ЛАКТОФЕРРИН И ПРОФИЛАКТИКА РАКОВЫХ ЗАБОЛЕВАНИЙ оригинал

ЛАКТОФЕРРИН И ПРОФИЛАКТИКА РАКОВЫХ ЗАБОЛЕВАНИЙ оригинал

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Lactoferrin and Cancer Disease Prevention
Article in Critical Reviews In Food Science and Nutrition · April 2009
DOI: 10.1080/10408390701856157 · Source: PubMed
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1
LACTOFERRIN AND CANCER DISEASE PREVENTION
Lígia Rodrigues, 1* José Teixeira, 1 Fernando Schmitt, 2,3 Marie Paulsson, 4 Helena
Lindmark Månsson, 4,5
1 IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Campus de
Gualtar, 4710-057 Braga, Portugal,
2 Instituto de Patologia e Imunologia Molecular – IPATIMUP, Universidade do Porto, Rua Dr. Roberto
Frias, 4200-465 Porto, Portugal,
3 Faculdade de Medicina, Universidade do Porto, Al. Prof. Hernâni Monteiro 4200 - 319 Porto,
Portugal,
4 Department of Food Technology, Engineering and Nutrition, Lund University, P.O. Box 124, S-221 00,
Lund, Sweden,
5 Swedish Dairy Association, Scheelevägen 18, SE-223 63 Lund, Sweden.
Running title: Cancer disease prevention
Keywords: lactoferrin, chemoprevention, infection, inflammation, cancer
Corresponding author:
* Lígia Rodrigues
IBB – Institute for Biotechnology and Bioengineering
Centre of Biological Engineering
Campus de Gualtar, 4710-057 Braga, Portugal.
Tel. 351-253-604400
Fax 351-253-678986
e-mail: Адрес электронной почты защищен от спам-ботов. Для просмотра адреса в браузере должен быть включен Javascript.

2
ABSTRACT
Lactoferrin (LF) is an iron-binding glycoprotein that composes the transferrin family and is
predominantly found in the products of the exocrine glands located in the gateways of the
digestive, respiratory and reproductive systems, suggesting a role in the non-specific defence
against invading pathogens. Additionally, several physiological roles have been attributed to
LF, namely regulation of iron homeostasis, host defence against infection and inflammation,
regulation of cellular growth and differentiation and protection against cancer development and
metastasis. These findings have suggested LF’s great potential therapeutic use in cancer disease
prevention and/or treatment, namely as a chemopreventive agent. This review looks at the
recent advances in understanding the mechanisms underlying the multifunctional roles of LF
and future perspectives on its potential therapeutic applications.

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INTRODUCTION
Over the past few decades, clinical and mechanistic studies have indicated many relations
between nutrition and health, thus evidence that diet is a key environmental factor affecting the
incidence of many chronic diseases is overwhelming. The precise extent of this contribution is
difficult to judge, but a reduction of 35% in the age standardized incidence of cancer in the
United States has been proposed to be achievable via “practicable dietary means” (Elliot and
Ong, 2002; Davis and Milner, 2004; Petricoin and Liotta, 2003). Indeed, several food derived
compounds we eat are among the most promising chemopreventive agents being evaluated.
Chemoprevention is defined as using chemicals with the goal of preventing, interrupting or
reversing the carcinogenic process. Since carcinogenesis is a multistage process, which usually
takes many years in humans, there is ample opportunity to intervene and prevent the
development of cancer. Chemopreventive agents are ultimately expected to be in widespread
use by the general population. Therefore, they need to be non-toxic, inexpensive and available
for use by oral via. With the demonstration of Tamoxifen ability to prevent breast cancer in
women, the feasibility of chemoprevention in humans has now been well established (Jordan
and Morrow, 1999) The full extent of biologically active components in our diet is unknown,
and our understanding of their mechanisms of action is even more limited (Petricoin and Liotta,
2003). Much of the available data has been derived from in vitro studies with purified
compounds in forms and concentrations to which the tissues in our bodies may never be
exposed (Davis and Milner, 2004; Petricoin and Liotta, 2003). Furthermore, nutrition research
has traditionally concentrated on single issues (such as reducing risk of cardiovascular disease
or cancer) in “at risk” individuals, whereas what we need to address is the question of all the
possible effects of specific food components in a genetically heterogeneous population. This is
especially important for determining unintended risk as well as unintended benefit (Elliot and
Ong, 2002; Davis and Milner, 2004).

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A promising field of research is clinical studies with cancer preventive proteins existing in milk
(McIntosh, 1993; Tsuda et al., 2002), namely lactoferrin (LF). LF known for its inhibitory
action on cell proliferation, as well as for its anti-inflammatory and antioxidant abilities, has
been described to have anti-carcinogenic properties in several in vivo and in vitro studies
(Baveye et al., 1999; Brock, 2002; Nuijens et al., 1996; Tsuda et al., 2000, 2002; Ward et al.,
2002; Ushida et al., 1999; Sekine et al., 1997a; Iigo et al., 1999; Wang et al., 2000; Matsuda et
al., 2006; Pan et al., 2007). Nevertheless, it is still poorly understood if orally administrated LF
is absorbed from the intestine, and if it exerts its protective effect at the site were it is expected,
especially in the case of diseases occurring at sites distant from the gastrointestinal tract.
Many food components in the form of biologically active proteins, peptides, lipids, and other
substances survive the digestive processes and can enter the circulation. Thus, it is possible that
absorbed intact peptides can exert biological activities during health and disease. Lactoferricin
(LFCin) is a peptide fragment produced by acid-pepsin hydrolysis of LF. Although LFCin has
attracted considerable interest because of its well established antimicrobial activity (Yamauchi
et al., 1993), recent evidence indicates that orally or subcutaneously administrated LFCin also
possesses potent in vivo activity against cancer cells, namely inhibiting metastasis, angiogenesis
and tumour growth (Cho et al., 2004; Yoo et al., 1997a). However, the mechanisms by which
LFCin exerts its action are still unknown.
Regardless of LF reported potential, very few studies have covered an integrated and systematic
approach to its effects as an anti-cancer agent on breast cancer (Benaissa et al., 2005; Teng et
al., 2004; Miyamoto et al., 2005; Giancotti, 2006), although some high-throughput techniques,
such as DNA microarrays and proteomics, have already been applied (Forozan et al., 2000;
Perou et al., 1999; Shan et al., 2002) without a consistent application of bioinformatics tools for
data interpretation. Therefore, it remains a large task to identify the mechanisms by which LF
and its digested fragments exert their action and to discover innovative means to supply them to
consumers.

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This review aims to provide an overview of LF functions and mechanisms of interaction that
could be exploited further in developing its potential therapeutic applications, namely in cancer
disease prevention and/or treatment.
LACTOFERRIN OCCURRENCE AND STRUCTURE
LF was firstly recognised as a single-chain iron-binding protein (Groves, 1960; Johanson, 1960;
Montreuil et al., 1960) and the names given to it, LF or lactotransferrin, firmly assigned it to the
family of proteins known as the transferrins. The archetypal members of the transferrin family
are LF, serum transferrin and ovotransferrin, with ovotransferrin being equivalent to avian
serum transferrin, differing only in its glycosylation (Baker, 1994). These proteins are all
glycoproteins that typically exhibit 50-70% pair wise sequence identity (Metz-Boutigue et al.,
1984; Pierce et al., 1991; Moore et al., 1997). Between LF and serum transferrins the sequence
identity is around 60% and between LF from different species it is around 70% (Baker, 1994;
Baker and Baker, 2005; Teng et al., 2002; Bowman et al., 1988), for example bovine LF (bLF)
shares 69% amino acid identity with human LF (hLF) (Shah, 2000; Pierce et al., 1991). LF is
predominantly found in the products of the exocrine glands located in the gateways of the
digestive, respiratory and reproductive systems. Thus, LF can be found in the secreted milk, in
tears, synovial fluids, saliva and seminal fluid (Lönnerdal, 2003). Table 1 shows a selection of
the LF amounts reported from these various biological fluids. Additionally, LF can be found in
blood and plasma LF is derived from neutrophils, which degranulate and synthesize the protein
during inflammation (Britigan et al., 1994; Levay and Viljoen, 1995; Iyer and Lönnerdal, 1993;
Van Snick et al., 1974). Some physico-chemical properties of LF are summarized in Table 2.
Structurally, LF is folded into two lobes, representing its N- and C-terminal halves (Baker et al.,
1994), that show sequence homology with each other and can each reversibly bind one ferric ion
along with a synergistic anion, usually bicarbonate (Baker, 1994; Steijns and Hooijdonk, 2000).
In these respects it closely resembles transferrin, although its affinity for iron is somewhat
higher, allowing iron to be retained at lower pH values (Mazurier and Spik, 1980; Peterson et

6
al., 2000). This gives LF a more potent iron withholding ability (Baker et al., 2002). The lobes
are connected by a peptide of 10-15 residues, which in LF forms a 3-turn α-helix, but in
transferrins is irregular and flexible. There are non-covalent interactions, mostly hydrophobic
where the two lobes pack together (Haridas et al., 1995).
LACTOFERRIN GENE EXPRESSION AND REGULATION
In situ fluorescence hybridization indicated that the hLF gene is mapped in the region 3p21.3
(Kim et al., 1998; Klintworth et al., 1997; Teng et al., 1987), organized in 17 exons and its total
length is about 24.5 kb. A comparison of the structure of the hLF gene with that of other species
revealed that it is more closely related to bLF than to murine LF (Furmanski et al., 1989; Geng
et al., 1998). The positions of the introns are well matched with those of the bLF gene except
only for exons 14 and 15. All the exon-intron boundaries of the gene conform to the GT/AG
rule. Although LF is highly conserved among several species, its expression is both ubiquitous
and species, tissue, and cell-type specific (Das et al., 1999; Furmanski et al., 1989; Grant et al.,
1999; Shigeta et al., 1996; Teng et al., 2002). It is differentially regulated through multiple
signalling pathways such as steroid hormone, growth factor, and kinase cascade pathways (Das
et al., 1997; Teng et al., 2002; Close et al., 1997).
Expression of the LF gene is both constitutive and inducible. During the differentiation of
myeloid cells, the LF gene responds to the developmental signals and is transcribed in the
myelocyte and metamyelocyte stages (Berliner et al., 1995; Khanna-Gupta et al., 2000). While
LF is constitutively expressed in the wet surface mucosa, LF is estrogen inducible in uterine
tissue (Pentecost and Teng, 1987; Teng et al., 1986, 1989). Moreover, the presence of LF in the
human reproductive tract has also been localized in the prostate and seminal vesicle of males
(Goodman and Young, 1981; Wichmann et al., 1989) and is a major coat protein of human
sperm (Hekman and Rumke, 1969).
Since the LF gene promoter consists of multiple response elements for various signalling
pathways such as the route for kinase cascade, the estrogenic compounds could act through

7
response elements other than ERE (estrogen response element) to activate the gene (Teng, 2006;
Giancotti, 2006) .
There are few studies on the regulation of LF gene expression other than that by hormones. In a
cultured mouse mammary epithelial cell system, LF expression was induced at high cell density
in the absence of exogenously added basement membranes or prolactin (Close et al., 1997),
although an earlier study suggested that LF expression in a mouse mammary gland explants is
stimulated by prolactin (Green and Pastewka, 1978). Close and co-workers (1997) demonstrated
that LF expression in mammary epithelial cells is mediated by changes in cell shape and actin
cytoskeleton. This observation offers an explanation for the suppression of LF expression in
monolayer culture and in malignant tissues in which inappropriate cell shapes and cytoskeletal
structures are manifested under these conditions. Multiple signalling pathways and diverse
regulatory proteins regulate LF gene expression in either a positive or negative manner (Teng,
2006). Although the mechanism of regulation is still unknown, the factors involved in LF gene
expression could provide clues as to where LF is needed and under what conditions.
LACTOFERRIN METABOLISM AND RECEPTORS
LF is produced in neutrophils and stored in specific granules and possibly in tertiary granules
(Iyer and Lönnerdal, 1993; Lönnerdal, 2003; Van Snick et al., 1974). The steroid-thyroid
receptor super family works in concert to modulate LF gene expression supporting the
hypothesis that LF levels are hormone dependent (Levay and Viljoen, 1995; Giancotti 2006;
Teng, 2006). LF, unlike myeloperoxidase and some other granular products, is not synthesized
as a larger precursor and was found to be unphosphorylated (Olsson et al., 1988). The
neutrophil LF within the granules can either be secreted into the surrounding tissues or blood
(Van Snick et al., 1974), or the granules can fuse with phagosomes (Maher et al., 1993).
Secretion from polymorphonuclear cells into the circulation is dependent on degranulation
factors, which in turn appear to be dependent on the activation of guanylate cyclase, cGMP and
proteinkinase C (calcium dependent). This occurs in both aerobic and anaerobic conditions, is

8
unaffected by the presence of hydrogen sulphide and is stimulated by IL-8 and surface bound
IgG (Maher et al., 1993; Kahler et al., 1988). Plasma LF levels generally increase in iron
overload, inflammation, infectious diseases, and during tumour development, demonstrating a
multifactor stimulatory mechanism for LF release from neutrophils (Levay and Viljoen, 1995,
Bullen et al., 2006).
LF removal from circulation appears to occur in one of two ways. First, LF can be removed
from the circulation, as well as from the interstitial spaces, by what would appear to be receptor-
mediated endocytosis into phagocytic cells such as macrophages, monocytes and other cells of
the RES (reticuloendothelial system), with subsequent transfer of the iron to ferritin (Olofsson
et al., 1977; Van Snick et al., 1974, 1976). However, some controversy with regard to the cells
involved in this way of LF removal still exists (Ismail and Brock, 1993). The alternative mode
of LF removal would be its direct uptake by the liver by liver endothelial cells and hepatocytes
(Hu et al., 1993). Bennet and Kokocinski (1979) showed that labelled LF was rapidly cleared
from the circulation by the liver and spleen. Nevertheless, it is still not established whether LF,
like transferrin, is recycled (Birgens et al., 1988). Further research is needed to fully understand
LF metabolism in the human adult. The kidneys appear to play a role in LF clearance from the
circulation since both LF and LF fragments were found in the urine of infants (Hutchens et al.,
1991). Both faecal and urinary elimination of LF, however, need further investigation because
significant controversy still exists (Desai et al., 2007; Hirata et al., 2007; Yamauchi et al.,
2006).
LF receptors have been identified in the gastrointestinal tract, on leukocytes and macrophages,
platelets, and on bacteria. The surface of the LF molecule has several regions with high
concentrations of positive charge responsible for one of the features that distinguishes LF from
other transferrins and for some unique properties (Lampreave et al., 1990; Zakharova et al.,
2000; Baker, 1994; Van Berkel et al., 1997). The most striking region of positive charge
comprises the N-terminus of the polypeptide chain (with the sequence GRRRRS in hLF) which
projects from the protein surface and the adjacent C-terminus of helix 1 where residues 27-30
have the sequence RKVR (Baker et al., 2002; Rochard et al., 1989). This region provides a site

9
for binding heparin (Van Berkel et al., 1997) and glycosaminoglycans (Mann et al., 1994) and is
very likely the site that binds DNA. It may also be important for the ability of LF to bind to
many cell types, possibly through binding to the negatively charged phospholipids groups of the
cell membranes (Yu and Schryvers, 1993). The similarity of the N-terminal sequence
(GRRRRS) to a common nuclear localisation signal sequence motif in eukaryotes could further
explain the observation that LF can enter the nucleus (He and Furnmanski, 1995). Contiguous
with the N-terminal patch is helix 1, which forms the main part of the bactericidal
“lactoferricin” domain (Bellamy et al., 1992), which is characterised by its display of surface
arginine residues. Despite their virtually identical fold, other transferrins do not share this
bactericidal activity, presumably because they lack the necessary surface features. The other
main concentration of positive charge is in the inter-lobe region, associated with the helix that
joins the two lobes. This also appeals as a likely DNA binding region, both because of the
charge and because of the cleft that is formed between the lobes, which could create a docking
site.
LACTOFERRIN FUNCTIONS
Because of its close resemblance to transferrin, initial research on LF function was directed
towards establishing functions related to its iron-binding properties, namely iron absorption,
antimicrobial activity, and modulation of iron metabolism during inflammation. However,
despite their structural similarities, LF differs from its serum counterpart in several important
aspects including location and functional activity. LF has been proposed to play a role in
intestinal iron absorption, regulation of cellular proliferation and differentiation, protection
against microbial infection, anti-inflammatory responses, regulation of myelopoiesis,
immunomodulation and cancer prevention (Pan et al., 2007; Ward et al., 2005; Naidu, 2002;
Min and Krochta, 2005; Steijns and Van Hooijdonk, 2000; Baveye et al., 1999; Nuijens et al.,
1996; Levay and Viljoen, 1995; Iyer and Lönnerdal, 1993; Sanchez et al., 1992). Table 3
summarizes some of the established physiological roles for LF and its mechanisms.

10
The reported antimicrobial activities (Santagati et al., 2005; Valenti et al., 1998; Levay and
Viljoen, 1995) of LF highlight the many possible modes by which it can contribute to host
protection against microbial infections at the mucosal surfaces, namely by growth inhibition as
a result of iron scavenging (Nemet and Simonovits, 1985; Brock, 1980), disruption of the
bacterial cell membranes (Yamauchi et al., 1993; Ellison and Giehl, 1991; Al-Nabulsi and
Holley, 2006) or blocking of cell-virus interactions (Andersen et al., 2001; Ikeda et al., 2000;
Siciliano et al., 1999; Giansanti et al., 2002).
Regarding LF anti-inflammatory activity several mechanisms of action by blocking or inhibiting
key mediators of the inflammatory response have also been proposed, such as binding to LPS
(Miyazawa et al., 1991); inhibition of several cytokines (TNF-α and IL-1β) (Machnicki et al.,
1993; Crouch et al., 1992; Slater and Fletcher, 1987; Baveye et al., 1999) or binding to bacterial
CpG motifs (Britigan et al., 2001). Moreover, LF was found to elevate the number and increase
the activity of T and B lymphocytes and NK cells (Dhennin-Duthille et al., 2000; Goretzki and
Mueller, 1998), stimulate the release of a number of cytokines (Hangoc et al., 1991), increase
phagocytic activity and cytotoxicity of monocytes/macrophages (Birgens et al., 1984; Van
Snick and Masson, 1976), accelerate the maturation of T and B cells, and elevate the expression
of several types of cellular receptors (Adamik et al., 1998; Bennett and Davis, 1981; Frydecka et
al., 2002; Zimecki et al., 1991, 1995). Many immunological mechanisms are critically
dependent upon cell–cell interactions; the number and affinity of interactions between two cells
can often affect the nature of downstream events. The ability of LF to bind to cell surfaces is
likely to affect these parameters, and could thus give rise to altered immune responses (Legrand
et al., 2005).
Lactoferrin and iron metabolism
Despite much research into the interactions of LF with cells and tissues, there is still no good
evidence that it plays any role as an iron-transport molecule or indeed is involved in
“mainstream” iron metabolism (Brock, 2002). Nevertheless, it is consensual that LF influences
the iron availability (Levay and Viljoen, 1995).

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Iron is an element necessary for many metabolic functions of cells including cell proliferation.
Therefore, cells and organisms have developed elaborate mechanisms of uptake and storage of
iron to facilitate the utilization of this element in metabolic processes and in cell proliferation
and at the same time to keep concentrations of free iron at a minimum. In normal physiological
processes, iron uptake, storage and utilization are carefully regulated. Cellular damage arising
from free radicals, generated by available iron, is kept at a low level by cellular defences and
DNA repair systems (Liehr and Jones, 2001).
While iron supplementation and iron enrichment of foods is widely accepted, there have been
persistent concerns about potential deleterious effects, because iron enhances colon and
mammary carcinogenesis in rodents and because elevated body iron stores increase the risk of
several cancers in humans (Weinberg, 1984, 1992; Selby and Friedman, 1988; Stevens et al.,
1988). There is also indirect, but substantial evidence supporting a role of iron metabolism in
breast cancer. For instance, a six-fold higher tissue ferritin concentration has been measured in
malignant carcinoma of the breast compared to normal or benign tissue (Weinstein et al., 1982;
Elliott et al., 1993). Furthermore, transferrin and tumour cell transferrin receptor proteins were
elevated in breast carcinoma compared to normal or benign cells (Faulk et al., 1980; Rossiello et
al., 1984).
All these data taken together indicate that iron enhances tumour development in humans.
Certainly, proliferating tumour cells require iron and therefore tumour tissue may have
increased transferrin receptor and ferritin levels.
Current evidence suggests that while LF plays no major role in normal iron homeostasis (Ward
et al., 2005), it may contribute to alterations in iron metabolism during infection or
inflammation. In addition, the iron-binding function of LF may contribute to other physiological
functions.
Lactoferrin and human cancer
Many functions have been described for LF over the years, but concerning its anti-tumour role
and mechanisms involved there is still a big controversy on whether LF’s iron-binding activity

12
is implicated or not. Lonnerdal and Iyer (1995) state that it is likely to be the iron-binding
properties of LF that contribute to its anti-tumour properties, since free iron may act as a
mutagenic promoter by inducing oxidative damage to nucleic acid structure (Weinberg, 1984,
1992,1996). It is thought that LF may bind iron locally in tissues, therefore reducing the risk of
oxidant-induced tumourigenesis (Stevens et al., 1988). Moreover, estrogenic hormones appear
to regulate the uptake of iron and its utilization in proliferative processes (Liehr and Jones,
2001; Teng, 2006). Nevertheless, much research needs to be carried out in future to outline in
more detail the regulation of iron metabolism by estrogen. Tumours may thus arise in cells,
which are damaged by such processes and which at the same time are stimulated to proliferate
by hormone receptor-mediated mechanisms. The possible contribution of iron to genetic lesions
and to cell proliferation indicates that this element plays a crucial role in the development of
estrogen-dependent neoplasms such as breast or uterine cancers (Weinstein et al., 1982; Elliot et
al., 1993; Faulk et al., 1980; Rossiello et al., 1984). Therefore, such hormone responsive tumour
development may be prevented by iron chelators (Ghio et al., 1992; Lund and Aust, 1990;
Giancotti, 2006). Another possibility is the restriction of dietary iron intake and dietary iron
supplements, or the diet supplementation with LF, as part of a strategy to reduce tumour
development. Such studies need to be carried out in the future.
On the other hand, several other mechanisms have been described for the LF anti-tumour role,
such as regulation of NK cell activity (Damiens et al., 1998; Matsuda et al., 2006), modulation
of expression of G1 proteins (Damiens et al., 1999; Xiao et al., 2004), inhibition of VEGF(165)-
mediated angiogenesis (Norrby et al., 2001), and enhancement of apoptosis (Yoo et al., 1997b;
Sakai et al., 2006).
Damiens and co-workers (1998) investigated LF involvement, at inflammatory concentrations,
in cancer progression. It was reported that LF has a significant effect on NK cell cytotoxicity
against haematopoietic and breast epithelial cell lines. By pre-treatment of either NK cells or
target cells with LF, it was demonstrated that the LF effect is due both to a modulation of NK
cell cytotoxicity and the target cell sensitivity to lysis. It was also shown that LF inhibits
epithelial cell proliferation by blocking the cell cycle progression. Furthermore, Damiens and

13
co-workers (1999) found that treatment of breast carcinoma cells MDA-MB-231 with hLF
induced growth arrest at the G1 to S transition of the cell cycle by modulating the expression
and the activity of key regulatory proteins.
The effect of orally administered iron-unsaturated bLF on angiogenesis induced by VEGF165
and IL-1-α in adult rats was assessed by Norrby and co-workers (2001). LF treatment was
found to significantly inhibit the VEGF165-mediated response in terms of microvessel spatial
extension, overall vascularity and incidence of crossover. Thus, the oral administration of LF
thus appears to be of potential interest as an anti-angiogenesis treatment modality in the clinical
setting.
Additionally, the activity of bovine LFCin to induce apoptosis in THP-1 human monocytic
leukemic cells was considered (Yoo et al., 1997b). The results achieved suggested that LFCin,
but not LF itself, is able to induce apoptosis in THP-1 human monocytic tumour cells, and that
its apoptosis-inducing activity is related to the pathway mediated by production of the
intracellular reactive oxygen species (ROS) and activation of Ca2+/Mg2+-dependent
endonucleases.
Finally, there have also been a number of animal studies showing that LF can inhibit
development of experimental tumours (Ushida et al., 1998; Tsuda et al., 2002).
Although the results achieved by several researchers point to a clear anti-tumour role of LF
(Benaissa et al., 2005), the mechanisms by which it is exerted are not fully understood, thus
further work on this subject is warranted.
LACTOFERRIN AS A CHEMOPREVENTIVE AGENT
Several studies have been done to evaluate the effect of orally administrated LF in healthy or
diseased human beings and animals (Tomita et al., 2002; Teraguchi et al., 2004). To the date, it
has become evident that the oral administration of LF exerts various beneficial effects against
diseases (Table 4), namely as a chemopreventive agent. For the practical use of
chemopreventive agents, it is important to know whether their action is limited to only

14
inhibition. Therefore, use of chemopreventive agents should follow appropriate indications
based on reliable information regarding beneficial preventive and adverse promoting or toxic
effects (Tsuda et al., 2000). A thorough follow-up of patients or populations during and after the
administration of chemopreventive agents is obviously necessary for effective assessment and
conclusions to be drawn. For general public use, the agents require safety approval based on a
long-term toxicity or carcinogenicity testing. Further development of appropriate in vivo animal
assay systems to provide reliable information regarding organotropism and adverse effects is
also critical for this purpose. To this regard, use of naturally occurring compounds is
advantageous because most of them are ingested routinely as food components. Fiber,
unsaturated fatty acid, carotenoids, flavonoids, phenolic compounds, especially polyphenols,
and now bLF could be promising compounds in this respect. bLF as a food ingredient is thought
to be safe, because there is a long dietary history of its use. People who live on dairy products
must have ingested bLF for a long time, as raw milk and natural cheese contain high amounts of
this protein. A human clinical study in chronic hepatitis C patients showed that high bLF oral
doses, up to 7.2 g/body/day were well tolerated (Okada et al., 2002).
Troost and co-workers (2001) demonstrated that over than 60% of administrated bLF survives
the passage through adult human stomach and enters the small intestine in an intact form. On
the other hand, analysis of the gastric contents revealed that LFCin B was formed at a molar
concentration corresponding to 4.5% of ingested bLF (Kuwata et al., 1998a). When the animals
were given free access to milk containing bLF at 40 mg/ml (482 μmol/l), the levels of bLF
fragments containing the LFCin B region in the contents of the stomach, upper small intestine,
and lower small intestine were approximately 200, 20 and 1 μmol/l, respectively (Kuwata et al.,
1998b; Kuwata et al., 2001). Some parts of ingested LF are likely to be not fully digested and to
be present in the lower gastrointestinal tract. These intact bLF and partially digested bLF
peptides, which retain biological activities, may exert various physiological effects in the
digestive tract.
Orally administrated LF exhibits several beneficial effects at sites other than the digestive tract.
In the case of infants or adults with injury in the intestine, it is possible that ingested LF enters

15
the blood circulation and acts systemically. However, neither bLF nor functional bLF fragments
(LFCin B-containing peptides and anti-bLF antibody-binding peptides) were at a level higher
than the detection limit (1 mg/ml) detected in the portal blood of normal adult rats orally given
bLF at a maximal dose of 5 g/kg (Wakabayashi et al., 2004). Therefore, LF-related molecules
are not likely to be transported from the intestine into the circulation. Hence, it is rational to
consider that oral administered bLF and its digested products act initially on the intestinal
immune system and augment the protective immunity systemically (Teraguchi et al., 2004).
The protective effect of LF against chemically induced carcinogenesis, tumour growth and/or
metastasis have been demonstrated in an increasing number of animal model experiments,
namely directed to specific organs, such as esophagus, tongue, lung, liver, colon and bladder
(Bezault et al., 1994; Wang et al., 2000; Shimamura et al., 2004; Yoo et al., 1997a; Iigo et al.,
1999; Tsuda et al., 2002; Ushida et al., 1999; Tanaka et al., 2000; Fujita et al., 2002; Masuda et
al., 2000; Sekine et al., 1997a; Varadhachary et al., 2004; Kuhara et al., 2000). Table 5
summarizes the effectiveness of orally administrated LF–related compounds on cancer. Despite
the evidence that LF possesses chemopreventive activity, little is known about (i) its anti-cancer
activity against established tumours; (ii) its ability to potentiate chemotherapy, as described with
other immunotherapeutics; or (iii) the immune mechanisms by which its anti-tumour activity is
mediated. Moreover, no human clinical studies on the potential chemopreventive effect have
been done so far.
As discussed above, it appears that like the other biological functions of LF, its anti-tumour role
is complex (Ward et al., 2005).
Several researchers suggested a direct effect of LF on tumour cell growth based on the fact that
both LF and a splice variant are downregulated or absent in many cancer cell lines and in
experimental tumours (Campbell et al., 1992; Panella et al., 1991; Penco et al., 1999; Teng et
al., 2004; Benaissa et al., 2005; Siebert et al., 1997; Breton et al., 2004; Rossiello et al., 1984).
Furthermore, Damiens and co-workers (1999) found that treatment of human breast carcinoma
cells MDA-MB-231 with hLF induced growth arrest at the G1 to S transition of the cell cycle
by modulating the expression and the activity of key G1 regulatory proteins, including the Cdk

16
inhibitors p21 and p27, which may be mediated in part by modulation of the Akt and MAPK
pathways. The reported effects of LF occur through a p53-independent mechanism both in
MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29.The
same conclusions were achieved in another study using head and neck cell lines (Xiao et al.,
2004). In vivo studies also suggest that the inhibition of tumour cell growth by LF may be
related to the ability of this protein to induce apoptosis of cancer cells by activating the FAS
signalling pathway in cancerous cells (Fujita et al., 2004a, b). Fujita and co-workers (2004a)
studied the effect of LF on the gene expression of 10 apoptosis-related molecules in colon
mucosa of azoxymethane (AOM)-treated rats during early and late stages of colon
carcinogenesis and found that FAS and pro-apoptotic BCL-2 members participate in the LF
action and may contribute to suppressive effects on tumour development in the rat colon.
Additionally, they studied (Fujita et al., 2004b) the influence of bLF on FAS-mediated
apoptosis with regard to expression of FAS, activation of caspase-8 and caspase-3, and DNA
fragmentation in the colon mucosa of AOM-treated rats and the results confirmed the
suggestion that apoptosis caused by elevated expression of FAS is involved in chemoprevention
by LF in colon carcinogenesis.
LF was also found to stimulate the production and activation of several immune cells, including
lymphocytes and NK cells (Legrand et al., 2004; Horwitz et al., 1984; Shau et al., 1992), in
addition to increasing the target cell sensitivity to NK lysis (Damiens et al., 1998), thus
immunomodulation may be critical to the anti-tumour role of LF. Damiens and co-workers
(1998) investigated LF involvement, at inflammatory concentrations, in cancer progression.
They reported that LF has a significant effect on NK cell cytotoxicity against haematopoietic
and breast epithelial cell lines. By pre-treatment of either NK cells or target cells with LF, it was
demonstrated that the LF effect is due both to a modulation of NK cell cytotoxicity and the
target cell sensitivity to lysis. Moreover, LF was found to exert an effect on target cells
depending on the cell phenotype, i.e. it does not modify the susceptibility to lysis of
haematopoietic cells such as Jurkat and K-562 cells, but does significantly increase that of the

17
breast and colon epithelial cells. Ultimately, it was demonstrated that LF inhibits epithelial cell
proliferation by blocking the cell cycle progression.
Marked inhibition of tumour growth and reduced lung colonization by B16-F10 melanoma
experimental metastasis were found in mice treated with hLF injected intraperitoneal as a result
of an enhanced NK cell activity (Bezault et al., 1994). Moreover, it has been shown that the
protective effect of oral administration of LF in several rodent cancer models is associated with
enhancement of the local intestinal mucosal immune response. In this regard, upregulation
and/or enhanced activation of NK cells, CD4+ T lymphocytes and CD8+ T-lymphocytes were
observed upon LF administration (Wang et al., 2000; Iigo et al., 1999; Kuhara et al., 2000). A
low absorption of LF from the intestine was reported, nevertheless an enhancement of the
systemic immune response was also observed.
Some authors proposed that the mechanism implicated in the protective effect of LF against
cancer cell development is mediated by IL-18 (Reddy, 2004), as LF was found to strongly
regulate IL-18 expression in intestinal epithelium (Iigo et al., 2004; Varadhachary et al., 2004;
Kuhara et al., 2000). Finally, LF was also shown to inhibit tumour-initiated angiogenesis in
vitro and in vivo, which may relate to the anti-angiogenic properties of IL-18 (Shimamura et al.,
2004; Yoo et al., 1997a; Cao et al. 1999; Norrby, 2004), LF was found to participate as a
regulator of angiogenesis, possibly by blocking endothelial function and inducing IL-18
production.
PRODUCTION OF LACTOFERRIN AND ITS DERIVATIVES
Many methods of isolating LF from milk have been reported (Groves, 1960; Johansson, 1969;
Law and Reiter, 1977; Wakabayashi et al., 2006), but it is quite difficult to scale up an
experimental method to achieve industrial production. There are many problems involved in the
manufacture of high-purity LF, including the need for mass production, reuse of the residual
milk materials after isolation of LF, a comparatively simple manufacturing process, and high
levels of stability and sanitation of the plant equipment. Nowadays, high-purity LF is obtainable

18
on a laboratory scale using gel filtration chromatography (Al-Mashikhi and Nakai, 1987),
immobilized monoclonal antibodies (Kawakami et al., 1987), chelating chromatography (Al-
Mashikhi et al., 1988), hydrophobic interaction chromatography, Cibacron Blue affinity
chromatography (Shimazaki and Nishio, 1991), immunoaffinity chromatography (Noppe et al.
2006), carboxymethyl cation exchange chromatography (Yoshida and Ye, 1991; Elagamy et al.,
1996), cation exchange membranes (Chiu and Etzel, 1997), adsorptive membrane
chromatography (Plate et al., 2006), simulated moving bed technology (Andersson and
Mattiasson 2006), semi-batch foaming process (Saleh and Hossain, 2001), and microfiltration
affinity purification (Chen and Wang, 1991). However, none of these techniques has been used
at commercial scales because of high processing costs (Zydney, 1998).
Recently, an approach of industrial isolation and purification of bovine LF (approximately 20-
30 ton annually worldwide) from cheese whey and skim milk was reported (Tamura, 2004). The
concentration of LF in cheese whey is roughly 100 mg/L and about 100-400 mg/l in bovine
milk. Since LF exists as a cationic protein in milk and whey, it is readily adsorbed to a cation-
exchange resin and then eluted using salt solutions. The eluted crude LF is then desalted and
concentrated using ultrafiltration and diafiltration membranes, after which it is subjected to
pasteurization. Purified LF powder with a purity of 95% or higher is finally obtained by freeze-
drying (Tomita et al., 2002). In an alternative process, microfiltration and spray-drying are
performed instead of pasteurization and freeze-drying, respectively. While making efforts to
develop a practical method for the pasteurization of LF, Abe and co-workers (1991) found that
LF is stable against heat treatment under acidic conditions, while heat treatment at a neutral pH
causes denaturation of the protein. It is considered that heating at a pH above 4 and to a
temperature of 90–100°C for 5–10 min as well as the UHT method are suitable and practical
methods for the pasteurization of LF. This pasteurization process was patented, and it has been
applied to the manufacture of a wide variety of commercial products containing LF.
Additionally, LF was also isolated from bovine colostrum by ultrafiltration and then purified
with a fast flow strong cation exchange chromatography system in a production scale (Lu et al.,
2007). Processes involving chromatography have some limitations at industrial scale such as

19
high cost and relatively low throughputs. Membrane filtration could represent an interesting
alternative to chromatography for LF fractionation as membrane processes are already well
integrated in the dairy industry. However, fouling and poor selectivity in protein separation have
been associated with such membrane filtration processes. Different strategies have been
investigated to overcome limitations associated with LF separation by membrane filtration
including variation of the hydrodynamic parameters (Chilukuri et al., 2001), modification of the
physico-chemical environment (Chaufer et al., 2000; Rabiller-Baudry et al., 2001; Brisson et al.,
2007), use of different membrane types (Mehra and Donnelly, 1993; Ulber et al., 2001) and
alteration of the membrane surface properties (Rabiller-Baudry et al., 2001).
Regarding LF derivatives, a pepsin hydrolysate of LF was produced by treatment with porcine
pepsin under acidic conditions (Saito et al. 1991). After hydrolysis has been completed, pepsin
was inactivated by heat treatment. Then the reaction mixture was filtered and concentrated by
reverse osmosis. Finally, the hydrolysate of LF was obtained by pasteurization and freeze-
drying for use in infant formula. In addition, lactoferricin can be purified from this LF
hydrolysate by two-step hydrophobic chromatography (Bellamy et al., 1992). The peptide is
eluted with an acidic buffer, the eluted solution is concentrated by reverse osmosis, and finally,
lactoferricin is produced by freeze-drying as a powder with over 95% purity. This production
process for lactoferricin has also been patented. The toxicity of purified LF was judged to be
extremely low in safety tests. From the results of single-dose, 4-week, and 13-week oral toxicity
tests, the dose of LF that caused no adverse effects was found to be 2000 mg./kg/day for rats of
both sexes (Yamauchi et al., 2000a). In addition, LF did not exhibit any mutagenic potential in a
bacterial reverse-mutation test (Yamauchi et al. 2000b). Based on the results of these safety tests
and the results of clinical studies, purified LF is considered to be a highly safe food additive.
Along with increased recognition of the biological effects of LF, as described below, the
applications of LF have been expanded. LF-supplemented infant formula, follow-up milk, skim
milk, yogurt, chewing gum, and nutritional supplements are being marketed. In addition to
foods, LF is also used in skin care cosmetics, in special therapeutic diets for the relief of
inflammation in dogs and cats, and in aquaculture feed (Wakabayashi et al., 2006).

20
CONCLUSION
Evidence that diet is a key environmental factor affecting the incidence of many chronic
diseases is overwhelming. Indeed, several food derived compounds we eat are among the most
promising chemopreventive agents being evaluated. To this regard, the use of naturally
occurring compounds, like for instance LF, is advantageous because most of them are ingested
routinely as food components and therefore they are safe. Nevertheless, the approval of a new
chemopreventive agent requires the development of appropriate in vivo animal assay systems to
provide reliable information regarding organotropism and adverse effects.
A host of interesting features of LF, namely its role on iron homeostasis, organ morphogenesis
and host defence against infection, inflammation and cancer, have led to a wide range of
potential applications in the medical field. Distinct mechanisms of action have been described
for each LF’s functions which possibly work in concert to potentiate its biological effect in vivo.
LF presents the capacity to bind to a broad variety of epithelial and immune cells, which
probably confers this protein the ability to regulate cellular signalling pathways and
consequently exert its protective functions, for example against cancer. Nevertheless, further
investigations on the cellular localization and downstream molecular events that follow LF
engagement with these receptors are needed. The protective effect of LF against chemically
induced carcinogenesis, tumour growth and/or metastasis have been demonstrated in an
increasing number of animal model studies, therefore suggesting its great potential therapeutic
use in cancer disease prevention and/or treatment. However, much available data has been
derived from studies where high doses of non homologous LF of unknown purity were used and
to which the tissues in our bodies may never be exposed, therefore all extrapolations require a
careful interpretation of the results.
Finally, despite the evidence that LF possesses chemopreventive activity, its anti-cancer activity
against established tumours, ability to potentiate chemotherapy as described with other

21
immunotherapeutics, and the immune mechanisms by which its anti-tumour activity is
mediated, is still largely unknown.
ACKNOWLEDGEMENT
The F.C.T. (Fundação para a Ciência e a Tecnologia) provided financial support for L.
Rodrigues through a post doctoral research grant SFRH/BPD/26064/2005.
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31
Table 1 Occurrence of lactoferrin (hLF – human lactoferrin; bLF – bovine lactoferrin) in biological fluids
Biological fluid
Amounts reported
(mg/ml)*
Colostral breast milk
> 7
Mature breast milk
> 1-2
Tear fluid
> 2.2
Seminal plasma
> 0.4-1.9
Synovial fluid
> 0.01-0.08
hLF
Saliva
> 0.007-0.01
Colostral whey
> 1.5
bLF
Milk
> 0.02-0.2
* Data adapted from Levay and Viljoen, 1995; Steijns and Van Hooijdonk, 2000; Babina et al., 2006; Shimazaki, 2000

32
Table 2 Lactoferrin (hLF – human lactoferrin; bLF – bovine lactoferrin) physico-chemical properties
bLF
hLF
References
Amino acid residues
689
690
Pierce et al., 1991; Baker, 1994
Molecular weight
77 ± 1.5
76.8 ± 1.6
Querinjean et al., 1971; Spik et al., 1994;
Castellino et al., 1970
Isoelectric point
8.5-9.0
8.4-9.0
Spik et al., 1994; Lampreave et al., 1990;
Zakharova et al., 2000

33
Table 3 Reported in vitro and in vivo physiological roles of lactoferrin
Physiological role
Mechanism
References
Iron absorption
Increasing solubility and receptor
mediated uptake
Kawakami et al., 1993; Mikogami et al., 1995; Levay
and Viljoen, 1995; Ward et al., 2005
Antioxidant
Iron scavenger
LF has the ability to bind to cell
membranes enhancing its ability
to prevent iron-mediated lipid
peroxidation
Matsue et al., 1994; 1995; Konishi et al., 2006;
Larkins, 2005
Antimicrobial
Growth
inhibition
by
iron
scavenging
or
membrane
disintegration
LF has a powerful iron-binding
capacity and shows a strong
interaction with other molecules
and cell surface
Nemet and Simonovits, 1985 ; Yamauchi et al., 1993 ;
Qiu et al., 1998 ; Weinberg, 2007 ; Santagati et al.,
2005; Lönnerdal, 2003 ; Lee et al., 2005
Antiviral
Prevention of virus attachment,
inhibition of virus replication,
blocking of cell-virus interactions
Yi et al., 1997 ; Siciliano et al., 1999 ; Andersen et al.,
2001 ; Ikeda et al., 2000 ; Giansanti et al., 2002 ;
Longhi et al., 2006; Mistry et al., 2007; Ammendolia
et al., 2007 ; Pan et al., 2006
Anti-inflammatory,
immune modulating
LPS binding, stimulation of NK
cells,
reduction
of
pro-
inflammatory cytokines, T-cell
maturation.
LF
modulates
the
migration,
maturation and function of the
immune cells at the cellular level
and at the molecular level
Zimecki et al., 1991, 1995 ; Hangoc et al., 1991 ;
Crouch et al., 1992 ; Yamauchi et al., 1993 ; Adamik
et al., 1998 ; Baveye et al., 1999 ; Dhennin-Duthille et
al., 2000 ; Britigan et al., 2001; Fischer et al. 2006;
Berlutti et al. 2006; Senkovich et al., 2007; Legrand
et al., 2005; Kruzel et al., 2006
Anti-cancer
Regulation of NK cell activity,
modulation of expression of G1
proteins,
inhibition
of
VEGF(165)-mediated
angiogenesis,
enhancement
of
apoptosis
Bezault et al., 1994 ; Sekine et al., 1997 ; Yoo et al.,
1997b ; Damiens et al., 1998, 1999 ; Norrby et al.,
2001 ; Mohan et al., 2006a; McKeown et al., 2006;
Kuhara et al., 2006 ; Baumrucker et al. 2006; Kim et
al., 2006; Giuffre et al., 2006 ; Kawakami et al., 2006

34
Table 4 Effectiveness of orally administered bLF in humans.
Disease
Efficacy
Administrated
agent and dose
References
Bacterial flora:
Faecal flora in low birth weight
infants
Faecal flora in infants
Increase of Bifidobacterium,
decrease of Clostridium
Increase of Bifidobacterium
bLF; 1mg/ml in
infant formula
Wakabayashi
et
al.,
2006
Roberts et al., 1992;
Logsdon et al., 2006
Infection (digestive tract):
Gastric infection with Helicobacter
pylori
Increase of eradication by
triple therapy
bLF; 0.2g/body
Di Mario et al., 2003
Infection (other than digestive tract):
Neutropenic patients
Chronic hepatitis C
Influenza
Decrease of incidence of
bacteremia and severity of
infection
Decrease of ALT and HCV
RNA in serum
Attenuation of pneumonia
through the suppression of
infiltration of inflammatory
cells in the lung
bLF; 0.8 g/body
bLF; 1.8 and 3.6
g/body
62.5 mg per mouse
Trumpler et al., 1989
Tanaka et al., 1999 ;
Iwasa et al., 2002 ;
Yamauchi et al., 2000c ;
Konishi et al. 2006 ;
Katsuaki, 2006
Shin et al., 2005

35
Table 5 Effectiveness of orally administered LF-related compounds on cancers
Cancer model
Efficacy
Animal
Administrated
agent and dose
References
Carcinogen-induced tumour in
colon,
lung,
esophagus,
bladder, liver, tongue
Inhibition of tumour
development
Rat
bLF
0.2 and 2% in diet
Sekine et al., 1997a, b;
Ushida
et
al.,
1999;
Tanaka
et
al.,
2000;
Masuda et al., 2000;
Fujita et al., 2002; Mohan
et al., 2006b
Spontaneously
developed
intestinal polyposis
Inhibition of polyp
development
ApcMin
mouse
bLF
2% in diet
Ushida et al., 1998
Tumour cell injection
Inhibition
of
lung
metastasis
Mouse
bLF
0.3 g/kg
Iigo et al., 1999
Tumour cell injection
Inhibition of tumour
development
Mouse
rhLF
1 g/kg
Varadhachary et al., 2004
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