical compounds. Since the Fe2+ ion remains bound to the LF molecule, it does not exert a catalytic effect in the reactions of activated oxygen metabolites formation.

Anti-inflammatory effect

LF has the ability to modulate the action of phagocytic blood cells - monocytes and neutrophils [Baveye S, 2000, Legrand D, 2005, Kruzel ML, 2002, Artym J, 2004], enhances the cytotoxic action of natural killers [Damiens E, 1998, Artym J, 2004], regulates the functions of the lymphocyte component of immunity [Mincheva-Nilsson L., 1997, Artym J, 2003, Artym J, 2005].

The important antibacterial and anti-inflammatory role of LF is also confirmed by clinical data [Vorland L., 1999, Zimecki M, 2001]. There is an opinion that LF can be considered as an acute-phase protein, since its level in the blood serum increases in many inflammatory diseases: bacterial and viral infections of newborns [Gutteberg TJ, 1984, 1984], rheumatoid arthritis, pulmonary tuberculosis, acute pneumonia and many others [Benini L., 1985, Baynes R., 1986, Nemtsova E.R., 1995]. In acute pneumonia, the dynamics of LF concentration in plasma and the ratio of LF level and the number of leukocytes in the blood are prognostic indicators [Baynes R., 1986}.

The antioxidant, antimicrobial and immunomodulatory properties of LF determine its anti-inflammatory effect, which is manifested at all phases of inflammation [Conneely OM, 2001]. The presence of LF at sites of inflammation can limit the degree of cell damage by oxygen radicals due to the antioxidant mechanisms described above [Ward PP, 2002].

The function of LF as an acute-phase protein is also manifested in secretions: a sharp increase in the concentration of LF is noted in saliva in inflammatory diseases of the salivary glands; in pancreatic juice - in inflammatory diseases of the pancreas, etc. [Arao S, 1999, Cafénberg G.H., 2000, Niehaus M.D., 2000].

MALIGNANT TUMORS AND LF

The discovery of the immunomodulatory effect of LF stimulated the study of its influence on the growth of malignant tumors.

Authors suggest

The authors suggest that the antitumor action of Lf is based on its immunomodulatory activity: a significant increase in IL-18 production in the gastrointestinal tract, activation of NK cells and an increase in the number of circulating CD8+ lymphocytes were noted. Indirect confirmation of this is the absence of a direct cytostatic effect of LF on tumor cells in culture.

Study results

The results of experimental studies of human and bovine LF on models of transplantable tumors in animals: melanoma B-16-B16, L5178, Y-ML25, rectal carcinoma C26 showed that, indeed, both subcutaneous and oral administration of LF at a dose of 1-5 mg/mouse inhibits the growth of the primary tumor node and the formation of distant metastases; intratumoral administration of bovine LF on a model of orthotopic tumors - squamous cell carcinoma and fibrosarcoma of the floor of the oral cavity leads to 50-54% inhibition of tumor growth compared to the control.

EFFECT ASSUMPTIONS

Despite the large number of studies aimed at studying the mechanism of the anticarcinogenic action of LF, it is not completely clear.

REDUCTION IN THE METABOLIZING ACTIVITY OF THE CYTOCHROME P450 ISOENZYME IA2

One of the assumptions is that LF affects the enzymes of phases I and II of procarcinogen metabolism [Tsuda H, 1998, 2002]. Fujita K, et al [Fujita K, 2002] studied the effect of Lactoferrin on the isoenzyme IA2 of cytochrome P450 on models of rat liver and rectal tumors induced by diethylnitrosamine (DEN) and 2-amino-3,8-dimethylimidazo[4,5-£1quinoxaline (MelQx). This isoenzyme is induced by MelQx and, in turn, metabolizes MelQx in the liver, converting it from a procarcinogen into a carcinogen. As a result, it was shown that LF, when used in combination with MelQx, normalizes the levels of mRNA and the protein of the cytochrome P450 isoenzyme IA2, and also reduces the amount of MeIQx-DLG adducts. The authors suggest that it is the reduction in the metabolizing activity of the cytochrome P450 isoenzyme IA2 in relation to procarcinogens that plays a leading role in the anticarcinogenic effect of Lactoferrin in this model.

INCREASE IN APOPTOSIS

It is also likely that chemoprophylaxis with lactoferrin is due to an increase in the expression of a number of proapoptotic proteins under the influence of this glycoprotein and, accordingly, an increase in apoptosis. This assumption is based on data from Japanese authors who studied the expression of genes of 10 molecules involved in apoptosis in the process of developing carcinogenesis in the colon induced in rats by azoxymethane in combination with LF [Fujita K, 2004, 2004].

They found increased expression of Fas, Bax and other proapoptotic proteins in the tissues of animals that were administered Lf, compared to the tissues of animals that were administered only the carcinogen. Similar results were obtained in vitro on various cell types [Tsuda H, 2002, Mader JS, 2005, Sakai T, 2005].

LACTOFERRIN RELEVANCE

The conclusions below demonstrate the relevance of creating a drug based on human Lactoferrin.

LACTOFERRIN ACTION

Several mechanisms are involved in the implementation of the anticarcinogenic and antitumor action of LF: 1) antioxidant [Shklar G, 1998], 2) activation of cytotoxic cells responsible for antitumor immunity [Wang W.P., 2000]; 3) antiangiogenic action [Norrby K., 2001]; induction of proapoptotic factors [Fujita K, 2004, 2004] and influence on xenobiotic metabolism enzymes [Fujita K, 2002, Tsuda I, 2002].

FINAL CONCLUSIONS

Thus, taking into account the above-described properties, functions and action of Lactoferrin, as well as its localization in tissues as a barrier protein and the role as an acute phase protein, it can be assumed that the use of Lactoferrin as a means of replacement therapy is very promising for detoxifying the body, reducing the microbial load and reducing the intensity of inflammatory reactions.