Reactive Halogen Species: Role in Living Systems and Current Research Approaches.

Reactive halogen species (RHS) are highly reactive compounds that are normally required for regulation of immune response, inflammatory reactions, enzyme function, etc. At the same time, hyperproduction of highly reactive compounds leads to the development of various socially significant diseases - asthma, pulmonary hypertension, oncological and neurodegenerative diseases, retinopathy, and many others. The main sources of (pseudo)hypohalous acids are enzymes from the family of heme peroxidases - myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. Main targets of these compounds are proteins and peptides, primarily methionine and cysteine residues. Due to the short lifetime, detection of RHS can be difficult. The most common approach is detection of myeloperoxidase, which is thought to reflect the amount of RHS produced, but these methods are indirect, and the results are often contradictory. The most promising approaches seem to be those that provide direct registration of highly reactive compounds themselves or products of their interaction with components of living cells, such as fluorescent dyes. However, even such methods have a number of limitations and can often be applied mainly for in vitro studies with cell culture. Detection of reactive halogen species in living organisms in real time is a particularly acute issue. The present review is devoted to RHS, their characteristics, chemical properties, peculiarities of interaction with components of living cells, and methods of their detection in living systems. Special attention is paid to the genetically encoded tools, which have been introduced recently and allow avoiding a number of difficulties when working with living systems.

Ceruloplasmin Reduces the Lactoferrin/Oleic Acid Antitumor Complex-Mediated Release of Heme-Containing Proteins from Blood Cells.

Our previous study showed that not only bovine lactoferrin (LF), the protein of milk and neutrophils, but also the human species forms complexes with oleic acid (OA) that inhibit tumor growth. Repeated injections of human LF in complex with OA (LF/8OA) to hepatoma-carrying mice decelerated tumor growth and increased animals' longevity. However, whether the effect of the LF/8OA complex is directed exclusively against malignant cells was not studied. Hence, its effect on normal blood cells was assayed, along with its possible modulation of ceruloplasmin (CP), the preferred partner of LF among plasma proteins. The complex LF/8OA (6 µM) caused hemolysis, unlike LF alone or BSA/8OA (250 µM). The activation of neutrophils with exocytosis of myeloperoxidase (MPO), a potent oxidant, was induced by 1 µM LF/8OA, whereas BSA/8OA had a similar effect at a concentration increased by an order. The egress of heme-containing proteins, i.e., MPO and hemoglobin, from blood cells affected by LF/8OA was followed by a pronounced oxidative/halogenating stress. CP, which is the natural inhibitor of MPO, added at a concentration of 2 mol per 1 mol of LF/8OA abrogated its cytotoxic effect. It seems likely that CP can be used effectively in regulating the LF/8OA complex's antitumor activity.

Photochemically induced formation of adhesive hydrogels from sodium alginate, acrylamide, and iron sandwich complexes.

Visible light irradiation of an aqueous solution of sodium alginate and organometallic complex [(C5H5)Fe(toluene)]BF4 transforms it into a rigid hydrogel due to crosslinking of the carboxylate groups by the iron ions. Irradiation of the same iron complex together with K2S2O8 initiates the polymerization of acrylamide, which provides an efficient method for light-controlled one-step preparation of alginate-polyacrylamide double network hydrogels, which are capable of gluing wet glass with 100-200 kPa shear strength.

Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Magnetic Nanoscalpel for the Effective Treatment of Ascites Tumors.

One of the promising novel methods for radical tumor resection at a single-cell level is magneto-mechanical microsurgery (MMM) with magnetic nano- or microdisks modified with cancer-recognizing molecules. A low-frequency alternating magnetic field (AMF) remotely drives and controls the procedure. Here, we present characterization and application of magnetic nanodisks (MNDs) as a surgical instrument ("smart nanoscalpel") at a single-cell level. MNDs with a quasi-dipole three-layer structure (Au/Ni/Au) and DNA aptamer AS42 (AS42-MNDs) on the surface converted magnetic moment into mechanical and destroyed tumor cells. The effectiveness of MMM was analyzed on Ehrlich ascites carcinoma (EAC) cells in vitro and in vivo using sine and square-shaped AMF with frequencies from 1 to 50 Hz with 0.1 to 1 duty-cycle parameters. MMM with the "Nanoscalpel" in a sine-shaped 20 Hz AMF, a rectangular-shaped 10 Hz AMF, and a 0.5 duty cycle was the most effective. A sine-shaped field caused apoptosis, whereas a rectangular-shaped field caused necrosis. Four sessions of MMM with AS42-MNDs significantly reduced the number of cells in the tumor. In contrast, ascites tumors continued to grow in groups of mice and mice treated with MNDs with nonspecific oligonucleotide NO-MND. Thus, applying a "smart nanoscalpel" is practical for the microsurgery of malignant neoplasms.

Effects of Synthetic Short Cationic Antimicrobial Peptides on the Catalytic Activity of Myeloperoxidase, Reducing Its Oxidative Capacity.

Cationic antimicrobial peptides (CAMPs) have gained attention as promising antimicrobial therapeutics causing lower or no bacterial resistance. Considerable achievements have been made in designing new CAMPs that are highly active as antimicrobials. However, there is a lack of research on their interaction with biologically important proteins. This study focused on CAMPs' effects on myeloperoxidase (MPO), an enzyme which is microbicidal and concomitantly damaging to host biomolecules and cells due to its ability to produce reactive oxygen and halogen species (ROS/RHS). Four CAMPs designed by us were employed. MPO catalytic activity was assessed by an absorbance spectra analysis and by measuring enzymatic activity using Amplex Red- and Celestine Blue B-based assays. The peptide Hm-AMP2 accelerated MPO turnover. Pept_1545 and Hm-AMP8 inhibited both the MPO chlorinating and peroxidase activities, with components of different inhibition types. Hm-AMP8 was a stronger inhibitor. Its Ki towards H2O2 and Cl- was 0.3-0.4 µM vs. 11-20 µM for pept_1545. Peptide tyrosine and cysteine residues were involved in the mechanisms of the observed effects. The results propose a possible dual role of CAMPs as both antimicrobial agents and agents that downregulate MPO activation, and suggest CAMPs as prototypes for the development of antioxidant compounds to prevent MPO-mediated ROS/RHS overproduction.

An IDH-independent mechanism of DNA hypermethylation upon VHL inactivation in cancer.

Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate - a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.

Core-Shell Fe3O4@C Nanoparticles for the Organic Dye Adsorption and Targeted Magneto-Mechanical Destruction of Ehrlich Ascites Carcinoma Cells.

The morphology, structure, and magnetic properties of Fe3O4 and Fe3O4@C nanoparticles, as well their effectiveness for organic dye adsorption and targeted destruction of carcinoma cells, were studied. The nanoparticles exhibited a high magnetic saturation value (79.4 and 63.8 emu/g, correspondingly) to facilitate magnetic separation. It has been shown that surface properties play a key role in the adsorption process. Both types of organic dyes-cationic (Rhodomine C) and anionic (Congo Red and Eosine)-were well adsorbed by the Fe3O4 nanoparticles' surface, and the adsorption process was described by the polymolecular adsorption model with a maximum adsorption capacity of 58, 22, and 14 mg/g for Congo Red, Eosine, and Rhodomine C, correspondingly. In this case, the kinetic data were described well by the pseudo-first-order model. Carbon-coated particles selectively adsorbed only cationic dyes, and the adsorption process for Methylene Blue was described by the Freundlich model, with a maximum adsorption capacity of 14 mg/g. For the case of Rhodomine C, the adsorption isotherm has a polymolecular character with a maximum adsorption capacity of 34 mg/g. To realize the targeted destruction of the carcinoma cells, the Fe3O4@C nanoparticles were functionalized with aptamers, and an experiment on the Ehrlich ascetic carcinoma cells' destruction was carried out successively using a low-frequency alternating magnetic field. The number of cells destroyed as a result of their interaction with Fe3O4@C nanoparticles in an alternating magnetic field was 27%, compared with the number of naturally dead control cells of 6%.

The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer.

Aptamers are short, single-stranded DNA or RNA oligonucleotide molecules that function as synthetic analogs of antibodies and bind to a target molecule with high specificity. Aptamer affinity entirely depends on its tertiary structure and charge distribution. Therefore, length and structure optimization are essential for increasing aptamer specificity and affinity. Here, we present a general optimization procedure for finding the most populated atomistic structures of DNA aptamers. Based on the existed aptamer LC-18 for lung adenocarcinoma, a new truncated LC-18 (LC-18t) aptamer LC-18t was developed. A three-dimensional (3D) shape of LC-18t was reported based on small-angle X-ray scattering (SAXS) experiments and molecular modeling by fragment molecular orbital or molecular dynamic methods. Molecular simulations revealed an ensemble of possible aptamer conformations in solution that were in close agreement with measured SAXS data. The aptamer LC-18t had stronger binding to cancerous cells in lung tumor tissues and shared the binding site with the original larger aptamer. The suggested approach reveals 3D shapes of aptamers and helps in designing better affinity probes.

Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity.

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

Aptamer-Conjugated Superparamagnetic Ferroarabinogalactan Nanoparticles for Targeted Magnetodynamic Therapy of Cancer.

Nanotechnologies involving physical methods of tumor destruction using functional oligonucleotides are promising for targeted cancer therapy. Our study presents magnetodynamic therapy for selective elimination of tumor cells in vivo using DNA aptamer-functionalized magnetic nanoparticles exposed to a low frequency alternating magnetic field. We developed an enhanced targeting approach of cancer cells with aptamers and arabinogalactan. Aptamers to fibronectin (AS-14) and heat shock cognate 71 kDa protein (AS-42) facilitated the delivery of the nanoparticles to Ehrlich carcinoma cells, and arabinogalactan (AG) promoted internalization through asialoglycoprotein receptors. Specific delivery of the aptamer-modified FeAG nanoparticles to the tumor site was confirmed by magnetic resonance imaging (MRI). After the following treatment with a low frequency alternating magnetic field, AS-FeAG caused cancer cell death in vitro and tumor reduction in vivo. Histological analyses showed mechanical disruption of tumor tissues, total necrosis, cell lysis, and disruption of the extracellular matrix. The enhanced targeted magnetic theranostics with the aptamer conjugated superparamagnetic ferroarabinogalactans opens up a new venue for making biocompatible contrasting agents for MRI imaging and performing non-invasive anti-cancer therapies with a deep penetrated magnetic field.

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.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Protective Effect of Dinitrosyl Iron Complexes with Glutathione in Red Blood Cell Lysis Induced by Hypochlorous Acid.

Hypochlorous acid (HOCl), one of the major precursors of free radicals in body cells and tissues, is endowed with strong prooxidant activity. In living systems, dinitrosyl iron complexes (DNIC) with glutathione ligands play the role of nitric oxide donors and possess a broad range of biological activities. At micromolar concentrations, DNIC effectively inhibit HOCl-induced lysis of red blood cells (RBCs) and manifest an ability to scavenge alkoxyl and alkylperoxyl radicals generated in the reaction of HOCl with tert-butyl hydroperoxide. DNIC proved to be more effective cytoprotective agents and organic free radical scavengers in comparison with reduced glutathione (GSH). At the same time, the kinetics of HOCl-induced oxidation of glutathione ligands in DNIC is slower than in the case of GSH. HOCl-induced oxidative conversions of thiolate ligands cause modification of DNIC, which manifests itself in inclusion of other ligands. It is suggested that the strong inhibiting effect of DNIC with glutathione on HOCl-induced lysis of RBCs is determined by their antioxidant and regulatory properties.

Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase.

Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called "halogenating cycle". Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.

DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress.

Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mM NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.

Fine Regulation of Neutrophil Oxidative Status and Apoptosis by Ceruloplasmin and Its Derivatives.

Timely neutrophil apoptosis is an essential part of the resolution phase of acute inflammation. Ceruloplasmin, an acute-phase protein, which is the predominant copper-carrying protein in the blood, has been suggested to have a marked effect on neutrophil life span. The present work is a comparative study on the effects of intact holo-ceruloplasmin, its copper-free (apo-) and partially proteolyzed forms, and synthetic free peptides RPYLKVFNPR (883-892) and RRPYLKVFNPRR (882-893) on polymorphonuclear leukocyte (PMNL, neutrophil) oxidant status and apoptosis. The most pronounced effect on both investigated parameters was found with copper-containing samples, namely, intact and proteolyzed proteins. Both effectively reduced spontaneous and tumor necrosis factor-α (TNF-α)-induced extracellular and intracellular accumulation of superoxide radicals, but induced a sharp increase in the oxidation of intracellular 2',7'-dichlorofluorescein upon short exposure. Therefore, intact and proteolyzed ceruloplasmin have both anti- and pro-oxidant effects on PMNLs wherein the latter effect is diminished by TNF-α and lactoferrin. Additionally, all compounds investigated were determined to be inhibitors of delayed spontaneous apoptosis. Intact enzyme retained its pro-survival activity, whereas proteolytic degradation converts ceruloplasmin from a mild inhibitor to a potent activator of TNF-α-induced neutrophil apoptosis.

Aptamer-Targeted Plasmonic Photothermal Therapy of Cancer.

Novel nanoscale bioconjugates combining unique plasmonic photothermal properties of gold nanoparticles (AuNPs) with targeted delivery using cell-specific DNA aptamers have a tremendous potential for medical diagnostics and therapy of many cell-based diseases. In this study, we demonstrate the high anti-cancer activity of aptamer-conjugated, 37-nm spherical gold nanoparticles toward Ehrlich carcinoma in tumor-bearing mice after photothermal treatment. The synthetic anti-tumor aptamers bring the nanoparticles precisely to the desired cells and selectively eliminate cancer cells after the subsequent laser treatment. To prove tumor eradication, we used positron emission tomography (PET) utilizing radioactive glucose and computer tomography, followed by histological analysis of cancer tissue. Three injections of aptamer-conjugated AuNPs and 5 min of laser irradiations are enough to make the tumor undetectable by PET. Histological analysis proves PET results and shows lower damage of healthy tissue in addition to a higher treatment efficiency and selectivity of the gold nanoparticles functionalized with aptamers in comparison to control experiments using free unconjugated nanoparticles.

In Vivo Cancer Cells Elimination Guided by Aptamer-Functionalized Gold-Coated Magnetic Nanoparticles and Controlled with Low Frequency Alternating Magnetic Field.

Biomedical applications of magnetic nanoparticles under the influence of a magnetic field have been proved useful beyond expectations in cancer therapy. Magnetic nanoparticles are effective heat mediators, drug nanocarriers, and contrast agents; various strategies have been suggested to selectively target tumor cancer cells. Our study presents magnetodynamic nanotherapy using DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles exposed to a low frequency alternating magnetic field for selective elimination of tumor cells in vivo. The cell specific DNA aptamer AS-14 binds to the fibronectin protein in Ehrlich carcinoma hence helps deliver the gold-coated magnetic nanoparticles to the mouse tumor. Applying an alternating magnetic field of 50 Hz at the tumor site causes the nanoparticles to oscillate and pull the fibronectin proteins and integrins to the surface of the cell membrane. This results in apoptosis followed by necrosis of tumor cells without heating the tumor, adjacent healthy cells and tissues. The aptamer-guided nanoparticles and the low frequency alternating magnetic field demonstrates a unique non-invasive nanoscalpel technology for precise cancer surgery at the single cell level.