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It is imperative to explore new biocompatible drugs with low toxicity for use in medicinal fields such as fighting tumors. Bovine lactoperoxidase (BLPO) stems from the most important enzymes in the bovine whey that provide a proper pattern for nano-formulation with nanomaterials. LPO is a suitable protein to be coated or adsorbed to alginate modified graphene oxide (GO-SA), which forms the modified GO-SA-LPO hybrid structure. This novel combination provides LPO stability with strong anticancer effects and boosts immunity response. The characterization results obtained from different techniques confirmed a successful LPO adsorption on the GO-SA composite surface. Moreover, nano-formulation of LPO with GO-SA composite exhibited a reduction in its size and overall charge. In addition, the experimental results showed greater LPO activity stability in the modified GO-SA-LPO nanocombination than free LPO after storage for 10 weeks at 4 °C. The in vitro study, a crucial step in the validation of our approach, demonstrated that the modified GO-SA-LPO nanocombination showed a potent anticancer selectivity toward colon cancer cell lines more than GO-SA composite or free form of LPO, which enhanced in a dose-dependent manner with high safety manner against normal cells. The apoptotic effect of this novel nanocombination was confirmed by the greatest variations in the expression of both well-known apoptosis genes (p53 and Bcl-2), severe changes in the cellular morphology, DNA fragmentation, and nuclear staining with fluorescence yellow and orange of the target cancer cells. Also, this superior efficacy of the modified GO-SA-LPO nanocombination was induced by suppressing some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL-6), and necrosis factor-kappa B (NF-ĸB). Our observations presented that the modified nanocombination of LPO may offer a novel remedy for treating colon tumors via induced apoptosis pathway, inflammation reduction, and immune response improvement., (© 2024. The Author(s).)

This study identifies promising potential of a novel and safer nanocombination of bovine milk lactoperoxidase (LPO) and lactoferrin (LF) to target breast cancer in vitro and in adult female albino rat model. Favorable selective anticancer effects of the prepared nanocombination were observed, in a dose-dependent manner, against both MCF-7 and MDA cell lines, sparing normal HFB-4 cells. The administration of LPO + LFNPs markedly improved the induced-breast cancer disorders, prolonged survival and reduced the values of serum TNF-α, IL1β, CD4 + , ALAT, ASAT, urea, creatinine, cholesterol and triglycerides with remarkable elevation in mammary SOD and GPx activity and GSH level. Moreover, the histopathological findings showed that LPO + LFNPs succeeded in prevention of mammary gland tumorigenesis. Superior efficacy of LPO + LFNPs was observed against pro-inflammatory cytokines through their anti-inflammatory and immunomodulatory properties. The treatment of LPO + LFNPs more significantly modulated the apoptosis and enhanced the expression of cell cycle regulator genes, which demonstrates a successful tumor therapy in vitro and in vivo. Therefore, this study provided evidence that the chemo-preventive feature of LPO + LFNPs may offer a novel alternative therapy for the treatment of breast cancer through enhances apoptosis pathway, improvement of immune response, reduction of inflammation and restoration of the impaired oxidative stress., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

There is an urgent need in the medicinal fields to discover biocompatible nanoformulations with low cytotoxicity, which provide new strategies for promising therapies for several types of tumors. Bovine lactoperoxidase (LP) and lactoferrin (LF) have recently attracted attention in medicine for their antitumor activities with recognized safety pattern. Both LP and LF are suitable proteins to be coated or adsorbed to Cu and Fe nanometals for developing stable nanoformulations that boost immunity and strong anticancer effects. New nanometals of Cu and Fe NPs embedded in LP and LF forming novel nanocombinations of LP-CNPs and LF-FNPs had a spherical shape with an average nanosize of about 21 nm. The combination of LP-CNPs and LF-FNPs significantly exhibited the highest growth inhibitory efficacy, in terms of effectively lowering the half-maximal inhibitory concentration (IC 50 ) values, against Caco-2, HepG2 and MCF7 cells comparing to nanometals, LP, LF and individual nanoproteins (LP-CNPs or LF-FNPs). The highest apoptotic effect of this nanocombination (LP-CNPs and LF-FNPs) was confirmed by the highest percentages of annexin-stained apoptotic cells and G0 population with the strongest alteration in the expression of two well-characterized apoptosis guards (p53 and Bcl-2) and the maximum suppression in the proliferation marker (Ki-67). Also, the in silico analysis predicted that LP-CNPs and LF-FNPs enhanced AMP-activated protein kinase (AMPK, p53 activator) activity and inhibited cancer migration-related proteases (cathepsin B and matrix metalloproteinase (MMP)-9). Our results offer for the first time that these novel nanocombinations of LP and LF were superior in their selectivity and apoptosis-mediating anticancer activity to Cu and Fe nanometals as well as the free form of these proteins or their individual nanoforms., (© 2022. The Author(s).)

Lactoperoxidase is a glycosylated protein with a molecular mass of 78 kDa, which being excreted in several mammalian secretions. Lactoperoxidase is included in many biological processes and well-known to have biocidal actions, attending as active antibiotics and antiviral agents. This wide-spectrum of biocidal activities mediates via a definite inhibitory system named lactoperoxidase system which acts a potent role in the innate immune response since its activity is not restricted by the antimicrobial effect, but might act a significant role in the hydrolysis of many toxins like aflatoxin. Hence with the current progresses in technology, nanoparticles can offer chances as an active candidate that might be utilized for stabilizing and potentiating the activity of LPO for use in several applications. Due to the variability functions of LPO, this enzyme considers an active target to be encapsulated or coated to NPs for developing novel nanocombinations with controlled surface characteristics. The development of approaches which might enhance conformational stabilization for several weeks of LPO via nanoformulation could improve the biopharmaceutical applicability of this bioactive ingredient. Nanoformulation of LPO enhances novel functions that can be useful in many biotechnological applications like food industry, cosmetic and pharmaceutical applications or to deliver and encapsulate bioactive components.

Interaction between nanoparticles (NPs) and protein is particularly important due to the formation of dynamic nanoparticle-protein complex. The current study indicated that silica NPs were able to induce conformational modification in the adsorbed lactoperoxidase (LPO) which in turns degrades the synthetic dyes. The maximum degradation efficiency was recorded for the LPO modified silica NPs in the presence of H 2 O 2 comparing with either free LPO or silica NPs. Degradation efficiency of crystal violet and commassie blue R250 after 6 h was assessed to be 100(%). Also, degradation efficiency of Congo red reached 90.6% and 79.3% in the presence and absence of H 2 O 2 , respectively, however methyl red degradation efficiency recorded 85%. The viability assay experiment indicated that the IC 50 value of the LPO modified silica NPs on human fibroblast cells reached 2.8 mg/ml after 48 h incubation. In addition to dye removal, the LPO modified silica NPs were able to inhibit the antibiotic resistant bacterial strains (Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp.) at concentrations up to 2.5 mg/ml with inhibition activity about 95%. These findings emphasized that the ability of LPO for degradation of the synthetic dyes after adsorption on silica NPs besides it could be a promising agent with potent inhibitory effect targeting a wide range of multidrug resistant bacteria.

Bovine lactoperoxidase (LPO) and lactoferrin (LF) are suitable proteins to be loaded or adsorbed to chitosan nanoparticles (NPs) for preparing stable nanoformulations with potent anticancer activity. In the present study, nanocombinations of LPO and LF revealed improvement in their stability and activity compared to single (free or nanoformulated) bovine proteins. The coating or loading of LPO-loaded NPs with LF resulted in the highest synergistic cytotoxicity effect against Caco-2, HepG-2, MCF-7 and PC-3 cells in comparison with other NPs and free proteins without causing toxicity toward normal cells. This synergistic improvement in the anticancer activity was apoptosis-dependent that was confirmed by severe alterations in cellular morphology, high percentage of annexin-stained cells and sub-G1 populations as well as nuclear staining with orange fluorescence of treated cancer cells. Additionally, significant alterations in the expression of well characterized cellular proliferation and apoptosis guards (NF-κB, Bcl-2 and p53) in these NPs-treated cancer cells compared to 5-fluorouracil (5-FU) treated cells. Our findings provide for the first time that these new synergistic nanoformulated forms of LPO and LF were superior in their selective apoptosis-mediating anticancer effect than free form of these proteins and 5-FU. LF coating or loading of LPO-loaded NPs present as promising therapy for cancer.

Lactoperoxidase is a milk hemoprotein that acts as a non-immunoglobulin protective protein and shows strong antimicrobial activity. Bovine milk contains about 15 and 7 times higher levels of lactoperoxidase than human colustrum and camel milk, respectively. Human, bovine, and camel lactoperoxidases (hLPO, bLPO, and cLPO, respectively) were purified as homogeneous samples with specific activities of 4.2, 61.3, and 8.7 u/mg, respectively. The optimal working pH was 7.5 (hLPO and bLPO) and 6.5 (cLPO), whereas the optimal working temperature for these proteins was 40 °C. The K m of hLPO, cLPO, and bLPO were 17, 16, and 19 mM, and their corresponding V max values were 2, 1.7, and 2.7 μmol/min ml. However, in the presence of H 2 O 2 , the K m values were 11 mM for hLPO and cLPO and 20 mM for bLPO, while the corresponding V max values were 1.17 for hLPO and 1.4 μmol/min ml for cLPO and bLPO. All three proteins were able to inhibit the herpes simplex virus type 1 (HSV-1) in Vero cell line model. The relative antiviral activities were proportional to the protein concentrations. The highest anti-HSV-1 activity was exhibited by bLPO that inhibited the HSV particles at a concentration of 0.5 mg/ml with the relative activity of 100%.

This article continues a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. Besides caseins, which are the major proteinaceous constituents of any milk that can be isolated by isoelectric precipitation, milk contains a set of soluble whey proteins, such as β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin, lactoperoxidase, glycomacropeptide, and proteose peptone (the last two are soluble casein derivatives). Lactoferrin and lactoperoxidase (LPO) are known to possess prominent biocidal activity, serving as efficient antibiotics and antiviral agents against a wide spectrum of bacteria, fungi, and viruses. LPO is a heme-containing peroxidase expressed as preproprotein. The mature protein has a single catalytic domain, structure of which is known for a protein isolated from several species. Functionally, LPO is a crucial component of the LPO system that includes LPO, hydrogen peroxide (H2O2), and thiocyanate (SCN(-)), being a well-studied, naturally occurring antimicrobial system in milk that is effective against many microorganisms and some viruses. Although various aspects of LPO structure and function are rather well studied and were subjects of several recent reviews, the abundance and potential functional roles of intrinsically disordered regions in this protein have never being addressed as of yet. The major goal of this article is to fill this gap and to show how intrinsic disorder is encoded in the amino acid sequence of LPO, and how intrinsic disorder is related to functions of this important milk protein.