Novel Indoline Spiropyrans Based on Human Hormones ß-Estradiol and Estrone: Synthesis, Structure, Chromogenic and Cytotoxic Properties.

The introduction of a switchable function into the structure of a bioactive compound can endow it with unique capabilities for regulating biological activity under the influence of various types of external stimuli, which makes such hybrid compounds promising objects for photopharmacology, targeted drug delivery and bio-imaging. This work is devoted to the synthesis and study of new spirocyclic derivatives of important human hormones-ß-estradiol and estrone-possessing a wide range of biological activities. The obtained hybrid compounds represent an indoline spiropyrans family, a widely known class of organic photochromic compounds. The structure of the compounds was confirmed by 1H and 13C NMR, IR, HRMS and single-crystal X-ray analysis. The intermolecular interactions in the crystals of spiropyran (3) were defined by Hirshfeld surfaces and 2D fingerprint plots, which were successfully acquired from CrystalExplorer (v21.5). All target hybrids demonstrated pronounced activity in the visible region of the spectrum. The mechanisms of thermal isomerization processes of spiropyrans and their protonated merocyanine forms were studied by DFT methods, which revealed the energetic advantage of the protonation process with the formation of a ß-cisoid CCCH conformer at the first stage and its further isomerization to more stable ß-transoid forms. The proposed mechanism of acidochromic transformation was confirmed by the additional NMR study data that allowed for the detecting of the intermediate CCCH isomer. The study of the short-term cytotoxicity of new spirocyclic derivatives of estrogens and their 2-formyl-precursors was performed on the HeLa cell model. The precursors and spiropyrans differed in toxicity, suggesting their variable applicability in novel anti-cancer technologies.

Novel molecular hybrids of indoline spiropyrans and α-lipoic acid as potential photopharmacological agents: Synthesis, structure, photochromic and biological properties.

Organic photochromic compounds are attracting great interest as photoswitchable components of various bioconjugates for using in photopharmacology, targeted drug delivery and bio-imaging. Here we report on the synthesis of two novel molecular hybrids of indoline spiropyrans and alpha-lipoic acid via an esterification reaction. Preliminary photochemical studies revealed photochromic activity of 5-methoxy-substituted spirocompounds in their acetonitrile solutions. Both hybrid spiropyrans along with their parent substances in the hybrids were tested for the short-term cytotoxicity on HeLa cell cultures. The results of cytotoxicity studies showed unpredictable biocompatibility of the hybrids in comparison with the parent hydroxyl-substituted spiropyrans and α-lipoic acid, especially at the relatively high concentration of 2 mM. Using flow cytometry, we demonstrated that the both hybrids induced antioxidant response in the model cells. After the 24 h treatment, the hybrids administered at lower (500 µM) concentration caused suppressed cytosolic ROS and/or induced cellular thiols. At higher concentration, one of the hybrids demonstrated properties qualitatively similar to alpha-lipoic acid, yet far more strong. Together, flow cytometry results suggested that both hybrids of spiropyrans possess emergent biochemical and signaling antioxidant properties, exceeding those of alpha-lipoic acid.

Loading of the Model Amino Acid Leucine in UiO-66 and UiO-66-NH2: Optimization of Metal-Organic Framework Carriers and Evaluation of Host-Guest Interactions.

Two metal-organic frameworks (MOFs), UiO-66 and UiO-66-NH2, were considered as containers for bioactive chemicals. We provide a synthesis technique, which allowed the production of these materials suitable for biomedical applications. Both MOFs were characterized as single-phase porous materials composed of nanoparticles (30-65 nm) with a ζ-potential of more than 40 mV in water suspension. D,L-Leucine was applied as a model molecule, which allowed us to trace the mechanism of the loading process. We showed that after synthesis, amino groups of UiO-66-NH2 are coordinated with solvent residuals. It results in a similar route of leucine loading in UiO-66 and UiO-66-NH2 samples. Using joint data of thermogravimetric analysis and calorimetry, infrared spectroscopy, and nitrogen adsorption, we revealed that methyl groups of leucine molecules are responsible for bonding of an MOF matrix. We proposed the formation of bonds between CH3 groups and benzene rings of linkers via CH-π interaction. We also assessed the toxicity of the synthesized MOFs toward HeLa cells at 50 µg/mL after 24 h incubation and revealed no negative effects on the viability of the cells, prompting further biomedical research in the areas of small-molecule delivery and cell signaling and metabolism modulation.

Effects of JUN and NFE2L2 knockdown on oxidative status and NFE2L2/AP-1 targets expression in HeLa cells in basal conditions and upon sub-lethal hydrogen peroxide treatment.

Although NFE2L2 transcription factor is considered to make the most significant contribution to the NFE2L2/AP-1-pathway-dependent antioxidants regulation in the human cell, AP-1 has the potential to provide significant backup and even play an equal role in the cell. Considering this, the present study is focused on revealing how JUN, an AP-1 component, and NFE2L2 contribute to regulation of four target genes containing AREs with embedded TREs-SQSTM1, FTH1, HMOX1 and CBR3 and to cellular oxidative status in general in basal conditions and under pro-oxidative influence. NFE2L2 and JUN were down-regulated in HeLa cells using siRNA-mediated knockdown approach. These cells were subsequently exposed to 400 µM hydrogen peroxide in the medium or equal volume of sterile water. They revealed some evidence of both backup functioning and competing between the two factors. Importantly, JUN demonstrated a high level of participation (inc. as a negative regulator) in functioning of the classic NFE2L2 targets and in cellular oxidative status establishment in general. One of the key findings was a dramatic increase in JUN expression following NFE2L2 knockdown in basal conditions. The both AP-1 and NFE2L2 sub-pathways equally determine the outcome of the NFE2L2/AP-1 pathway activation induced by various stimuli, and the outcome is stimulus type- and stimulus-intensity-specific and results from either of the two eventually dominating sub-pathways.

Individual expression features of GPX2, NQO1 and SQSTM1 transcript variants induced by hydrogen peroxide treatment in HeLa cells.

Pathway activity assessment-based approaches are becoming highly influential in various fields of biology and medicine. However, these approaches mostly rely on analysis of mRNA expression, and total mRNA from a given locus is measured in the majority of cases. Notably, a significant portion of protein-coding genes produces more than one transcript. This biological fact is responsible for significant noise when changes in total mRNA transcription of a single gene are analyzed. The NFE2L2/AP-1 pathway is an attractive target for biomedical applications. To date, there is a lack of data regarding the agreement in expression of even classical target genes of this pathway. In the present paper we analyzed whether transcript variants of GPX2, NQO1 and SQSTM1 were characterized by individual features of expression when HeLa cells were exposed to pro-oxidative stimulation with hydrogen peroxide. We found that all the transcripts (10 in total) appeared to be significantly individually regulated under the conditions tested. We conclude that individual transcripts, rather than total mRNA, are best markers of pathway activation. We also discuss here some biological roles of individual transcript regulation.

Meta-stable state photoacid containing ß-estradiol fragment with photomodulated biological activity and anti-cancer stem cells properties.

Photopharmacology is a young and rapidly developing field of research that offers significant potential for new insights into targeted therapy. While it primarily focuses on cancer treatment, it also holds promise for other diseases. The key feature of photopharmacological agents is the presence of a photosensitive and biologically active component in the same molecule. In our current study, we synthesized a spiropyran-based meta-stable state photoacid containing a fragment of ß-estradiol. This compound exhibits negative photochromism and photocontrolled fluorescence under visible-light irradiation due to the initial stabilization of its self-protonated form in solution. We conducted comprehensive biological studies on the HeLa cells model to assess the short- and long-term cytotoxicity of the photoacid, its metabolic effects, its influence on signaling and epithelial-mesenchymal transition super-system pathways, and the proportion of the population enriched with cancer stem cells. Our findings reveal that this derivative demonstrates low cytotoxicity to HeLa cells, yet it is capable of dramatically reducing malignant cells side population enriched in cancer stem cells. Additionally, appropriate structural modification lead to an increase in some other biological effects compared to ß-estradiol. In particular, our substance possesses rare properties of AP-1 suppression and demonstrates some pro-oxidant and metabolic effects, which can be regulated by visible light irradiation. As a result, the new estradiol-based photoacid may be considered a promising multi-acting photopharmacological agent for the next-generation anti-cancer research & development.

Probiotic biomarkers and models upside down: From humans to animals.

Probiotics development for animal farming implies thorough testing of a vast variety of properties, including adhesion, toxicity, host cells signaling modulation, and immune effects. Being diverse, these properties are often tested individually and using separate biological models, with great emphasis on the host organism. Although being precise, this approach is cost-ineffective, limits the probiotics screening throughput and lacks informativeness due to the 'one model - one test - one property' principle. There is а solution coming from human-derived cells and in vitro systems, an extraordinary example of human models serving animal research. In the present review, we focus on the current outlooks of employing human-derived in vitro biological models in probiotics development for animal applications, examples of such studies and the analysis of concordance between these models and host-derived in vivo data. In our opinion, human-cells derived screening systems allow to test several probiotic properties at once with reasonable precision, great informativeness and less expenses and labor effort.

Physicochemical Properties of Magnetic Nanoparticles: Implications for Biomedical Applications In Vitro and In Vivo.

Magnetic and superparamagnetic iron oxide nanoparticles are emerging as promising candidates for various applications in biology and medicine, and especially in oncology. These applications, however, require that a specific set of physical, chemical, and biological properties be combined in a given sample of nanoparticles for them to act as intended. Some of these properties are fundamental: They strictly determine the nanoparticles' behavior both in vitro and in vivo. These properties are the charge, the solution stability and zeta potential, and the coating of the nanoparticles. A certain combination of these properties may satisfy a researcher in an in vitro study, but other properties should also be considered when in vivo applications are planned. For in vivo experiments, additional determinants of the quality of nanoparticles are their size, shape, modifications with targeting moieties, and degradation/excretion pathways. All these properties are in the focus of the present review.

Low toxic maghemite nanoparticles for theranostic applications.

BACKGROUND: Iron oxide nanoparticles have numerous and versatile biological properties, ranging from direct and immediate biochemical effects to prolonged influences on tissues. Most applications have strict requirements with respect to the chemical and physical properties of such agents. Therefore, developing rational design methods of synthesis of iron oxide nanoparticles remains of vital importance in nanobiomedicine. METHODS: Low toxic superparamagnetic iron oxide nanoparticles (SPIONs) for theranostic applications in oncology having spherical shape and maghemite structure were produced using the fast microwave synthesis technique and were fully characterized by several complementary methods (transmission electron microscopy [TEM], X-ray diffraction [XRD], dynamic light scattering [DLS], X-ray photoelectron spectroscopy [XPS], X-ray absorption near edge structure [XANES], Mossbauer spectroscopy, and HeLa cells toxicity testing). RESULTS: TEM showed that the majority of the obtained nanoparticles were almost spherical and did not exceed 20 nm in diameter. The averaged DLS hydrodynamic size was found to be ~33 nm, while that of nanocrystallites estimated by XRD was16 nm. Both XRD and XPS studies evidenced the maghemite (γ-Fe2O3) atomic and electronic structure of the synthesized nanoparticles. The XANES data analysis demonstrated the structure of the nanoparticles being similar to that of macroscopic maghemite. The Mossbauer spectroscopy revealed the γ-Fe2O3 phase of the nanoparticles and vibration magnetometry study showed that reactive oxygen species in HeLa cells are generated both in the cytoplasm and the nucleus. CONCLUSION: Quasispherical Fe3+ SPIONs having the maghemite structure with the average size of 16 nm obtained by using the fast microwave synthesis technique are expected to be of great value for theranostic applications in oncology and multimodal anticancer therapy.

A Proteomics Analysis Reveals 9 Up-Regulated Proteins Associated with Altered Cell Signaling in Colon Cancer Patients.

Colorectal cancer is the second most common cancer in women and third most common cancer in men. Cell signaling alterations in colon cancer, especially in aggressive metastatic tumors, require further investigations. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes' grade B) and metastatic (Dukes' grade C&D) colon cancer. Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The Panorama™ Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages 'samr', 'gplots', 'supclust' (pelora, wilma algorithms), 'glmnet' for the differential expression analysis, supervised clustering and penalized logistic regression. Significance analysis of microarrays revealed 9 significantly up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, pan cytokeratin, and 1 significantly down-regulated protein (GAP1) in tumoral mucosa. Pan-cytokeratin and APP were up-regulated in tumor versus non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all employed regression/clustering methods. Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.

Individual expression features of GPX2, NQO1 and SQSTM1 transcript variants induced by hydrogen peroxide treatment in HeLa cells

Abstract Pathway activity assessment-based approaches are becoming highly influential in various fields of biology and medicine. However, these approaches mostly rely on analysis of mRNA expression, and total mRNA from a given locus is measured in the majority of cases. Notably, a significant portion of protein-coding genes produces more than one transcript. This biological fact is responsible for significant noise when changes in total mRNA transcription of a single gene are analyzed. The NFE2L2/AP-1 pathway is an attractive target for biomedical applications. To date, there is a lack of data regarding the agreement in expression of even classical target genes of this pathway. In the present paper we analyzed whether transcript variants of GPX2, NQO1 and SQSTM1 were characterized by individual features of expression when HeLa cells were exposed to pro-oxidative stimulation with hydrogen peroxide. We found that all the transcripts (10 in total) appeared to be significantly individually regulated under the conditions tested. We conclude that individual transcripts, rather than total mRNA, are best markers of pathway activation. We also discuss here some biological roles of individual transcript regulation.