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.
The effect of low concentrations of miRNA on the ability of GeneChip miRNA 4.0 hybridization chips to evaluate their representation in the sample was studied. It is shown that the evaluation of the expression of 61 miRNAs is statistically significantly associated with the multiplicity of plasma dilution. Only 12 miRNAs showed very high Pearson correlation coefficient (>0.95) and they all decreased in response to dilution. High abundance of has-miR-4532 miRNA in plasma was demonstrated. This miRNA was never detected during sequencing of similar samples. It was concluded that in case of miRNA expression <1.12±0.33 units in log2 scale, dilution was not followed by further decrease in the signal intensity in GeneChip miRNA 4.0 chips.
We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton's jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm2. These results substantiate medical use of lasers for expansion of cells intended for transplantation.
The effects of laminins 332 and 411 (LM-332 and LM-411) on the epithelial—mesenchymal transformation of colorectal cancer cells (lines HT-29, HCT-116, and RKO) with different metastatic potential were studied. Culturing of RKO cells on both laminins was associated with modification of the cell shape, which became more spindle-like or stellate, and with higher expression of EMT-associated transcription factors SNAI1 and ZEB1. In addition, culturing on LM-332 led to a decrease in the expression of laminin α5 chain (LAMA5), while culturing on LM-411 led to an increase in the expression of a cell—cell junction component (DSP). Culturing of HT-29 cells on LM-332 was associated with the formation of more close contacts between the cells and by a higher expression of epithelial markers (CDH1 and DSP genes) and a decrease in SNAI1 expression. Culturing of HCT-116 cells on both laminins led to a decrease in FN1 expression, on LM-332 — to an increase in laminin α4 chain (LAMA4) expression, and on LM-411 — to a lesser expression of LAMA4 and transcription factors SNAI2 and ZEB1. These data indicated that colorectal cancer cell adhesion to laminins contributed to the probability of epithelial—mesenchymal transformation of cells. The direction of this transformation seemed to depend on the initial characteristics of the cells.
Hypoxia of trophoblast cells is an important regulator of normal development of the placenta. However, some pathological states associated with hypoxia, e.g. preeclampsia, impair the functions of placental cells. Oxyquinoline derivative inhibits HIF-prolyl hydroxylase by stabilizing HIF-1 transcription complex, thus modeling cell response to hypoxia. In human choriocarcinoma cells BeWo b30 (trophoblast model), oxyquinoline increased the expression of a core hypoxia response genes along with up-regulation of NOS3, PDK1, and BNIP3 genes and down-regulation of the PPARGC1B gene. These changes in the expression profile attest to activation of the metabolic cell reprogramming mechanisms aimed at reducing oxygen consumption by enabling the switch from aerobic to anaerobic glucose metabolism and the respective decrease in number of mitochondria. The possibility of practical use of the therapeutic properties of oxyquinoline derivatives is discussed.
The search for novel parameters to predict the risk of relapse in breast cancer was conducted. Significant correlation between the risk of relapse and α-2A adrenergic receptor (ADRA2A) expression was revealed using public microarray datasets. This relationship was confirmed by validation on independent microarray dataset. It was found that when assessing the risk of BC relapse, the accuracy of prediction based solely on the expression of ADRA2A gene is close to that made using OncotypeDX and MammaPrint test systems. In this case, addition of only one or two supplemental prognostic markers (for instance, expression of SQLE gene or SQLE andDSCC1genes) to ADRA2A ensures the accuracy of prediction not inferior to reliability of these test systems.
The paper presents a formalized statement of the problem of selecting parameters and construction of a genomic classifier for medical test systemswith mathematical methods of machine learning without the use of biological and medical knowledge. A method is proposed to solve this problem. The results of testing the method using microarray datasets containing information on genome-wide transcriptome of the samples of estrogen positive breast tumors are discussed. Testing showed that the quality of classification provided by the constructed test system and implemented on the basis of assessments of expression of 12 genes is not inferior to the quality of classification carried out by such test systems as OncotypeDX and MammaPrint.
Natural response to hypoxia critically depends on rapid stabilization of hypoxia-inducible factor (HIF). Under normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress activity of HIF-prolyl hydroxylases leading to HIF activation in the cell. Here we show that 24-h incubation of BeWo b30 choriocarcinoma cells (a model of trophoblast in the placental barrier) with oxyquinoline derivative leads to a decrease in transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for FITC-dextran (70 kDa) remains unchanged. These findings suggest that the overall barrier function is preserved, while the structure of intercellular tight junctions can undergo minor changes. Using Affymetrix Human Transcriptome Array 2.0, we showed that the treatment with oxyquinoline derivative was followed by a decrease in the expression of claudins 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3), and angiomotinlike protein 1 (AMOTL1).
The hypoxic response critically depends on the rapid stabilization of the Hypoxia-Inducible Factor (HIF). In normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while the hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress the activity of HIF-prolyl hydroxylases, leading to the stabilization of HIF. Here we show that the 24 hour incubation of the trophoblast model choriocarcinoma cells BeWo b30 line with the oxyquinoline derivative leads to a decrease in the transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for the FITC-dextran (70 kDa) remains unchanged. These observations indicate that the overall barrier function remain preserved, while the structure of intercellular tight junctions may undergo remodeling. Using Affymetrix Human Transcriptome Array 2.0, we show that the treatment with oxyquinoline derivative lead to a decrease in the expression of claudines 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3) and angiotomin-like protein 1 (AMOTL1), which possibly explain the observed changes in TEER.
Hypoxia of trophoblast cells is an important regulating factor in the process of normal placenta development. However, the effect of hypoxia on the placenta in a number of pathological conditions, such as preeclampsia, leads to impaired cellular functions. A oxyquinoline derivative is capable of inhibiting HIF-prolyl hydroxylases, thereby stabilizing the transcription complex of HIF-1 and activating the cell response to hypoxia. BeWo b30 human choriocarcinoma cells are used to model trophoblast, which forms the basis for placenta barrier. Oxyquinoline effect leads both to an increased expression of a number of the genes that form the core response to hypoxia, and upregulated expression of NOS3, PDK1, and BNIP3 genes and downregulated expression of the PPARGC1B gene. This indicates the activation of mechanisms of metabolic cell reprogramming aimed at reducing oxygen consumption by reducing the number of mitochondria and switching from aerobic glucose metabolism to anaerobic. Possible applications of the obtained results is under discussion.
We studied the expression of peroxiredoxin genes (PRDX1, PRDX2, PRDX3, and PRDX6) in human erythroleukemia K652, human breast carcinoma MCF-7, and human ovarian carcinoma SKOV-3 cells during cisplatin resistance development. It was found that drug resistance formation was accompanied by a significant increase in the expression of PRDX1, PRDX2, PRDX3, PRDX6 genes in all cancer cell strains, which confirms the important contribution of redox-dependent mechanisms into the development of cisplatin resistance of cancer cells.