Modulators of the Nrf2 Signaling Pathway Enhance the Cytotoxic Effect of Standard Chemotherapeutic Drugs on Organoids of Metastatic Colorectal Cancer.

The activity of known modulators of the Nrf2 signaling pathway (bardoxolone and brusatol) was studied on cultures of tumor organoids of metastatic colorectal cancer previously obtained from three patients. The effect of modulators was studied both as monotherapy and in combination with standard chemotherapy drugs used to treat colorectal cancer. The Nrf2 inhibitor brusatol and the Nrf2 activator bardoxolone have antitumor activity. Moreover, bardoxolone and brusatol also significantly enhance the effect of the chemotherapy drugs 5-fluorouracil, oxaliplatin, and irinotecan metabolite SN-38. Thus, bardoxolone and brusatol can be considered promising candidates for further preclinical and clinical studies in the treatment of colorectal cancer.

Assessing the Efficacy of Anti-Cancer Drugs on Organoid Models Derived from Prostate Cancer.

It was proven that tumor organoids effectively mirror the phenotypic and genetic traits of the original biomaterial. It was reported that outcomes from drug testing in organoid cultures can accurately represent the clinical response observed in patients. In this study, an organoid culture was derived from biopsy material of prostate cancer (PC). Subsequently, clinical practice drugs, docetaxel and enzalutamide, were tested on this organoid culture. Various techniques for evaluating the efficacy of drugs in vitro were compared. The half-maximal inhibitory concentration of docetaxel was found to be markedly lower compared to that of enzalutamide. However, when tested at clinically relevant concentrations and incubation times, enzalutamide was more effective than docetaxel. Therefore, it is crucial to optimize the testing conditions for drugs on in vitro cultures for their subsequent application in clinical practice.

[Impedance Spectroscopy and Transcriptome Analysis of Choriocarcinoma BeWo b30 as a Model of Human Placenta].

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.

Changes in the Metastatic Properties of MDA-MB-231 Cells after IGFBP6 Gene Knockdown Is Associated with Increased Expression of miRNA Genes Controlling INSR, IGF1R, and CCND1 Genes.

Metastatic cascade is associated with the process of epithelial-mesenchymal transition accompanied by changes in cell proliferation, migration, adhesion, and invasiveness mediated by the insulin-like growth factor (IGF) signal pathway. IGFBP6 protein binds IGF and prevents its interaction with receptors. IGFBP6 gene knockdown through RNA-interference inhibits cell migration and increased the rate of proliferation of breast cancer MDA-MB-231 cells. IGFBP6 knockdown cells are characterized by increased expression of MIR100 and MIRLET7A2 genes encoding hsa-miR-100-3p, hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7a-2-3p miRNA. The target genes of these microRNAs are IGF2, IGF1R, INSR, and CCND1 associated with IGF signaling pathway and proliferative and migratory activity during the metastatic cascade. A significant decrease in the expression of INSR and CCND1 genes was demonstrated by PCR and microarray analysis.

Transcriptome Guided Drug Combination Suppresses Proliferation of Breast Cancer Cells.

One of actively developing trends in modern pharmacology is the use of the transcriptome analysis for drug repositioning. We have previously detected two molecular markers of relapses in patients with malignant breast tumors: ELOVL5 and IGFBP6. Poor prognosis is associated with low expression of these markers. Here we analyze the effects of simvastatin and a new potential proteasome inhibitor K7174 inducing expression of IGFBP6 and EVOVL5 on the proliferation of breast cancer cells MDA-MB-231 and DU4475. Compound K7174 potentiates the inhibitory effect of simvastatin on the proliferation of DU4475 cells characterized by low expression of ELOVL5-IGFBP6 pair, but not on the proliferation of MDA-MB-231 cells with high expression of these markers.

Comparison of Profiles of Extracellular MicroRNA Secreted by Caco-2 Cells from the Apical Side of the Membrane under Static and Microcirculation Conditions.

Extracellular microRNA are one of the indicators of the functional state of cells. Culturing of Caco-2 cells under the conditions of microcirculation in a Homunculus microfluidic device allows better simulating natural environment of the body in comparison with static culturing. Impedance spectroscopy (BioClinicum Research Center) was used for non-invasive estimation of the monolayer quality and changes in the cell apical membrane due to the formation of microvilli. Under static conditions, Caco-2 cells release more microRNA from the apical membrane than under microcirculation conditions, while secretion of miR-320a, miR-24-3p, and miR-221-3p microRNA under static conditions can indicate stress of the cells and activation of inflammatory response. Under microcirculation conditions, the expression of laminin-α1 (LAMA1) was lower than under static conditions, which indicates deeper differentiation of cells.

Application of Impedance Spectroscopy for the Control of the Integrity of In Vitro Models of Barrier Tissues.

We compared available methods for monitoring the integrity of in vitro models of barrier tissues and studied the possibility of using impedance spectroscopy to solve this problem. It was demonstrated (theoretically and experimentally) that TEER measurements are not sufficiently sensitive to detect small defects in the cell barrier that significantly affect its permeability. For obtaining reliable results, it is necessary to set a sufficiently high threshold TEER, which leads to the loss of many intact samples. At the same time, impedance spectroscopy has all advantages of the classical method of measuring TEER (it is rapid and non-invasive method), while its application in combination with the methods of machine learning allows reliable detection of defects in the cell barrier.

Non-Invasive Evaluation of Extracellular Matrix Formation in the Intestinal Epithelium.

Differentiation of colorectal cancer Caco-2 cells was assessed using Affymetrix Human Gene 1.0 ST arrays and by the main electrical parameters measured by bioimpedance spectroscopy. Transepithelial electrical resistance (TEER) was maximum on day 7, then decreased by day 11, and remained stable. The baseline resistance was maximum on day 4, minimum on day 7, but then gradually increased over 2 weeks, which can be explained by the formation of the basement membrane components or the apical mucous layer. Caco-2 cells express components of laminin-111 and laminin-511. A synchronous increase in the expression of mucin 3 mRNA (MUC3A/MUC3B) and mucin 17 mRNA (MUC17) and reduced expression of miR-21 and miR-622 microRNA genes were observed. Possible use of the described approach for studying the formation of extracellular matrix is discussed.

[Expression of SLC30A10 and SLC23A3 Transporter mRNAs in Caco-2 Cells Correlates with an Increase in the Area of the Apical Membrane].

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.

[Ribosome Inactivation and the Integrity of the Intestinal Epithelial Barrier].

The mistletoe lectin viscumin (MLI) is a ribosome-inactivating protein from Viscum album widely used in cancer therapy. Its antitumor properties are due to its immunomodulating action, previously demonstrated in experiments involving intravenous, subcutaneous, and oral administration of viscumin. To investigate whether viscumin has a cytotoxic effect on the intestinal epithelium, its safety was assessed using (i) impedance spectroscopy to measure the integrity of the colorectal adenocarcinoma Caco-2 cell monolayer after exposure to viscumin and (ii) a novel technique of determining the portion of viscumin-inactivated ribosomes. It was shown that inactivation of at least 20% of the ribosomes within 6 h did not lead to disruption of the Caco-2 cell monolayer or alter the physicochemical parameters of enterocyte membranes.

New Fluorescent Reporter Systems for Evaluation of the Expression of E- and N-Cadherins.

During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.

Role of IGFBP6 Protein in the Regulation of Epithelial-Mesenchymal Transition Genes.

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.

In Vitro Model for Studying of the Role of IGFBP6 Gene in Breast Cancer Metastasizing.

IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.

Elimination of a Viscumin-Ferromagnetic Nanoparticles Conjugate from the Tumor Nodule in Mice.

External magnetic field is characterized by low toxicity and existence of magnetic properties, which contributes to an interest in the development of products from ferromagnetic nanoparticles (FNP) for antitumor therapy. Previously we synthesized a conjugate of ferromagnetic magnetite nanoparticles and viscumin (mistletoe lectin I, MLI), which exhibits the antitumor activity. Studying the pharmacological properties of this conjugate (FNP-MLI) was directed to the evaluation of FNP-MLI elimination after intratumor injection in mice. The elimination rate of FNP-MLI was much lower than that of native plant MLI. The presence of FNP-MLI was not accompanied by undesired changes in the tumor tissue. The use of a FNP-MLI conjugate allowed us to prolong the time of MLI presence in tissues without increasing the dose of exogenous lectin. These features contribute to the prolongation of an immunomodulatory effect of MLI.

Enzyme-Substrate Reporters for Evaluation of Substrate Specificity of HIF Prolyl Hydroxylase Isoforms.

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.

Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10-11-10-7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Structure-activity relationship for branched oxyquinoline HIF activators: Effect of modifications to phenylacetamide "tail".

HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 µM concentrations for all studied derivatives of branched tail oxyquinolines.

Nitroxide synthase activity as a marker of early stages of experimental cartilage dysmetabolism.

Time course of nitroxide synthase activity in the knee joint cartilage was studied in animals with experimental anterior instability of the knee joint. A significant increase in nitroxide synthase activity in chondrocytes was paralleled by a progressive decrease in glycosaminoglycan content in the cartilaginous matrix and subsequent destruction of the cartilage cytoarchitectonics.

[Nitric oxide synthase and apoptosis activity in chondrocytes of articular cartilage in posttraumatic knee instability]. / Aktivnost' nitroksidsintazy i apoptoza v khondrotsitakh sustavnogo khriashcha pri posttravmaticheskoi nestabil'nosti kolennogo sustava.

The dynamics of nitric oxide synthase (NOS) and apoptosis activity was studied in the in chondrocytes of articular cartilage of the patients with posttraumatic knee instability. Statistically significant increase of chondrocyte NOS activity was detected in the earliest period after the trauma (1.5 months). This was accompanied by the appearance of a great number of apoptotic cells in the zones of the cartilage with high NOS activity. Remarkably, these changes developed at the same time with statistically significant decrease of total matrix glycosaminoglycan content. These results suggest that there is a NOS-dependent signaling way of apoptosis that could participate in the development of early dystrophic changes in the cartilage.