Inhibitory Effects Of Bovine Lactoferrin Research Articles

Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma

Lactoferrin (LF), an iron-binding glycoprotein, has been reported to have anticancer properties. However, the molecular mechanisms behind its anticancer effects on oral squamous cell carcinoma (OSCC) have not yet been elucidated. Therefore, we aimed to clarify the effects of LF on invasion of OSCC, and its underlying molecular mechanism. OSCC cell lines, HSC2 and HOC313, were treated with bovine LF (bLF). The effects of bLF on cell invasion were examined by a chamber migration assay, wound healing assay, and Boyden chamber method with a basement-membrane-analogue. Expression levels of MMP-1, MMP-3, and AP-1 were examined using RT-PCR, qRT-PCR, and western blotting. Roles of LRP1, a receptor of bLF, on cell invasion were analyzed using siLRP1 knockdown cells. Furthermore, to clarify the importance of LRP1 in invasion, the effects of bLF on tPA-induced invasion of OSCC cells were examined. The invasion assays showed that bLF suppressed invasion of the OSCC cells. Moreover, bLF down-regulated AP-1, and resulted in reductions of MMP-1 and MMP-3. With SiLRP1 knockdown, OSCC cells failed to induce their invasion, and bLF was not able to exert its effects on invasion. Furthermore, bLF remarkably inhibited tPA-induced cell invasion. These findings suggest the importance of LRP1 in bLF-suppressed invasion of OSCC cells via the reduction of AP-1 and MMP production.

Evaluation of antimicrobial properties of bovine lactoferrin against foodborne pathogenic microorganisms in planktonic and biofilm forms (in vitro)

Lactoferrin is one of the most important components in milk. Lactoferrin is an 80 kDa iron-binding glycoprotein that involves a diverse range of biological activities. However, the antimicrobial properties are its most widely studied function. The aim of this study was to assay the antimicrobial and antibiofilm activities of bovine lactoferrin against five of the most important foodborne pathogenic bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Listeria innocua, in planktonic and biofilm forms. Bovine lactoferrin was purchased, MIC and MBC were determined for the mentioned bacterial strains and the effect of lactoferrin concentrations on biofilm formation was evaluated using microtiter plate method. Based on the results, lactoferrin had a greater effect on gram-positive bacteria, L. monocytogenes and L. innocua were the most sensitive strains. MIC for L. monocytogenes, L. innocua, S. aureus and P. aeruginosa was 6, 6, 8 and 14 mg/mL, respectively, Nevertheless, MIC for gram negative E. coli was not observed, even at 16 mg/mL. The result demonstrated that MBC for L. monocytogenes, L. innocua and S. aureus were 10, 12 and 16 mg/mL, respectively. However, MBC for P. aeruginosa and E. coli were not observed for the lactoferrin concentrations evaluated. The highest biofilm formation inhibitory effects of bovine lactoferrin were against L. monocytogenes and L. innocua. To our knowledge, this is the first report on the antibiofilm activity of bovine lactoferrin against foodborne pathogenic bacteria. Our findings suggest lactoferrin to possibly use as an anti-biofilm agent in both food and pharmaceutical industries.

Molecular mechanisms underlying the inhibitory effects of bovine lactoferrin on osteosarcoma

Osteosarcoma (OS) is one the most common primary malignancies of the bone in children and young adults with high metastasis. The use of non-toxic naturally derived compounds is one of present strategies in OS therapy to reduce secondary effects and chemo-resistance. Lactoferrin (LF), a transferrin protein derived from milk, currently appears to be an anticancer agent. However, its suppressive effects on OS have not been fully investigated. Therefore, we aimed to examine the molecular mechanism underlying the inhibitory effects of bovine LF (bLF) on OS. OS cell lines (NOS1, U2OS, MG63, and 143B) and an osteoblastic (ST2) were treated with bLF. Effects of bLF on OS-cell proliferation and migration were examined by proliferation and wound-healing assays. Expression levels of low-density-lipoprotein-receptor-related protein 1 (LRP1) and cytokines including interleukin-1 beta (IL-1β), IL-6, and receptor-activator of nuclear factor kappa-Β ligand (RANKL) were measured using western blotting. Osteoclast formation was examined by co-culture of 143B, ST2, and bone marrow cells. We found that bLF down-regulated IL-1β, IL-6, and RANKL expression and suppressed phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 in 143B cells; bLF also drastically suppressed 143B-activated RANKL production in ST2 cells. This may have contributed to the reduction in the number of differentiated osteoclasts. Taken together, these data reveal that bLF down-regulates NF-κB to attenuate proliferation, migration, and bone resorption in OS and the OS-microenvironment. This study provides new findings and the precise underlying mechanisms of the inhibitory effects of bLF on OS. bLF can be a possible therapeutic agent for OS patients.

Molecular mechanism of inhibitory effects of bovine lactoferrin on the growth of oral squamous cell carcinoma.

BackgroundLactoferrin (LF), a member of the transferrin family, recently has been demonstrated to have anticancer effects on various cancers including oral squamous cell carcinoma (OSCC). However, little is known about the underlying mechanisms of its effects on OSCC. Therefore, we aimed to investigate the mechanism of the suppressive effects of bovine LF (bLF) on the growth of OSCC cells.MethodsIn the current study, HSC2, HSC3, HSC4 and normal human oral keratinocytes (RT7) cell lines were tested with bLF 1, 10, and 100 μg/ml. The effects and detail mechanisms of bLF on proliferation and apoptosis of cells were investigated using flow cytometry and western blotting.ResultsWe found that bLF (1, 10, and 100 μg/ml) induced activation of p53, a tumor suppressor gene, is associated with the induction of cell cycle arrest in G1/S phase and apoptosis in OSCC. Moreover, bLF downregulated the phosphorylation of Akt and activated suppressor of cytokine signaling 3 (SOCS3), thereby attenuating multiple signaling pathways including mTOR/S6K and JAK/STAT3. Interestingly, we revealed that bLF exerted its effect selectively against HSC3 but not on RT7 via different effects on the phosphorylation status of NF-κB and Akt.ConclusionThis is the first report showing that bLF selectively suppresses proliferation through mTOR/S6K and JAK/STAT3 pathways and induction of apoptosis in OSCC. This study provides important new findings, which might be useful in the prevention and treatment of OSCC.

Inhibitory Effect of Bovine Lactoferrin on Catechol-O-Methyltransferase.

Lactoferrin (LF) is a well-known multifunctional protein. In this study, we report the inhibitory potency of bovine LF (bLF) on catechol-O-methyltransferase (COMT), which catalyzes methylation of catechol substrates. We found that bLF binds to and inhibits COMT using its N-terminal region. An N-terminal peptide fragment obtained from bLF by trypsin digestion showed a higher inhibitory activity than intact bLF. A synthetic fragment of the bLF N-terminal residues 6–50, with two pairs of disulfide bonds, also showed higher inhibitory activity than intact bLF. Enzyme kinetic studies proved that bLF did not compete with S-adenosylmethionine (the methyl donor substrate) as well as methyl acceptor substrates such as dihydroxybenzoic acid, (−)-epicatechin, norepinephrine, or l-3,4-dihydroxyphenylalanine. The inhibitory potency of bLF decreased against a COMT preparation pretreated with dithiothreitol, suggesting that the oxidation status of COMT is relevant to interaction with bLF. We further confirmed that COMT activity in the cell extracts form Caco-2 and HepG2 cells was inhibited by bLF and by the synthesized fragment. Enzyme kinetic study indicated that bLF functions as a non-competitive inhibitor by binding to an allosteric surface of COMT.

Molecular Mechanisms of the Inhibitory Effects of Bovine Lactoferrin on Lipopolysaccharide-mediated Osteoclastogenesis

Lactoferrin (LF) is an important modulator of the immune response and inflammation. It has also been implicated in the regulation of bone tissue. In our previous study we demonstrated that bovine LF (bLF) reduces LPS-induced bone resorption through a reduction of TNF-α production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-α and RANKL (receptor activator of nuclear factor κB ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-α and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NFκB DNA binding activity as well as IκBα and IKKβ (IκB kinase β) phosphorylation. MAP kinase activation was also inhibited by bLF pretreatment. However, bLF pretreatment failed to block the degradation of IRAK1 (interleukin-1 receptor-associated kinase 1), which is an essential event after its activation. Remarkably, we found that bLF pretreatment inhibited LPS-mediated Lys-63-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6). We also found that bLF is mainly endocytosed through LRP1 (lipoprotein receptor-related protein-1) and intracellular distributed bLF binds to endogenous TRAF6. In addition, bLF inhibited IL-1β- and flagellin-induced TRAF6-dependent activation of the NFκB signaling pathway. Collectively, our findings demonstrate that bLF inhibits NFκB and MAP kinase activation, which play critical roles in chronic inflammatory disease by interfering with the TRAF6 polyubiquitination process. Thus, bLF could be a potent therapeutic agent for inflammatory diseases associated with bone destruction, such as periodontitis and rheumatoid arthritis.

Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells.

Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.

Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice.

In order to determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF), and related compounds on tumor growth and metastasis, bovine LF (bLF), and bLF hydrolysate and lactoferricin (bLFcin), active products generated by acid-pepsin hydrolysis were administered orally to BALB/c mice bearing subcutaneous (s.c.) implants of the highly metastatic colon carcinoma 26 (Co 26Lu). bLF and the bLF hydrolysate demonstrated significant inhibition of lung metastatic colony formation from s.c. implanted tumors without appreciable effects on tumor growth. bLFcin displayed a tendency for inhibition of lung metastasis. On the other hand, bLF did not exert marked anti-metastatic activity in athymic nude mice bearing Co 26Lu, though bLF had a tendency to inhibit the lung metastatic colony formation associated with anti-asialoGM1 antibody (Ab) treatment. AsialoGM1+ and CD8+ cells in white blood cells were increased after treatment with bLF. In vitro, the viability of Co 26Lu-F55 cells was markedly decreased when co-cultured with white blood cells from mice administrated bLF p.o., but recovered on treatment with anti-asialoGM1 Ab or anti-CD8 mAb and complement. The results suggest bLF and related compounds might find application as tools in the control of metastasis and that asialoGM1+ and CD8+ cells in the blood are important for their inhibitory effects.

Metal complexes of bovine lactoferrin inhibit in vitro replication of herpes simplex virus type 1 and 2.

The inhibitory effect of bovine lactoferrin (BLf) saturated with ferric, manganese or zinc ions, on the infection of Vero cells by human herpes simplex virus type 1 (HSV1) and 2 (HSV2) was investigated. Viral infectivity determined by intracellular antigen synthesis and plaque formation was efficiently inhibited by metal saturated lactoferrins in a dose-dependent manner. Effective BLf concentrations which reduced the infection by 50% ranged from 5.2 to 31 micrograms ml-1 and were far below the cytotoxicity threshold. Fe3+BLf and Mn2+BLf exhibited selectivity indexes higher than Zn2+BLf and apoBLf for both viruses and the effect was mainly directed towards the early steps of infection. The slight viral inhibition shown by the citrate complexes of the different metals could indicate that the antiviral effect was not significantly influenced by Fe3+, Mn2+ or Zn2+ ions delivered by BLf into the cells.

Growth inhibitory effect of bovine lactoferrin to Toxoplasma gondii tachyzoites in murine macrophages: tyrosine phosphorylation in murine macrophages induced by bovine lactoferrin.

Previous studies have shown that lactoferrin induces growth inhibitory effects in mouse macrophages against intracellular Toxoplasma gondii, and these effects were not mediated by the oxygen-dependent and inorganic nitrogen-dependent pathway. To clarify the mechanism of anti-Toxoplasma gondii activity induced by lactoferrin, we examined whether lactoferrin promoted the phosphorylation of tyrosine residues in macrophage proteins. In immunoblotting assays using anti-[phosphorylated tyrosine] monoclonal antibody, phosphorylation of tyrosine residues was detected in protein(s) of approximately 30 kDa in macrophages incubated with lactoferrin. Inhibition of the lactoferrin-induced tyrosine-phosphorylation by genistein led to loss of the lactoferrin-induced growth inhibitory effect against the parasites. These findings suggest that lactoferrin induces tyrosine-phosphorylation in macrophages, and the tyrosine-phosphorylation seems to be associated with the induction of the growth inhibitory activity exerted against intracellular Toxoplasma gondii.

Growth inhibitory effect of bovine lactoferrin on Toxoplasma gondii tachyzoites in murine macrophages: Role of radical oxygen and inorganic nitrogen oxide in Toxoplasma growth-inhibitory activity

To study the effector pathway of Toxoplasma growth-inhibitory activity induced by lactoferrin in murine macrophage, the role of reactive oxygen intermediates (O 2 −) and inorganic nitric oxide (NO) was examined. Production of O 2 − was diminished in cultures of macrophages supplemented with lactoferrin and the effect of lactoferrin was dose and time dependent. Production of NO was enhanced in cultures of macrophages supplemented with interferon-γ, but not with lactoferrin. These findings suggest that this Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages is not mediated by O 2 − or NO molecules. A competitive inhibitor of the l-arginine dependent effector pathway, N G-monomethyl- l-arginine (N GMMA), virtually abolished the inhibitory effects induced by interferon-γ. Similarly, the inhibitory activity induced by lactoferrin was also diminished in cultures supplemented with N GMMA. From these findings, it appears that the Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages may be mediated by an l-arginine-dependent effector pathway that does not involve NO production.

Growth inhibitory effects of bovine lactoferrin to Toxoplasma gondii parasites in murine somatic cells.

Lactoferrin (LF) is known to have broad spectrum antimicrobial properties. In regards to its defense mechanism against parasitic infection, it has shown phagocytic activity in the destruction of amastigotes, an intracellular parasitic form of Trypanosoma cruzi in macrophages. The effect of bovine lactoferrin on the intracellular growth Toxoplasma gondii parasites was examined in murine macrophage and embryonal cells. Co-cultures of host cells with the parasites were supplemented with either lactoferrin, apo-lactoferrin, holo-lactoferrin or transferrin in the culture media for varying periods. The growth activity of intracellular parasites in the host cells was determined by the measurement of selective incorporation of 3H-uracil. Supplement of lactoferrin had no effect on the penetration activity of the parasites, while development of intracellular parasites was inhibited linearly in concentration of lactoferrin. Supplement of apo-lactoferrin and holo-lactoferrin, but not transferrin showed similar effects. These suggest that lactoferrin induces the inhibitory effects on the development of intracellular parasites. Pretreatment of lactoferrin to the macrophages, however, did not show any inhibitory effects. Whereas, mouse embryonal cells preincubated with lactoferrin suppressed the intracellular growth. Thus, the action of lactoferrin to macrophages would be different from that of mouse embryonal cells.