Structural features of Prussian Blue-related iron complex - FeT of activity to peroxidate unsaturated fatty acids.

Aim: FeT is a complex of Fe3+, ferricyanide and tartrate, similar in structure to Prussian Blue. Its synthesis was planned to produce a potential antiproliferative drug. Methods: Dynamic light scattering was applied to study nanostructures formed by FeT complexes, while their biological activity was tested following changes in cell proliferation using cultured T24 human bladder cancer cells. Results: The antiproliferative activity of FeT derived from its ability to peroxidate unsaturated fatty acids, which can cause cell death through oxidative stress and/or ferroptosis. FeT molecules associate into drop-like nanostructures in water solutions, between 10-130 nm, which can bind albumin. Conclusion: Fatty acid peroxidation is significantly activated by light. The characteristics and reactivity of FeT represent a prospective application in medicine.

Three generations of mTOR kinase inhibitors in the activation of the apoptosis process in melanoma cells.

Many signaling pathways are involved in the mammalian target of rapamycin (mTOR), and this serine/threonine kinase regulates the most important cellular processes such as cell proliferation, autophagy, and apoptosis. The subject of this research was the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on the expression of pro-survival proteins, activity of caspase-3, proliferation, and induction of apoptosis in melanoma cells. The following inhibitors were used: protein kinase inhibitors such as AKT-MK-2206, MEK-AS-703026, mTOR-everolimus and Torkinib, as well as dual PI3K and mTOR inhibitor-BEZ-235 and Omipalisib, and mTOR1/2-OSI-027 inhibitor in single-mode and their combinations with MEK1/2 kinase inhibitor AS-703026. The obtained results confirm the synergistic effect of nanomolar concentrations of mTOR inhibitors, especially the dual PI3K and mTOR inhibitors (Omipalisib, BEZ-235) in combination with the MAP kinase inhibitor (AS-703026) in the activation of caspase 3, induction of apoptosis, and inhibition of proliferation in melanoma cell lines. Our previous and current studies confirm the importance of the mTOR signal transduction pathway in the neoplastic transformation process. Melanoma is a case of a very heterogeneous neoplasm, which causes great difficulties in treating this neoplasm in an advanced stage, and the standard approach to this topic does not bring the expected results. There is a need for research on the search for new therapeutic strategies aimed at particular groups of patients. Effect of three generations of mTOR kinase inhibitors on caspase-3 activity, apoptosis and proliferation in melanoma cell lines.

Albumin Binds Doxorubicin via Self-Assembling Dyes as Specific Polymolecular Ligands.

Congo red (CR) type self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin. SRLS also complex with some drugs with a flat, ring-shaped structure with aromatic characteristics, intercalating them into their ribbon structure. The combination of interaction with proteins and drug binding by SRLS enables the use of such systems for immunotargeting. It is especially interesting in the case of chemotherapeutic agents. The present experiments aimed to show that the model carrier system composed of supramolecular albumin and Congo red efficiently binds doxorubicin (Dox) and that the drug can be released at reduced pH. The presented results come from the studies on such complexes differing in the molar ratio of CR to Dox. The following methods were used for the analysis: electrophoresis, dialysis, gel filtration, spectral analysis, and analysis of the size of the hydrodynamic radius using the dynamic light scattering method (DLS). The applied methods confirmed the formation of the CR-Dox complex, with large dimensions and changed properties compared with free CR. The presented results show that albumin binds both CR and its complex with Dox. Various CR-Dox molar ratios, 5:1, 2:1, and 1:1, were analyzed. The confirmation of the possibility of releasing the drug from the carriers thus formed was also obtained. The presented research is important due to the search for optimal solutions for the use of SRLS in drug immunotargeting, with particular emphasis on chemotherapeutic agents.

Interaction of Supramolecular Congo Red and Congo Red-Doxorubicin Complexes with Proteins for Drug Carrier Design.

Targeted immunotherapy has expanded to simultaneous delivery of drugs, including chemotherapeutics. The aim of the presented research is to design a new drug carrier system. Systems based on the use of proteins as natural components of the body offer the chance to boost safety and efficacy of targeted drug delivery and excess drug removal. Congo red (CR) type supramolecular, self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin and antibodies complexed with antigen. CR can intercalate some chemotherapeutics including doxorubicin (Dox). The goal of this work was to describe the CR-Dox complexes, to analyze their interaction with some proteins, and to explain the mechanism of this interaction. In the present experiments, a model system composed of heated immunoglobulin light chain Lλ capable of CR binding was used. Heat aggregated immunoglobulins (HAI) and albumin were chosen as another model system. The results of experiments employing methods such as gel filtration chromatography and dynamic light scattering confirmed the formation of the CR-Dox complex of large size and properties different from the free CR structures. Electrophoresis and chromatography experiments have shown the binding of free CR to heated Lλ while CR-Dox mixed structures were not capable of forming such complexes. HAI was able to bind both free CR and CR-Dox complexes. Albumin also bound both CR and its complex with Dox. Additionally, we observed that albumin-bound CR-Dox complexes were transferred from albumin to HAI upon addition of HAI. DLS analyses showed that interaction of CR with Dox distinctly increased the hydrodynamic diameter of CR-Dox compared with a free CR supramolecular structure. To our knowledge, individual small proteins such as Lλ may bind upon heating a few molecules of Congo red tape penetrating protein body due to the relatively low cohesion of the dye micelle. If, however, the compactness is high (in the case of, e.g., CR-Dox) large ribbon-like, micellar structures appear. They do not divide easily into smaller portions and cannot attach to proteins where there is no room for binding large ligands. Such binding is, however, possible by albumin which is biologically adapted to form complexes with different large ligands and by tightly packed immune complexes and heat aggregated immunoglobulin-specific protein complex structures of even higher affinity for Congo red than albumin. The CR clouds formed around them also bind the CR-Dox complexes. The presented research is essential in the search for optimum solutions for SRLS application in immuno-targeting therapeutic strategies, especially with the use of chemotherapeutics.

Controlled Release of Doxorubicin from the Drug Delivery Formulation Composed of Single-Walled Carbon Nanotubes and Congo Red: A Molecular Dynamics Study and Dynamic Light Scattering Analysis.

The controlled delivery and release of drug molecules at specific targets increases the therapeutic efficacy of treatment. This paper presents a triple complex which is a new potential drug delivery system. Triple complex contains single-walled carbon nanotubes, Congo red, and doxorubicin. Nanotubes are built of a folded graphene layer providing a large surface for binding Congo red via "face-to-face" stacking which markedly increases the binding capacity of the carrier. Congo red is a compound that self-associates to form supramolecular ribbon-like structures, which are able to bind some drugs by intercalation. The nanotube-Congo red complex can bind the model drug doxorubicin. Thus, a new triple carrier system was obtained. The aim of this paper is to present studies on the controlled release of a model anticancer drug from a triple carrier system through pH changes. The specific aim of the study was to model the structure of the obtained experimental systems and to compare the changes in the average energy of interaction between its components induced by pH changes. The studies also aimed to compare the intensity of pH-dependent changes in hydrodynamic diameters of individual components of the triple carrier system. The effect of pH changes on the stability of the analyzed systems was examined using the molecular modeling method and dynamic light scattering. The decrease in pH influenced the structure and stability of the analyzed triple systems and ensured efficient drug release. The changes in hydrodynamic diameters of the obtained fractions were examined with the use of dynamic light scattering and were confirmed by computer simulation methods. The formulation presented in this paper shows potential for a therapeutic application owing to its high drug binding capacity and pH-dependent release. This ensures prolonged local action of the drug. The results reveal that the studied complex fulfills the basic requirements for its potential use as drug carrier, thus reducing side effects and enhancing pharmacological efficacy of drugs.

The Epigenetic Modifier 5-Aza-2-deoxycytidine Triggers the Expression of CD146 Gene in Prostate Cancer Cells.

BACKGROUND/AIM: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT). Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro. MATERIALS AND METHODS: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing. RESULTS: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype. CONCLUSION: The demethylating agent 5-aza-2-deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.

Self-Assembled Supramolecular Ribbon-Like Structures Complexed to Single Walled Carbon Nanotubes as Possible Anticancer Drug Delivery Systems.

Designing an effective targeted anticancer drug delivery method is still a big challenge, since chemotherapeutics often cause a variety of undesirable side effects affecting normal tissues. This work presents the research on a novel system consisting of single walled carbon nanotubes (SWNT), dispersed with Congo Red (CR), a compound that forms self-assembled ribbon-like structures (SRLS) and anticancer drug doxorubicin (DOX). SWNT provide a large surface for binding of planar aromatic compounds, including drugs, while CR supramolecular ribbon-like assemblies can be intercalated by drugs, like anthracycline rings containing DOX. The mechanism of interactions in SWNT-CR-DOX triple system was proposed based on electrophoretic, spectral, Dynamic Light Scattering and scanning electron microscopy analyzes. The profile of drug release from the investigated system was evaluated using dialysis and Differential Scanning Calorimetry. The results indicate that ribbon-like supramolecular structures of CR bind to SWNT surface forming SWNT-CR complexes which finally bind DOX. The high amount of nanotube-bound CR greatly increases the capacity of the carrier for the drug. The high capacity for drug binding and possible control of its release (through pH changes) in the analyzed system may result in prolonged and localized drug action. The proposed SWNT-CR-DOX triple system meets the basic criteria that justifies its further research as a potential drug carrier.

Dispersion of single-wall carbon nanotubes with supramolecular Congo red - properties of the complexes and mechanism of the interaction.

A method of dispersion of single-wall carbon nanotubes (SWNTs) in aqueous media using Congo red (CR) is proposed. Nanotubes covered with CR constitute the high capacity system that provides the possibility of binding and targeted delivery of different drugs, which can intercalate into the supramolecular, ribbon-like CR structure. The study revealed the presence of strong interactions between CR and the surface of SWNTs. The aim of the study was to explain the mechanism of this interaction. The interaction of CR and carbon nanotubes was studied using spectral analysis of the SWNT-CR complex, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and microscopic methods: atomic force microscopy (AFM), transmission (TEM), scanning (SEM) and optical microscopy. The results indicate that the binding of supramolecular CR structures to the surface of the nanotubes is based on the "face to face stacking". CR molecules attached directly to the surface of the nanotubes can bind further, parallel-oriented molecules and form supramolecular and protruding structures. This explains the high CR binding capacity of carbon nanotubes. The presented system - containing SWNTs covered with CR - offers a wide range of biomedical applications.

Silver ions as em marker of congo red ligation sites in amyloids and amyloid-like aggregates.

Congo red (CR) is a known selective amyloid ligand. The focus of our work is identification (by EM imaging) of dye binding sites and their distribution in amyloids and amyloid-like aggregates formed in vitro. In order to produce the required contrast, CR has been indirectly combined with metal via including Titan yellow (TY) by intercalation which exhibits a relatively strong affinity for silver ions. The resulting combined ligand retains its ability to bind to proteins (which it owes to CR) and can easily be detected in EM studies thanks to TY. We have found, however, that in protein aggregates where unfolding is stabilized by aggregation and therefore is irreversible, TY alone may serve as both, the ligand and the metal carrier. The formation of ordered structures in amyloids was studied using IgG light chains with amyloidogenic properties, converted into amyloids by shaking. The resulting EM images were subjected to interpretation on the basis of the authors' earlier research on the CR/light chain complexation process. Our results indicate that dimeric light chains, which are the subject of our study, produce amyloids or amyloid-like complexes with chain-like properties and strong helicalization tendencies. Cursory analysis suggests that the edge polypeptide loops belonging to unstable light chains form intermolecular bridges which promote creation of loose gel deposits, or are otherwise engaged in the swapping processes leading to higher structural ordering.

The use of supramolecular structures as protein ligands.

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.

Influence of the electric field on supramolecular structure and properties of amyloid-specific reagent Congo red.

Among specific amyloid ligands, Congo red and its analogues are often considered potential therapeutic compounds. However, the results of the studies so far have not been univocal because the properties of this dye, derived mostly from its supramolecular nature, are still poorly understood. The supramolecular structure of Congo red, formed by π-π stacking of dye molecules, is susceptible to the influence of the electric field, which may significantly facilitate electron delocalization. Consequently, the electric field may generate altered physico-chemical properties of the dye. Enhanced electron delocalization, induced by the electric field, alters the total charge of Congo red, making the dye more acidic (negatively charged). This is a consequence of withdrawing electrons from polar substituents of aromatic rings-sulfonic and amino groups-thus increasing their tendency to dissociate protons. The electric field-induced charge alteration observed in electrophoresis depends on dye concentration. This concentration-dependent charge alteration effect disappears when the supramolecular structure disintegrates in DMSO. Dipoles formed from supramolecular fibrillar species in the electric field become ordered in the solution, introducing the modified arrangement to liquid crystalline phase. Experimental results and theoretical studies provide evidence confirming predictions that the supramolecular character of Congo red is the main reason for its specific properties and reactivity.

Albumin binds self-assembling dyes as specific polymolecular ligands.

Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

The use of the Congo red-related dye DBACR to recognize the heavy chain-derived abnormality of myeloma immunoglobulins.

INTRODUCTION: The aim of this study was to differentiate heavy and light chain-derived instability of monoclonal myeloma immunoglobulins by complexation of matched supramolecular dyes. These are composed of several micellar pieces of self-assembled dye molecules which may penetrate the protein interior of the binding locus with polypeptide chains. These dyes were used to elicit, by precipitation, the postulated higher aggregation tendency of the heavy chain derived from its higher hydrophobicity. MATERIALS AND METHODS: Agarose gel electrophoresis was used to create conditions for dye complexation and to reveal the precipitation. RESULTS: Congo red derivatives with aromatic ring substitutes, BACR and DBACR, of increased penetrating capability were chosen to provoke the precipitation of abnormal immunoglobulins by displacing association-prone polypeptide chains from the protein interior. CONCLUSIONS: The results of this study confirm the heavy chain-related propensity of some monoclonal immunoglobulins to aggregate and precipitate. The simplicity of the technique may improve clinical diagnosis and facilitate predictions of disease complications.

Instability of monoclonal myeloma protein may be identified as susceptibility to penetration and binding by newly synthesized Congo red derivatives.

Monoclonal myeloma proteins often have an abnormal, unstable structure, and tend to aggregate with fatal clinical consequences. A method for early clinical identification of this aggregation tendency is impatiently awaited. This work proposes the use of supramolecular dyes as specific ligands to reveal protein instability. Disclosure of excessive polypeptide chain flexibility in unstable monoclonal proteins, leading to increased susceptibility to penetration by foreign compounds, appeared possible when new supramolecular Congo red-derived dyes with different protein-binding capabilities were used for complexation. Two basic protein instability levels, local and global, were differentiated by comparing the extent of protein loading with dye and the subsequent electrophoretic migration rate of the complexes. A simple electrophoretic test is proposed for assessment of the instability of monoclonal proteins in clinical conditions.

In vivo accumulation of self-assembling dye Congo red in an area marked by specific immune complexes: possible relevance to chemotherapy.

Supramolecular micellar structures have been proposed as carriers in aim-oriented drug transportation to a target marked by specific immune complexes. In this study, the self-assembling dye Congo red was used as a model supramolecular carrier and its accumulation in the target was studied in vivo. The target was created in vivo as the local specific inflammation provoked by subcutaneous injection of antigen to the ear of a previously immunized rabbit. The color caused by accumulation of Congo red after its intravenous injection was registered by pictures of the ear with suitably filtered visible light shining through it to distinguish Congo red against the background color of hemoglobin. The results confirmed the expected accumulation and retention of Congo red in the inflammation area marked by deposits of specific immune complexes. The role of albumin and its possible interference with transportation of drugs through the blood by supramolecular carriers was also subjected to preliminary examination. The results revealed that albumin collaborates rather than interferes with drug transportation; this is another factor making the use of supramolecular carriers for aim-oriented chemotherapy highly promising.

Protein distorsion-derived mechanism of signal discrimination in monocytes revealed using Congo red to stain activated cells.

The supramolecular dye Congo red was used to check whether monocyte activation may be mediated by a torsion-dependent mechanism preventing transduction of weak random signals in cell contacts in a way corresponding to the discrimination mechanism found in complement fixation by immune complexes. Tight cell-cell contacts generating torsional effects may be expected to produce alteration of receptor structure, making them accessible for binding of supramolecular dyes. In this study, Congo red was used to observe the binding accessibility of (1) monocytes (human) induced by contact with cancer cells (HCV29T, human), (2) monocytes (mouse) stimulated by interaction with heat-aggregated IgG and (3) monocytes (mouse) activated by rosetting in the presence of an SRBC-anti-SRBC system. Microscopic studies confirmed the activation of monocytes manifested by their clustering and Congo red binding, but only tightly clustered cells appeared to attach the dye on the surface. Usually not the whole cell surface is found to be engaged in dye complexation. Staining occurs predominantly on the interfaces of reacting cells, making probable the suggestion that cell adhesion receptors are involved in dye binding. The cells in the central areas of tight clusters undergo accelerated death. In the presence of Congo red they are easily recognized as intensely fluorescent. The characteristic localization of dead cells in the central area of clusters indicates that death is not random but results from cell activation. The role of Congo red in this process remains to be clarified. The staining characteristics of monocytes after application of Congo red probably discloses the initial step in signal transduction generated by torsional movements in receptor proteins.

The structural abnormality of myeloma immunoglobulins tested by Congo red binding.

BACKGROUND: Frequently observed structural deviations of myeloma-derived immunoglobulins affect polypeptide chain packing and domain stability, enhancing their tendency to aggregate, with all the clinical consequences. Congo red complexation with myeloma immunoglobulins is proposed in this work as a general test to disclose the instability of these proteins. The large ribbon-like supramolecular ligands of Congo red form complexes with proteins by adhesion to beta-conformation polypeptide chains, if allowed to make contact with their backbone interfaces. This can occur in the case of myeloma-derived immunoglobulins with deficient polypeptide chain packing. MATERIAL/METHODS: Specially adapted two-dimensional agarose electrophoresis of serum proteins, which allows the transient contact of Congo red and serum proteins during migration, was used to reveal the presence of protein components amenable to ligand penetration and binding. The combination of electrophoresis and Congo red binding to proteins permits the removal of loosely attached dye and evaluation of the effective complexation properties of the immunoglobulin fraction directly in the serum. RESULTS: Comparative studies of dye complexation with two L chains having different reactivities with Congo red confirmed that dye binding depended on protein instability in the conditions used. Myeloma proteins revealed different binding capabilities in the test used here. CONCLUSIONS: The complexes formed by the supramolecular dye Congo red with myeloma immunoglobulins differ in stability. Those of high stability indicate the abnormal protein structure thought to produce clinical symptoms. This work proposes an easy technique to differentiate the stability of complexes.

The structure and protein binding of amyloid-specific dye reagents.

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.