In hepatocellular carcinoma (HCC), the presence of cancer stem cells (CSCs) have been linked to drug resistance, epithelial-mesenchymal transition (EMT), and cancer relapse. This study investigates the expression profile of ZEB1, ZEB2, ABCG2 in HCC-CSCs, and the role of EMT promoter ZEB2 in cells treated with resveratrol. The expression of ZEB1, ZEB2 and ABCG2 transcripts were analyzed in CD133^(+)/CD44^(+) cells isolated from the PLC/PRF/5 cell line. ZEB2-dependent ABCG2 gene expression and the effects of resveratrol on proliferation, cell cycle and apoptosis were explored in SNU398 cell clones. An inverse correlation between ZEB1/ZEB2 and ABCG2 levels were observed both in CSCs and in ZEB2-knock-down cells. The resveratrol treatment significantly decreased cell viability, while promoting cell cycle arrest in ZEB2-independent manner. Interestingly, resveratrol-treated cells with low levels of ZEB2 were resistant to apoptosis. The interplay of expression levels of ABCG2 and ZEB family EMT transcription factors may play a role in establishing CSC-like phenotype in HCC cells resistant to resveratrol.

BRCA1 (breast cancer 1) protein is involved in the genome stability maintenance participating in homologous recombination-dependent DNA repair. Disruption of BRCA1 functioning is associated with breast and ovarian cancer. Despite the important role of BRCA1 in DNA repair in all cell types, the development of BRCA1-associated cancer takes place mainly in estrogen-dependent tissues such as breast and ovarian ones. Using breast cancer cell line MCF-7 it was demonstrated in in vitro experiments that the estrogen 17β-estradiol (E2), phytoestrogens (genistein and apigenin) and antiestrogens (tamoxifen and fulvestrant) inhibited estrogen receptor (ERα) expression while only genistein influenced BRCA1 increasing its expression. In hypoxia, that is an important factor of solid tumors progression, the decrease of BRCA1 and ERα expression was demonstrated in MCF-7 cells. Therefore, hypoxia influences both BRCA1-dependent DNA repair and hormonal regulation of breast cancer cell growth. Taken together, obtained results demonstrate a relationship between BRCA1 and steroid hormones signal transduction pathways in breast cancer cells and point out to the importance of complex BRCA1 and ERa expression regulation mechanisms studies including epigenetic gene expression regulation.

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.

Selenium is an essential trace element, the deficiency of which leads to the development of several serious diseases, including male infertility, prostate cancer, etc. It has been shown that oxidative stress contributes to the progression of prostate cancer, and antioxidants such as selenium and vitamin E can significantly reduce the risk of this disease. Sodium selenite, one of the selenium compounds that induce the formation of reactive oxygen species, is considered as a potential anticancer agent. The SS concentrations that lead to a decrease in the viability of human prostate adenocarcinoma cells (line Du-145) have been selected, and the effect of sodium selenite on the expression of mRNA of the SELV, SELW, and TGR selenocysteine proteins in these cells has been analyzed.

Embryonic stem cells (ESCs) have the capacity for self-renewal and pluripotency. Due to high proliferative activity, ESCs use a specific pathway of the formation of ATP molecules, which can lead to the development of the adaptive metabolic response under the conditions of energy deficiency (which is different from the response of differentiated cells). It is known that metabolic signals are integrated with the cell cycle progression; however, the signaling pathways that connect the availability of nutrients with the regulation of cell cycle in ESCs are insufficiently studied. We have studied the effect of the AICAR agent, which imitates an increase in AMP level and induces the activation of the metabolic sensor AMPK, on proliferation, cell cycle distribution, and pluripotency of mouse ESCs (mESCs). It has been demonstrated that cells treated with AICAR do not stop at the control G1/S point of the cell cycle, since they do not accumulate P21/WAF1 (G1/S checkpoint regulator), despite P53 activation. On the contrary, AICAR increases the rate of mESC proliferation, which correlates with increased expression of pluripotency marker genes (OCT3/4, NANOG, SOX2, KLF4, ESRRB, PRDM14). In addition, an increase in the transcription of the HIFlα gene (a key regulator of the cell proliferation and viability, as well as glucose metabolism under stress) was detected. An increase in the expression of glycolytic enzyme genes (LDHA, ALDOA, PCK2, GLUT4) under the effect of AICAR indicates a change in mESC metabolism towards increased glycolysis. Thus, AICAR-dependent AMPK activation as one of possible mechanisms of the mESC adaptive response to the emergence of energetic imbalance is not accompanied by a cell cycle arrest at the G1/S checkpoint, but involves the processes of increasing glycolytic activity.

High metastatic ability and poor clinical outcome are the most known clinical features of the triple-negative breast tumors. Given that the tumor cells undergoing epithelial-mesenchymal transition (EMT) often gain malignant and invasive features, we have investigated the possibility of EMT reversal in triple-negative breast cancer cells by targeting the epigenetic-modifying enzymatic complexes named histone deacetylases (HDACs) and examined the possible mechanism underlying the HDACs-based inversion in model MDA-MB-231 cells. Cells were treated with a maximal tolerable 200 nM concentrations of classical HDACs inhibitor Trichostatin A (TSA) for 48 h and afterwards the invasiveness and immigration of the cells were evaluated in TransWell Invasion Scratch Wound Healing assays. Then, in treated and control cells, quantitative real time-PCR reacions were performed for assessing the gene expression of EMT biomarkers E-cadherin, Vimentin and transcriptional factor Slug. After TSA treatment, the invasion and migration properties MDA-MB-231 cells significantly decreased, gene expression of E-cadherin was significantly up-regulated, while the levels of Slug and Vimentin encoding mRNAs were suppressed. We conclude that inhibition of HDACs in triple-negative breast cancer cells may lead to inversion of EMT and the decrease of invasiveness by down-regulating the gene expression of Slug. Since EMT is known as a pre-metastatic process, triple-negative breast tumors, the EMT reversal effects of HDACs inhibition may reduce tumor cell metastasis.

Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 μg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.

Using real-time RT-PCR in combination with bioinformatics, we have shown for the first time that the treatment of HCT-116 and HT-29 colon cancer cells with two anti-cancer agents (doxycycline or 3,3'-diindolylmethane) results in profound changes in the intracellular content of several lncRNAs (by up to 100 times). Since many of these RNAs are secreted by tumors into the bloodstream, the obtained results provide a basis for developing more sensitive protocols for serological monitoring of tumor relapse and metastasis, as well as for search of new anti-cancer drugs.

MiR-21 is the most studied cancer-promoting oncomiR, which target numerous tumor suppressor genes associated with proliferation, apoptosis, and invasion. Here we have studied the synthesis of miR-21 and quantified the mRNA and protein levels for miR-21 potential target genes, i.e., Acat1, Armcx1, and Pten, in the livers of female Wistar rats after their treatment with either 1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) or benzo[a]pyrene (BP). The most important finding appears to be the significant decrease in the miR-21 level the day after treatment with DDT with subsequent rebound. These changes are accompanied by an increase and subsequent drop in the levels of mRNAs and proteins of the Acat1, Armcx1, and Pten genes. These observations indicate the involvement of miR-21 in the posttranscriptional regulation of the Acat1, Armcx1, and Pten genes in response to xenobiotics. We hypothesize that the toxic effects of xenobiotics may be indirect and may manifest by inducing epigenetic changes, particularly through the regulation of miRNAs and their target genes.

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

In murine bone-marrow stromal microenvironment cells and in human multipotent mesenchymal stromal cells (MMSCs), proinflammatory cytokine interleukin-1 beta (IL-1β) serves as a growth factor. In murine bone tissue, IL-1β expression increases in vivo after irradiation. Here, we have presented our evaluation of the effects of exogenous IL-1β on the expression of NF-kB transcription factors in human MMSCs and stromal layer cells of murine long-term bone marrow cultures (LTBMCs). The cytokine signaling pathway was also activated in murine LTBMC by braking electron radiation in doses of 3-12 Gy. The level of expression of genes that code for IL-1β, IL-1β type-I receptor and NF-kB and IKK protein families have been studied at different time points post exposure. In both human and murine stromal cells, exogenous IL-1β led to an increase in the level of expression of its own gene, while levels of expression of NF-kB and IKK gene families were not substantially changed. Nevertheless, in human cells, a significant correlation between levels of expression of IL-1β and all NF-kB family genes was detected. It points to a similarity in IL-1β signal pathways in mesenchymal and hematopoietic cells, where the posttranslational modifications of NF-kB transcription factors play a major role. The irradiation of murine LTBMC resulted in a transient increase in the expression of genes that code NF-kB transcription factors and IL-1β. These results indicate an important role of Rel, Rela, Relb, and Nfkb2 genes in the induction of IL-1β signal pathway in murine stromal cells. An increase in IL-1β expression after the irradiation of stromal cells may be related to both the induction of inflammation due to massive cell death and to a profound stimulation of the expression of this proinflammatory cytokine expression.

In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

Breast cancer is one of the most common cancers in women worldwide. Tamoxifen (TAM) provided a successful treatment for ER-positive (ER+) breast cancer for many years. However, ER+ patients with metastatic diseases respond poorly to TAM therapy and many initial responders eventually relapse. Emerging evidence indicates that long non-coding RNAs (lncRNAs) may have a critical role in the regulation of cellular processes such as cancer progression and metastasis, though the function of lncRNAs in TAM-resistance is still unclear. To investigate the role of CCAT2 in the development of resistance to TAM treatment of breast cancer, we established TAM-resistant models in MCF-7 and T47D cells. The present study demonstrates that TAM-resistant cells show a higher level of CCAT2 expression compared with TAM-sensitive cells. Biologically, CCAT2 knockdown could inhibit proliferation and induce apoptosis in TAM-resistant cells exposed to TAM. Furthermore, knockdown of CCAT2 improves the sensitivity to TAM in TAM-resistant cells. Overall, the study results provide a novel therapeutic approach for TAM-resistant patients through depleting CCAT2 expression.

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H(2)O(2)), IEC-6 cells were subjected to 20 μmol/L H(2)O(2) and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes.

Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment.

The ratio of the expression levels of the immediate early genes c-jun and c-fos that encode components of the AP-1 transcription complex determines the direction of changes in the expression of genes controlled by the complex, including changes induced by glucocorticoids. The aim of the present work was to assess the levels of mRNA encoded by genes c-jun and c-fos and the ratio of expression levels of these genes in various regions of the neonatal rat brain after the administration of dexamethasone, a selective ligand of the glucocorticoid receptor. The level of mRNA encoded by the immediate early gene c-fos in the hippocampus and prefrontal cortex of 3-day-old rat pups was elevated at 30, 60, and 120 min after dexamethasone administration. The basal level of c-fos gene expression in the brainstem was higher than in the cortex and hippocampus, and administration of the hormone was followed by a reduction in the amount of transcript detectable in the brainstem after 2 h. As a result, the ratio of c-jun to c-fos transcript levels in the brainstem of neonatal rats was doubled after dexamethasone administration. The dexamethasone-induced shift of the ratio of c-jun to c-fos transcript levels in the brainstem of neonatal rats towards a predominance of c-jun reported for the first time in the present work may induce the expression of genes that contain AP-1 response elements in the promoters, since the glucocorticoid receptor can be involved in protein-protein interactions with the Jun/Jun homodimer of the AP-1 complex.

Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

The following hypothesis has been proposed: IF an SNP can significantly increase the expression of an oncogene by increasing the affinity of the TATA-binding protein (TBP) to its promoter, THEN this SNP can also reduce the apparent bioactivity of inhibitors of this oncogene during antitumor chemotherapy and vice versa. In the context of this hypothesis, the previously proposed method (http://beehive.bionet.nsc. ru/cgi-bin/mgs/tatascan/start.pl) was applied to analyze all SNPs found within the [-70; -20] regions (which harbor all proven TBP-binding sites) of the promoters of VEGFA, EGFR, ERBB2, IGF1R, FLT1, KDR, and MET oncogenes according to the human reference genome, hg19. For 83% of these SNPs, their effect on TBP affinity to the oncogene promoters required for assembly of preinitiation complexes was not significant. rs36208385, rs36208384, rs370995111, rs372731987, rs111811434, rs369547510, rs76407893, rs369728300, and rs72001900 can potentially serve as SNP markers to reduce the apparent bioactivity of oncogene inhibitors, while rs141092704, rs184083669, rs145139616, rs200697953, rs187746433, rs199730913, rs377370642, rs114484350, rs374921120, rs146790957, rs376727645, and rs72001900 can be the markers for enhancing this activity.

The major problem in prostate cancer treatment is the development of drug resistance and especially important, cross-resistance. The mechanisms of drug resistance, which are divided into ligand-dependent (requiring the presence of androgens in the cell) and independent (not requiring the presence of androgens) are reviewed. The mechanisms are mainly represented with mutations of the androgen receptor and expression of aberrant constitutively active splice variants, as well as up-regulation of genes involved in androgens synthesis.

To evaluate the ameliorative effect of curcumin on dietary aflatoxin-induced changes in the expression of genes in Nile tilapia Oreochromis niloticus liver, the fish were fed with a diet contaminated by 200 ppb of atlatoxin B1 (AFB1) with and without curcumin (5 mg/kg diet) for 16 weeks in addition to a negative and positive controls fed with the basal diet and basal diet supplemented with curcumin, respectively. Further, two recovery groups with and without curcumin were tested after 2 more weeks. Relative mRNA expression of genes involved in antioxidant function (superoxide dismutase, SOD), biotransformation (cytochrome P4501A, CYPA) and immune response (interleukin-1β, IL-1β and transforming growth factor β, TGF-β) were assessed using RT-PCR. Also, fish weight gain and survival rate were determined. Results revealed that AFB1 significantly-reduced the survivability and weight gain, while curcumin inclusion improved them. Fish fed with AFB1-contaminated diet showed the up-regulation of CYP1A and down-regulation of SOD, IL-1β and TGF-β. This expression pattern was still evident in the recovery group without curcumin, but to a lesser extent. Supplementation of curcumin ameliorated the overall gene expression close to the control levels. It appears that curcumin exhibited protective effects on AFB1-induced liver toxicity in O. niloticus by moderating oxidative stress, toxin biotransformation, immune response, and hence growth performance.