Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7.

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

Binding Mechanism of Nitro Musks to Human Lactoferrin: Multispectral Approach, Docking and Molecular Dynamics Simulation.

Nitro musks are highly bioaccumulative and potentially carcinogenic, commonly used as additives in fabric softeners, detergents, and other household products. Furthermore, these substances have been detected in breast milk and human adipose tissue, posing a risk of direct exposure to pregnant women and infants. Human lactoferrin (HLF) is abundant in colostrum, and plays an important role in the non-specific immune system of the human body. In this study, the mechanisms of action of two nitro musk compounds, typical examples of synthetic musks, with HLF were investigated using molecular docking, dynamics simulation and multispectral methods. The fluorescence findings demonstrated that nitro musks quenched the intrinsic fluorescence of human lactoferrin through static quenching. Thermodynamic analysis of the binding parameters suggested that hydrophobic interactions acted synergistically in the formation of the complex. Moreover, analyses utilizing multispectral techniques, such as Fourier transform infrared (FTIR) spectroscopy, validated that the microenvironment and structure of HLF were altered in the presence of nitro musks. Finally, molecular docking and molecular dynamics simulations were employed to explore the specific binding mode of nitro musks with HLF and to assess the stability of the complex. These findings may provide a reference for assessing health risks to pregnant women and infants.

Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin.

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins.

Interactions between polycyclic musks and human lactoferrin: Multi-spectroscopic methods and docking simulation.

Galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-γ-2-benzopyrane; HHCB) and Tonalide (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene; AHTN) are "pseudo-persistent" pollutants that can cause DNA damage, endocrine disruption, organ toxicity, and reproductive toxicity in humans. HHCB and AHTN are readily enriched in breast milk, so exposure of infants to HHCB and AHTN is of concern. Here, the molecular mechanisms through which HHCB and AHTN interact with human lactoferrin (HLF) are investigated using computational simulations and spectroscopic methods to identify indirectly how HHCB and AHTN may harm infants. Molecular docking and kinetic simulation studies indicated that HHCB and AHTN can interact with and alter the secondary HLF structure. The fluorescence quenching of HLF by HHCB, AHTN was static with the forming of HLF-HHCB, HLF-AHTN complex, and accompanied by non-radiative energy transfer and that 1:1 complexes form through interaction forces. Time-resolved fluorescence spectroscopy indicated that binding to small molecules does not markedly change the HLF fluorescence lifetime. Three-dimensional fluorescence spectroscopy indicated that HHCB and AHTN alter the peptide chain backbone structure of HLF. Ultraviolet-visible absorption spectroscopy, simultaneous fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and circular dichroism spectroscopy indicated that HHCB and AHTN change the secondary HLF conformation. Antimicrobial activity experiments indicated that polycyclic musks decrease lactoferrin activity and interact with HLF. These results improve our understanding of the mechanisms involved in the toxicities of polycyclic musks bound to HLF at the molecular level and provide theoretical support for mother-and-child health risk assessments.

Albumin fusion with human lactoferrin shows enhanced inhibition of cancer cell migration.

The fusion of human serum albumin (HSA) with human lactoferrin (hLF) (designated as hLF-HSA) has improved the pharmacokinetic properties and anti-proliferative activities of hLF against cancer cells. In this study, we evaluated the anti-migratory activities of hLF and hLF-HSA against the human lung adenocarcinoma PC-14 cell line using wound healing and Boyden chamber assays. Despite the unexpected hLF-induced migration, hLF-HSA clearly demonstrated the complete inhibition of PC-14 cell migration. To examine the mechanism underlying the enhanced PC-14 cell migration by hLF alone but suppressed migration by hLF-HSA, we focused on the matrix metalloproteinase (MMP) family of endopeptidases because MMPs are often reported to play important roles in facilitating the migration and metastasis of cancer cells. Furthermore, hLF is a transactivator of MMP1 transcription. As expected, treatment of cells with hLF and hLF-HSA led to the upregulation and downregulation of MMP1, respectively. In contrast, MMP9 expression levels, which are often associated with cancer migration, were unchanged in the presence of either protein. An MMP inhibitor attenuated hLF-induced migration of PC-14 cells. Therefore, specific enhancement and suppression of MMP1 expression by hLF and hLF-HSA have been implicated as causes of a marked increase and decrease in PC-14 cell migration, respectively. In conclusion, the fusion of HSA with hLF (hLF-HSA) promoted its anti-migratory effects against cancer cells. Therefore, hLF-HSA is a promising anti-cancer drug candidate based on its improved anti-migratory activity towards cancer cells.

Lactoferrin Modulates Induction of Transcription Factor c-Fos in Neuronal Cultures.

Lactoferrin (Lf) is a multifunctional protein from the transferrin family. Of particular interest is the ability of Lf to affect a wide range of neuronal processes by modulating the expression of genes involved in long-term neuroplasticity. The expression of the immediate early gene c-fos that is rapidly activated in response to external influences, and its product, transcription factor c-Fos, is widely used as a marker of long-term neuronal plasticity. The present study aims to examine the effect of human Lf on the induction of transcription factor c-Fos in the primary mouse neuronal cultures after stimulation and to determine the cellular localization of human Lf and its colocalization with induced c-Fos protein. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 7 of culturing, human Lf was added to the medium. After 24 h (day 8 in culture), c-Fos protein was induced in cells by triple application of 50 mM KCl. c-Fos content was analyzed using the immunofluorescent method 2 h after stimulation. Stimulation promoted exogenous Lf translocation into the nuclei of cultured neuronal cells, which correlated with increased induction of transcription factor c-Fos and was accompanied by nuclear colocalization of these proteins. These results attest to the potential of Lf as a modulator of neuronal processes and open up new prospects in studying the mechanisms of the regulatory effects of lactoferrin on cell function.

Enhanced Bioactive Potential of Functionalized Injectable Platelet-Rich Plasma.

Injectable platelet-rich fibrin (iPRF) is a frequently used platelet concentrate used for various medical purposes both in veterinary and human medicine due to the regenerative potential of hard and soft tissues, and also because of its antimicrobial effectiveness. This in vitro study was carried out to assess the cumulative antimicrobial and antibiofilm effect of iPRF functionalized with a multifunctional glycoprotein, human lactoferrin (Lf). Thus, the ability to potentiate cell proliferation was tested on keratinocytes and evaluated by the CCK8 test. The combinations of iPRF and Lf induced an increase in the proliferation rate after 24 h. The average cell viability of treated cultures (all nine variants) was 102.87% ± 1.00, and the growth tendency was maintained even at 48 h. The highest proliferation rate was observed in cultures treated with 7% iPRF in combination with 50 µg/mL of Lf, with an average viability of 102.40% ± 0.80. The antibacterial and antibiofilm activity of iPRF, of human lactoferrin and their combination were tested by agar-well diffusion (Kirby-Bauer assay), broth microdilution, and crystal violet assay against five reference bacterial strains. iPRF showed antimicrobial and antibiofilm potential, but with variations depending on the tested bacterial strain. The global analysis of the results indicates an increased antimicrobial potential at the highest concentration of Lf mixed with iPRF. The study findings confirmed the hypothesized enhanced bioactive properties of functionalized iPRF against both Gram-positive and Gram-negative biofilm-producing bacteria. These findings could be further applied, but additional studies are needed to evaluate the mechanisms that are involved in these specific bioactive properties.

Lactoferrin Has a Protective Effect on Mouse Brain Cells after Acute Gamma Irradiation of the Head.

We studied the effect of human lactoferrin on cells of the hippocampal dentate gyrus of 2-2.5-month-old male C57BL/6 mice after acute gamma irradiation of the head in a dose of 8 Gy from a 60Co source. Immediately after irradiation some animals received an intraperitoneal injection of human lactoferrin (4 mg/mouse). The appearance of TUNEL+ cells in the subgranular zone 6 h after irradiation was accompanied by a corresponding decrease in the number of Ki-67- and DCX-immunoreactive cells. Administration of lactoferrin had a protective effect on mouse brain cells, which manifested in a decrease in the number of TUNEL+ cells (by 77% relative to the irradiation alone) and an increase in the number of proliferating cells (from 16 to 61% relative to control animals) and immature neurons (from 14 to 22% relative to control animals) in the dentate gyrus of the hippocampus.

Human lactoferrin from breast milk: characterization by HPLC and its in vitro antibiofilm performance.

Preterm infants are at high risk of infection due to opportunistic bacteria as Pseudomonas aeruginosa, causing infections among infants in neonatal intensive care units. Human lactoferrin (hLf) is a multifunctional protein and one of the most abundant in breast milk, and plays an important role in prevention of different infections in neonates. This work offers a strategy to obtain a lyophilisate of purified lactoferrin from breast milk. In addition, a reliable HPLC method for quantification of lactoferrin with a linear quantification range of 0.040-0.140 mg/mL with selectivity, accuracy and repeatability, is described. Lyophilized hLf was obtained by purification through a heparin affinity column followed by ultrafiltration with a 30 kDa membrane. The final solution was lyophilized and the product was analyzed using HPLC method, recovering about 70% of initial lactoferrin in the sample. This molecule was elucidated through FTIR spectroscopy and SDS-PAGE electrophoresis. In addition, the capacity against biofilm formation of P. aeruginosa was demonstrated with 75% of inhibition at 6 mg/mL. These results suggest that lyophilized hLf can be obtained by purification of breast milk and that it can provide antibiofilm activity against P. aeruginosa.

Discovery of Lactoferrin as a Stimulant for hADSC-Derived EV Secretion and Proof of Enhancement of Resulting EVs through Skin Model.

Extracellular vesicles (EVs) are secreted from hADSCs in low concentrations, which makes it difficult to utilize them for the development of therapeutic products. To overcome the problem associated with low concentration, we proposed human lactoferrin (hLF) as a stimulant for the secretion of hADSC-derived EVs. hLF has been reported to upregulate intracellular Ca2+, which is known to be capable of increasing EV secretion. We cultured hADSCs in hLF-supplemented media and analyzed the changes in intracellular Ca2+ concentration. The characteristics of hADSC-derived EVs secreted by hLF stimulation were analyzed through their number, membrane protein markers, and the presence of hLFs to EVs. The function of hADSC-derived EVs was investigated through their effects on dermal fibroblasts. We found that hLF helped hADSCs effectively uptake Ca2+, resulting in an increase of EVs secretion by more than a factor of 4. The resulting EVs had enhanced proliferation and collagen synthesis effect on dermal fibroblasts when compared to the same number of hADSC-derived EVs secreted without hLF stimulation. The enhanced secretion of hADSC-derived EVs increased collagen synthesis through enhanced epidermal penetration, which resulted from increased EV numbers. In summary, we propose hLF to be a useful stimulant in increasing the secretion rate of hADSC-derived EVs.

Effects of recombinant human lactoferrin on calcium signaling and functional responses of human neutrophils.

Lactoferrin is a non-heme iron-binding glycoprotein with multiple health-beneficial functions including antimicrobial, antioxidant, anticarcinogenic, and immunomodulatory effects. There is emerging evidence that neutrophils may serve as targets of lactoferrin in vivo, and here we show how recombinant human lactoferrin (rhLf) can contribute to this regulation. Indeed, our results demonstrate that rhLf binds efficiently to human neutrophils and induces a variety of early cellular responses such as mobilization of intracellular Ca2+, remodeling of actin cytoskeleton, and degranulation (release of lysozyme and myeloperoxidase). In addition, rhLf facilitates lectin-induced H2O2 production and stabilization of lectin-induced cellular aggregates. The role of calcium signaling seems to be essential for rhLf-induced activation of neutrophils, as Ca2+-chelators inhibit degranulation response while lectin-induced H2O2 production correlates significantly with cytoplasmic Ca2+ elevation. Taken together, our findings justify that rhLf can activate neutrophil functions in a calcium-dependent manner and hence, can potentiate innate immune responses.

Development of dairy herd of transgenic goats as biofactory for large-scale production of biologically active recombinant human lactoferrin.

The primary male-goats Lac-1 (human lactoferrin gene construct hLF5) and Lac-2 (human lactoferrin gene construct hLF3) with genome containing human lactoferrin gene were bred and the sperm bank of primary male-goats and their male descendents (F1-F7) was created. The herd of goats (200 transgenic females) that produced recombinant human lactoferrin (rhLF) in their milk at levels up to 16 g/L was obtained. The rhLF from milk of transgenic goats, natural human lactoferrin (hLF) from woman milk and natural goat lactoferrin (gLF) from milk of non-transgenic goats were purified using cation-exchange chromatography. It has been shown that rhLF is a glycoprotein and its physicochemical characteristics of rhLF are similar to hLf as revealed by different analytical methods including electron paramagnetic resonance, spectrophotometry, differential scanning calorimetry, mass spectrometry and peptide mapping. The high expression level of rhLF achieved in milk of transgenic goats provides a solid basis for developing an efficient and cost-effective downstream processing. The rhLF exhibited a prominent biological activity suggesting it as a promising biopharmaceutical and food supplements.

Using response surface methodology optimize culture conditions for human lactoferrin production in desert Chlorella.

To optimize the expression conditions for human lactoferrin production, we have constructed the transgenic chlorella with human lactoferrin named as GTD8A1-HLF, the original chlorella was separated from Gurbantunggut Desert in Xinjiang China. To further improve the production of human lactoferrin, a sequential methodology was used to optimize human lactoferrin production by GTD8A1-HLF. First, a screening trial using a Plackett-Burman design was done, and variables with statistically significant effects on human lactoferrin bio-production were identified. These were further optimized by central composite design experiments and response surface methodology. Finally, we found that the maximum human lactoferrin production (52.70 mg/L) was achieved under the following optimized conditions: Initial pH 5.0, NaNO3 concentration of 0.600 mol/L, FeSO4 concentration of 0.006 mol/L, and a CuSO4 concentration of 0.002 mol/L, with the other medium components constituting the basal culture medium BBM. The yield of HLF protein under optimized culture conditions was approximately 4-fold higher than that obtained by using the basal culture medium BBM. The findings are significant for the potential industrial use of GTD8A1-HLF.

Expression and characterization of recombinant human lactoferrin in edible alga Chlamydomonas reinhardtii.

Lactoferrin (LF) is a naturally occurring iron-binding glycoprotein with a variety of biological functions. It has increasing demand every year and huge market potential. In this study, we explored the feasibility of expressing human LF (hLF) in edible algae C. reinhardtii. A codon-optimized hLF gene was synthesized, inserted into pCAMBIA-1301C and transformed into C. reinhardtii SP strain. In total, 7 hLF-expressing clones were selected with clone 121 exhibiting the highest expression level. The hLF-containing algal extract significantly inhibited the growth of bacteria such as Escherichia coli and Klebsiella variicola. During acute toxicity experiment no acute toxicity was detected, especially on changes of the body weight and histopathology of organs. The recombinant hLF possessed a similar or modestly reduced stability compared to commercial hLF standard. Our data indicated that expression of hLF in C. reinhardtii is feasible and paved a way to commercial production of lactoferrin using edible Chlamydomonas expression system. Abbreviations: atrazine chlorohydrolase gene (atzA); bovine serum albumin (BSA); human LF (hLF); lactoferrin (LF); Luria-Bertani (LB); quantitative reverse transcriptase PCR (qRT-PCR) ; SDS polyacrylamide gel electrophoresis (SDS-PAGE); Tris-acetate phosphate (TAP); western blotting (WB).

Role of lactoferrin and lactoferrin-derived peptides in oral and maxillofacial diseases.

The oral cavity harbors different taxonomic groups, the evolutionary coexistence of which develops the oral ecosystem. These resident microorganisms can alter the balance between the physiologic and pathologic conditions that affect the host, both locally and systemically. This highly sophisticated nature of the oral cavity poses a significant therapeutic challenge. Numerous human and animal studies have been conducted to potentiate the efficacy and competence of current treatments of pathologic conditions as well as to develop novel therapeutic modalities. One of these studies is the use of the potent antimicrobial agent lactoferrin (LF), which was originally derived from the host immune system. LF is an 80-kDa glycoprotein that has a free iron sequestration mechanism with evident antimicrobial, anti-tumor, and immunomodulatory properties. A wide range of active peptides have been isolated from the N-terminal region of LF, which possess antimicrobial activities. In this review, we discuss the role of LF and LF-derived peptides under a heterogeneous group of oral and maxillofacial conditions, including bacterial, fungal, viral infections; head and neck cancers; xerostomia; and implantology-bone-related manifestations.

Evaluation of behavioral profiles in mice fed with milk supplemented diets derived from human lactoferrin gene-modified cows.

To date, many safety assessments of genetically modified (GM) food have been done, but there was still considerable skepticism about the safety of genetic modified foods because no study could be designed to discover all of the potential effects. Since behavioral endpoints could provide one of the most sensitive strategies to reveal subtle functional deficits. In the present study, behavioral profiles in mice fed with milk derived from human lactoferrin gene-modified cows were investigated to enrich the toxicological data of GM food. Conventional milk and GM milk were added to diets at a proportion of 7.5%, 15% and 30%(w/w). After the mice consuming different diets for 30 days, a battery of behavioral tests were conducted to evaluate motor, sensory and cognitive functions. No significant differences were observed in spontaneous activity, grip strength and nociception between the treatment groups. And animals of different groups exhibited similar performance in rotarod, dark box, step-down and MORRIS water maze task. The study suggested that mice fed with conventional milk or human lactoferrin gene-modified milk had similar motor, sensory and cognitive functions.

Transport of Human Lactoferrin into Mouse Brain: Administration Routes and Distribution.

We studied different ways of transport of human lactoferrin to the brain of C57Bl/6 mice after its administration via different routes, analyzed its distribution in the brain, and determined the phenotype of lactoferrin-containing cells. Colocalization of lactoferrin and markers of various cell types was estimated by fluorescent immunohistochemical analysis. Lactoferrin was detected in mouse brain sections after its intranasal, sublingual, and intraperitoneal administration, but not after conjunctival administration. After intranasal administration, lactoferrin rapidly penetrated into the brain and accumulated in the cytoplasm of vascular endothelial cells in the neocortex, striatum, hippocampus, and thalamus. After application of protein solution onto fixed floating sections, highly specific binding of lactoferrin was found in the nuclei of neurons, astrocytes, and microglia cells, but not in the nuclei of endothelial cells of mouse brain.

Effects of Recombinant Human Lactoferrin on Osteoblast Growth and Bone Status in Piglets.

Lactoferrin (LF), an ~80 kDa iron-binding glycoprotein, modulates many biological effects, including antimicrobial and immunomodulatory activities. Recently, it was shown that LF also regulates bone cell activity, suggesting its therapeutic effect on postmenopausal bone loss. However, a minimal amount is known regarding the effects of recombinant human LF (rhLF) supplementation on bone status in young healthy infants. We found osteoblast cell differentiation was significantly promoted in vitro. Furthermore, treatment of human osteoblast cells with rhLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein kinase (p44/p42 MAPK, ERK1/2). In order to investigate the effects of rhLF on bone status in vivo, we used a piglet model, which is a useful model for human infants. Piglets were supplemented with rhLF milk for 30 days. Bone formation markers, Serum calcium concentration, bone mineral density (BMD), bone mineral content (BMC), tibia bone strength, and the overall metabolite profile analysis showed that rhLF was advantageous to the bone growth in piglets. These findings suggest that rhLF supplementation benefits neonate bone health by modulating bone formation.

Effect of bovine lactoferrin and human lactoferrin on the proliferative activity of the osteoblast cell line MC3T3-E1 in vitro.

We conducted a comparative in vitro study on the proliferative effects of natural human lactoferrin (nhLF) and bovine lactoferrin (bLF) on osteoblasts. We investigated cell proliferation, cell survival, cell cycle, and mRNA and protein expression of proliferating cell nuclear antigen. Results indicated that treatment with 100 µg/mL of bLF or nhLF promoted the proliferation and sustenance of osteoblasts, and increased the length of the G2/M and S phases compared with the untreated osteoblasts. Results of real-time quantitative PCR and Western blot showed that mRNA and protein expression of proliferating cell nuclear antigen by osteoblasts treated with bLF or nhLF were greater than those of the untreated control. At the same concentration, bLF demonstrated a greater effect on osteoblast proliferation than did nhLF. This study provides insights of significance in the utlization of bLF in healthy food formulas.

[Acute oral toxicity and 90 days feeding test of recombinant human lactoferrin].

OBJECTIVE: To study the edible safety of recombinant human lactoferrin( rh LF) expressed from transgenic cow mammary gland bioreactor. METHODS: According to the food additive safety toxicology evaluation procedures and method, acute oral toxicity in rats and 90 day subchronic toxicity test in mice were done to evaluate the edible safety of rh LF. RESULTS: Acute oral toxicity test indicated that rh LF was no toxic effect during the observation period, mouse acute oral LD50 of recombinant human lactoferrin was greater than 20 000 mg/kg. 90 days feeding test indicated that there was no-observedadverse-effect-level after givening 300 times rh LF recommended dose of animals body, toxicological parameters NOAEL was 10. 00 g/( kg·d). CONCLUSION: According to the acute toxic dose graduation standard, rh LF was nonpoisonous. Rh LF was no-observedadverse-effect-level and no subchronic toxicity after givening 300 times rh LF recommended dose of animals body. According to the result, rh LF was no potential food safety risk.