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Objective. Homeobox genes play a fundamental role in the embryogenesis, but some of them have been linked to oncogenesis. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of homeobox genes such as PAX6 (paired box 6), PBX3 (PBX homeobox 3), PBXIP1 (PBX homeobox interacting protein 1), MEIS1 (MEIS homeobox 1), and MEIS2 in ERN1 knockdown U87 glioma cells with the intent to reveal the role of ERN1 (endoplasmic reticulum to nucleus signaling 1) signaling pathway on the endoplasmic reticulum stress dependent regulation of homeobox genes. Methods. The control (transfected by empty vector) and ERN1 knockdown (transfected by dominant-negative ERN1) U87 glioma cells were exposed to glucose and glutamine deprivations for 24 h. The cells RNA was extracted and reverse transcribed. The expression level of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes was evaluated by a real-time quantitative polymerase chain reaction analysis and normalized to ACTB. Results. It was found that glucose deprivation down-regulated the expression level of PAX6, MEIS1, and MEIS2 genes in control glioma cells, but did not significantly alter PBX3 and PBXIP1 genes expression. At the same time, ERN1 knockdown significantly modified the sensitivity of all studied genes to glucose deprivation. Other changes in gene expression were detected in control glioma cells under the glutamine deprivation. The expression of PBX3 and MEIS2 genes was down- while PAX6 and PBXIP1 genes up-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on the majority of studied genes expression in U87 glioma cells. Conclusion. The results of the present study demonstrate that the exposure of U87 glioma cells under glucose and glutamine deprivations affected the expression of the majority of the studied homeobox genes and that the sensitivity of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes expression under these experimental conditions is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling.

Objective. The aim of the present study was to investigate the expression of pyruvate dehydrogenase genes such as PDHA1, PDHB, DLAT, DLD, and PDHX in U87 glioma cells in response to glutamine and glucose deprivations in control glioma cells and endoplasmic reticulum to nucleus signaling 1 (ERN1) knockdown cells, the major endoplasmic reticulum (ER) stress signaling pathway, to find out whether there exists a possible dependence of these important regulatory genes expression on both glutamine and glucose supply as well as ERN1 signaling. Methods. The expression level of PDHA1, PDHB, DLAT, DLD, and PDHX genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by empty vector) and cells with inhibition of ERN1(transfected by dnERN1) after cells exposure to glucose and glutamine deprivations. Results. The data showed that the expression level of PDHA1, PDHB, DLAT, and DLD genes was down-regulated (more profound in PDHB gene) in control glioma cells treated with glutamine deprivation. At the same time, ERN1 knockdown modified the impact of glutamine deprivation on the expression level of all these genes in glioma cells: suppressed the sensitivity of PDHB and DLD genes expression and removed the impact of glutamine deprivation on the expression of PDHA1 and DLAT genes. Glucose deprivation did not significantly change the expression level of all studied genes in control glioma cells, but ERN1 knockdown is suppressed the impact of glucose deprivation on PDHX and DLD genes expression and significantly enhanced the expression of PDHA1 and PDHB genes. No significant changes were observed in the sensitivity of PDHX gene expression to glutamine deprivation neither in control nor ERN1 knock-down glioma cells. The knock-down of ERN1 removed the sensitivity of DLAT gene expression to glucose deprivation. Conclusion. The results of this investigation demonstrate that the exposure of control U87 glioma cells under glutamine deprivation significantly affected the expression of PDHA1, PDHB, DLAT, and DLD genes in a gene specific manner and that impact of glutamine deprivation was modified by inhibition of the ER stress signaling mediated by ERN1. At the same time, glucose deprivation affected the expression of PDHA1, PDHB, PDHX, and DLD genes in ERN1 knockdown glioma cells only. Thus, the expression of pyruvate dehydrogenase genes under glutamine and glucose deprivation conditions appears to be controlled by the ER stress signaling through ERN1.

Objective. Nanographene oxide, an oxidation derivative of graphene, is considered to be one of the nanomaterials attractive for biomedical applications, although this nanomaterial is toxic. The increasing exploitation of graphene-based materials necessitates a comprehensive evaluation of the potential impact of these materials on the human health. Moreover, it is necessary to investigate in detail the mechanisms of its toxic effect on living cells particularly at the genome level. The present study aimed to evaluate the impact of low doses of nanographene oxide on the expression of key regulatory genes in normal human astrocytes. Methods. Normal human astrocytes, line NHA/TS, were exposed to low doses of nanographene oxide (1 and 4 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of NAMPT, TSPAN13, BCAR3, BRCA1, PTGS2, P4HA1, and P4HA2 mRNAs as well as microRNAs were measured by quantitative polymerase chain reaction. Results. It was found that the low doses of nanographene oxide induced a dysregulation in the expression of the key regulatory genes in normal human astrocytes in dose-dependent (1 and 4 ng/ml) and gene-specific manner. Nanographene oxide also strongly suppressed the expression of NAMPT, BCAR3, and TSPAN13 genes and significantly up-regulated BRCA1, PTGS2, P4HA1, and P4HA2 ones with a more significant effect in P4HA1 and P4HA2 genes. The expression of miR-96-5p and miR-145-5p was also down-regulated in astrocytes treated with nanographene oxide in a dose-dependent manner. Conclusion. The data obtained demonstrate that the low doses of nanographene oxide disturbed the genome functions by changing the expression levels of key regulatory genes in gene-specific and dose-dependent manner. Moreover, a higher dose of nanographene oxide induced more pronounced changes in expression of genes indicating for both genotoxic and neurotoxic possible effects in the normal human astrocytes.

Objective. Single-walled carbon nanotubes (SWCNTs) are able to cross the blood-brain barrier, penetrate through the cell membrane, and accumulate in the cell nucleus, which purposefully allows their use in the health sciences as imaging probes and drug carriers in the cancer therapy. The aim of this study was to investigate the effect of low doses of SWCNTs on the expression of microRNAs associated with the cell proliferation and the brain development in zebrafish (Danio rerio) embryos. Methods. The zebrafish embryos (72 h post fertilization) were exposed to low doses of SWCNTs (2 and 8 ng/ml of medium) for 24 or 72 h. The microRNAs (miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206) expression levels were measured by quantitative polymerase chain reaction analysis. Results. It was found that low doses of SWCNTs elicited dysregulation in the expression of numerous cell proliferation and brain development-related microRNAs (miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206) in dose- (2 and 8 ng/ml of medium) as well as malformations in the zebrafish embryos brain development in a time-dependent (24 and 72 h) manner. Conclusion. Taken together, the present data indicate that the low doses of SWCNTs disturbed the genome functions and reduced the miR-19, miR-21, miR-96, miR-143, miR-145, miR-182, and miR-206 expression levels in dose- and time-dependent manners and interrupted the brain development in the zebrafish embryos indicating for both the genotoxic and the neurotoxic interventions.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

Background: Endoplasmic reticulum to nucleus signaling 1 (ERN1) is a major signaling pathway of endoplasmic reticulum stress and is crucial for malignant tumor growth. Objective: The article aims to discuss the recent progress in the discovery of endoplasmic reticulum stress targets and their involvement in tumor growth. Methods: Literature from the PubMed database related to the endoplasmic reticulum stress involvement in the tumor growth and chemoresistance was searched and reviewed. Results: The endoplasmic reticulum stress plays an important part in malignant tumor growth and is involved in invasion and metastasis. Inhibition of protein kinase and endoribonuclease activities of the ERN1 signaling protein significantly reduces tumor growth through down-regulation of angiogenesis and cell proliferation but activates the invasion. ERN1 knockdown affects the expression of many genes associated with the regulation of apoptosis, cell proliferation, and survival as well as reprograms the hypoxic regulation of most gene expressions. Simultaneously, inhibition of ERN1 endoribonuclease only has a stronger suppressive effect on tumor growth and decreases the invasiveness. Conclusion: Present review summarizes the recent advances in inhibiting ERN1 signaling that regulates tumor growth. Further understanding of the regulatory mechanisms of genome reprogramming upon inhibition of ERN1 signaling may help discover new possibilities for developing novel effective therapeutics.

Graphical abstract, Highlights • Low doses of SWCNTs affect the expression of numerous genes in normal astrocytes. • Low doses of SWCNTs interrupt the expression of various microRNAs in astrocytes. • SWCNTs affect the gene expressions through induction of endoplasmic reticulum stress. • Low doses of SWCNTs disturb the genome stability showing the genotoxic effects. • SWCNTs at low doses may have potential exposure-related risks for human health., The unique properties of single-walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the biomedical devices. They are able to cross the blood–brain barrier, enter cells and accumulate in cell nuclei. We studied the effect of these carbon nanoparticles on the expression of genes associated with endoplasmic reticulum stress and proliferation, cell viability and cancerogenesis as well as microRNAs in normal human astrocytes. We have shown that treatment of normal human astrocytes by small doses of SWCNTs (2 and 8 ng/ml of medium for 24 hrs) affect the expression of DNAJB9, IGFBP3, IGFBP6, CLU, ZNF395, KRT18, GJA1, HILPDA, and MEST mRNAs as well as several miRNAs, which have binding sites at 3′-UTR of these mRNAs. These changes in the expression profile of individual mRNAs introduced by SWCNTs are dissimilar in magnitude and direction and are the result of both transcriptional and posttranscriptional mechanisms of regulation. It is possible that these changes in gene expressions are mediated by the endoplasmic reticulum stress introduced by carbon nanotubes and reflect the disturbance of the genome stability. In conclusion, the low doses of SWCNTs disrupt the functional integrity of the genome and possibly exhibit a genotoxic effect.

Objective. The aim of the present investigation was to study the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other functionally active proteins in U87 glioma cells under silencing of polyfunctional chaperone HSPB8 for evaluation of the possible significance of this protein in intergenic interactions. Methods. Silencing of HSPB8 mRNA was introduced by HSPB8 specific siRNA. The expression level of HSPB8, IRS1, HK2, GLO1, HOMER3, MYL9, NAMPT, PER2, PERP, GADD45A, and DEK genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Results. It was shown that silencing of HSPB8 mRNA by specific to HSPB8 siRNA led to a strong down-regulation of this mRNA and significant modification of the expression of IRS1 and many other genes in glioma cells: strong up-regulated of HOMER3, GLO1, and PERP and down-regulated of MYL9, NAMPT, PER2, GADD45A, and DEK gene expressions. At the same time, no significant changes were detected in the expression of HK2 gene in glioma cells treated by siRNA, specific to HSPB8. Moreover, the silencing of HSPB8 mRNA enhanced the glioma cells proliferation rate. Conclusions. Results of this investigation demonstrated that silencing of HSPB8 mRNA affected the expression of IRS1 gene as well as many other genes encoding tumor growth related proteins. It is possible that the dysregulation of most of the studied genes in glioma cells after silencing of HSPB8 is reflected by a complex of intergenic interactions and that this polyfunctional chaperone is an important factor for the stability of genome function and regulatory mechanisms contributing to the tumorigenesis control.

Objective. The aim of the present investigation was to study the expression of genes encoding homeobox proteins ZEB2 (zinc finger E-box binding homeobox 2), TGIF1 (TGFB induced factor homeobox 1), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, LHX6, NKX3-1 (NK3 homeobox 1), and PRRX1 (paired related homeobox 1) in U87 glioma cells in response to glucose deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of it possible significance in the control of glioma growth through ERN1 signaling and chemoresistance. Methods. The expression level of homeobox family genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation condition by real-time quantitative polymerase chain reaction. Results. It was shown that the expression level of ZEB2, TGIF1, PRRX1, and LHX6 genes was up-regulated in control glioma cells treated by glucose deprivation. At the same time, the expression level of three other genes (NKX3-1, LHX1, and LHX2) was down-regulated. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation condition on the expression almost all studied genes. Thus, treatment of glioma cells without ERN1 enzymatic activity by glucose deprivation condition lead to down-regulation of the expression level of ZEB2 and SPAG4 as well as to more significant up-regulation of PRRX1 and TGIF1 genes. Moreover, the expression of LHX6 and NKX3-1 genes lost their sensitivity to glucose deprivation but LHX1 and LHX2 genes did not change it significantly. Conclusions. The results of this investigation demonstrate that ERN1 knockdown significantly modifies the sensitivity of most studied homeobox gene expressions to glucose deprivation condition and that these changes are a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to glioma cell growth and possibly to their chemoresistance.

Objective. The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations. Methods. The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction. Results. It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3’-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells. Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.

Objective. The aim of the present study was to investigate the effect of adipokine NAMPT (nicotinamide phosphoribosyltransferase) silencing on the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other proliferation related proteins in U87 glioma cells for evaluation of the possible significance of this adipokine in intergenic interactions. Methods. The silencing of NAMPT mRNA was introduced by NAMPT specific siRNA. The expression level of NAMPT, IGFBP3, IRS1, HK2, PER2, CLU, BNIP3, TPD52, GADD45A, and MKI67 genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Anti-visfatin antibody was used for detection of NAMPT protein by Western-blot analysis. Results. It was shown that the silencing of NAMPT mRNA led to a strong down-regulation of NAMPT protein and significant modification of the expression of IRS1, IGFBP3, CLU, HK2, BNIP3, and MKI67 genes in glioma cells and a strong up-regulation of IGFBP3 and IRS1 and down-regulation of CLU, BNIP3, HK2, and MKI67 gene expressions. At the same time, no significant changes were detected in the expression of GADD45A, PER2, and TPD52 genes in glioma cells treated by siRNA specific to NAMPT. Furthermore, the silencing of NAMPT mRNA suppressed the glioma cell proliferation. Conclusions. Results of this investigation demonstrated that silencing of NAMPT mRNA with corresponding down-regulation of NAMPT protein and suppression of the glioma cell proliferation affected the expression of IRS1 gene as well as many other genes encoding the proliferation related proteins. It is possible that dysregulation of most of the studied genes in glioma cells after silencing of NAMPT is reflected by a complex of intergenic interactions and that NAMPT is an important factor for genome stability and regulatory mechanisms contributing to the control of glioma cell metabolism and proliferation.

Objective. The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation. Methods. The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction. Results. It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition. Conclusions. Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.

Objective. The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia. Methods. The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction. Results. It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells. Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.

Numerous data demonstrate that key regulatory factors, enzymes and receptors including HSPA5, MEST, SLC1A3, PDGFC, and ADM represent poly-functional, endoplasmic reticulum stress-dependent proteins, which control variable metabolic pathways. The expression level of genes of these proteins in the blood and subcutaneous adipose tissue of obese adolescents and adult men with and without insulin resistance was studied. It was shown that in blood of obese adolescents without insulin resistance the expression level of SLC1A3, HSPA5, MEST, and PDGFC genes was significantly increased, but development of insulin resis­tance led to down-regulation of these genes expression except HSPA5 gene as compared to the control group as well as to the group of obese adolescents without insulin resistance. At the same time, the expression level of ADM gene did not change significantly in obese adolescents without insulin resistance, but the development of insulin resistance led to down-regulation of this gene expression. In subcutaneous adipose tissue of obese adult men without insulin resistance the level of SLC1A3 gene expression was decreased, although ADM, MEST, and HSPA5 genes – increased. It was also shown that the development of insulin resistance in obese men affected the expression level of ADM and SLC1A3 genes only. Results of this investigation provide evidence that insulin resistance in obese adolescents and adult men is associated with specific changes in the expression of genes, which related to proliferation and development of obesity and insulin resistance as well as to endoplasmic reticulum stress and contribute to the development of obesity complications.

We have studied the effect of glutamine deprivation on the expression of genes encoding for ubiquitin specific peptidases (USP) and ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ATG7) in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1). It was shown that exposure of control glioma cells (transfected by empty vector) upon glutamine deprivation led to suppression of USP1 and ATG7 mRNA expression and up-regulated USP25 mRNA. At the same time, glutamine deprivation did not significantly change USP4, USP10, USP14, and USP22 gene expressions in these cells. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells increased effect of glutamine deprivation on the expression of USP1 gene and introduced sensitivity of USP4 and USP14 genes to this condition. Therefore, glutamine deprivation affected the expression level of most studied genes in gene specific manner in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth.

We have studied the effect of IRE1 signaling enzyme knockdown as well as hypoxia on the expression of genes encoding the important tumor growth related proteins (BRCA1, DEK, BCL2L1, COL6A1, TPD52, HOMER3, and GNPDA1) in U87 glioma cells. It was shown that the expression level of breast cancer 1 early onset (BRCA1) and tumor protein D52 (TPD52) mRNAs are strongly up-regulated in U87 glioma cells by down-regulation of IRE1 expression in comparison with the control cells. At the same time the expression level of collagen, type VI, alpha 1 (COL6A1), DEK oncogene (DEK), glucosamine-6-phosphate deaminase 1 (GNPDA1) and homer homolog 3 (HOMER3) was significantly down-regulated in glioma cells under these experimental conditions. It was also shown that hypoxia up-regulated the expression level of COL6A1 and TPD52 mRNAs and down-regulated – BRCA1, DEK, and GNPDA1 mRNAs in control glioma cells and that down-regulation of IRE1, which control cell proliferation and tumor growth, modified the effect of hypoxia on the expression of COL6A1, DEK, BCL2L1, HOMER3, and GNPDA1 genes. The present study demonstrated that hypoxia affected the expression of most studied genes in IRE1-dependent manner.

Objective. The aim of the present study was to examine the effect of chromium disilicide and titanium nitride nanoparticles on the expression level of genes encoding important regulatory factors (IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK/NUAK2, CD36, and PECAM1/CD31) in mouse liver for evaluation of possible toxic effects of these nanoparticles. Methods. Male mice received 20 mg chromium disilicide nanoparticles (45 nm) and titanium nitride nanoparticles (20 nm) with food every working day for 2 months. The expression of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver was studied by quantitative polymerase chain reaction. Results. Treatment of mice with chromium disilicide nanoparticles led to down-regulation of the expression of IGFBP2, IGFBP5, PECAM1, and SNARK genes in the liver in comparison with control mice, with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3 and CD36 genes was increased in mouse liver upon treatment with chromium disilicide nanoparticles. We have also shown that treatment with titanium nitride nanoparticles resulted in down-regulation of the expression of IGFBP2 and SNARK genes in the liver with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3, IGFBP4, and CD36 genes was increased in the liver of mice treated with titanium nitride nanoparticles. Furthermore, the effect of chromium disilicide nanoparticles on IGFBP2 and CD36 genes expression was significantly stronger as compared to titanium nitride nanoparticles. Conclusions. The results of this study demonstrate that chromium disilicide and titanium nitride nanoparticles have variable effects on the expression of IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver, which may reflect the genotoxic activities of the studied nanoparticles.

We have studied the effect of chromium disilicide and titanium nitride nanoparticles on the expression level of genes encoding important regulatory enzymes and factors (NAMPT, UPS7, E2F8, FAS/TNFSF6, TBX3, and IL13RA2) in mouse liver for evaluation of possible toxic effects of these nanoparticles. It was shown that treatment of mice by titanium nitride nanoparticles (20 nm; 20 mg with food every working day for 2 months) led to up-regulation of the expression of NAMPT, FAS, TBX3, and IL13RA2 genes and to down-regulation of USP7 and E2F8 genes in the liver tissue. Changes for TBX3 and IL13RA2 genes were more significant than for other genes. Furthermore, treatment of mice by chromium disilicide nanoparticles (45 nm; 20 mg with food every working day for 2 months) led to more significant changes in the expression of USP7, E2F8, FAS, and TBX3 genes in comparison to the effect of titanium nitride nanoparticles. At the same time, effect of titanium nitride nanoparticles on the expression of NAMPT gene in the liver tissue was stronger as compared to chromium disilicide nanoparticles. Additionally, treatment of mice by chromium disilicide nanoparticles did not change significantly the expression of IL13RA2 gene in the liver. The present study demonstrates that chromium disilicide and titanium nitride nanoparticles had variable effects on the expression of most studied genes in a gene specific manner, which possibly reflect genotoxic activities of studied nanoparticles, but molecular mechanisms of observed changes in gene expressions warrant further investigation.

We have studied the effect of hypoxia on the expression of genes encoding for LONP1/PRSS15 and cathepsins in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1). It was shown that hypoxia up-regulated the expression of CTSA, CTSB, CTSD, CTSF, CTSK, and LONP1 genes and down-regulated the expression of CTSC, CTSL, CTSO, and CTSS genes in control glioma cells (transfected by empty vector). Inhibition of ІRE1 signaling enzyme function in these glioma cells modified effect of hypoxia on the expression of most studied genes: eliminated the effect of hypoxia on CTSA, and LONP1 genes, introduced reverse changes on CTSD and CTSS genes, reduced – on CTSF and CTSK genes, and enhanced – on CTSB and CTSL genes. Therefore, hypoxia affected the expression level of most studied genes in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which control cell proliferation and tumor growth.

We have studied the effect of inhibition of IRE1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and a controller of cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in U87 glioma cells. It was shown that hypoxia leads to up-regulation of the expression of IL13RA2, CD24, ING1, ING2, ENDOG, and POLG genes and to down-regulation – of KRT18, TRAPPC3, TSFM, and MTIF2 genes at the mRNA level in control glioma cells. Changes for ING1 and CD24 genes were more significant. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes. In particular, it increases sensitivity to hypoxia of the expression of IL13RA2, TRAPPC3, ENDOG, and PLOG genes and suppresses the effect of hypoxia on the expression of ING1 gene. Additionally, it eliminates hypoxic regulation of KRT18, CD24, ING2, TSFM, and MTIF2 genes expressions and introduces sensitivity to hypoxia of the expression of BET1 gene in glioma cells. The present study demonstrates that hypoxia, which often contributes to tumor growth, affects the expression of almost all studied genes. Additionally, inhibition of IRE1 can both enhance and suppress the hypoxic regulation of these gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation must be further clarified.

Objective. The development of obesity and its metabolic complications is associated with dys-regulation of various intrinsic mechanisms, which control basic metabolic processes via changes in the expression of numerous regulatory genes. The main goal of this work was to study the association between the expression of insulin-like growth factors (IGF1 and IGF2) and IGF-binding proteins and insulin resistance in obese adolescents for evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression of IGF1, IGF2, and IGFBPs mRNA was measured in blood of obese adolescents with normal insulin sensitivity and insulin resistance in comparison with the normal (control) individuals. Results. In the blood of obese adolescents with normal insulin sensitivity the expression of IGFBP4, IGFBP5 and HTRA1 genes was down-regulated, but IGFBP2 and IGFBP7 genes up-regulated as compared to control (normal) group. At the same time, no significant changes in IGF1 and IGF2 gene expressions in this group of obese adolescents were found. Insulin resistance in obese adolescents led to up-regulation of IGF2, IGFBP2, and IGFBP7 gene expressions as well as to down-regulation of the expression of IGF1, IGFBP5 and HTRA1 genes in the blood in comparison with the obese patients, which have normal insulin sensitivity. Furthermore, the level of IGFBP4 gene expression was similar in both groups of obese adolescents. Conclusions. Results of this investigation provide evidence that insulin resistance in obese adolescents is associated with gene specific changes in the expression of IGF1, IGF2, IGFBP2, IGFBP5, IGFBP7, and HTRA1 genes and these changes possibly contribute to the development of glucose intolerance and insulin resistance.

We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase 2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment. Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.

We investigated the impact of IRE1/ERN1 (inositol requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) knockdown on hypoxic regulation of the expression of a subset of proliferation and migration-related genes in U87 glioma cells. It was shown that hypoxia leads to up-regulation of the expression of MEST and SNAI2, to down-regulation – of MYBL1, TCF8 and GTF2F2 genes at the mRNA level in control glioma cells. At the same time hypoxia did not affect the expression of TCF3 and GTF2B transcription factor genes. In turn, inhibition of IRE1 modified the effect of hypoxia on the expression of all studied genes, except MYBL1 and GTF2B. For instance, IRE1 knockdown decreased sensitivity to hypoxia of the expression of MEST, TCF8 and SNAI2 genes and increased sensitivity to hypoxia of GTF2F2 expression. At the same time, IRE1 inhibition introduced sensitivity to hypoxia of the expression of TCF3 gene in glioma cells. The present study demonstrated that the inhibition of IRE1 in glioma cells affected the hypoxic regulation of the expression of studied genes in various directions, though hypoxic conditions did not abolish the effect of IRE1 inhibition on the expression of respective genes. To the contrary, in case of SNAI2, GTF2F2 and MEST hypoxic conditions magnified the effect of IRE1 inhibition on the expression of respective genes in glioma cells.

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

We have studied gene expression of insulin-like growth factor binding proteins in U87 glioma cells upon glutamine deprivation depending on the inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress. We have shown that exposure of control glioma cells upon glutamine deprivation leads to down-regulation of NOV/IGFBP9, WISP1 and WISP2 gene expressions and up-regulation of CYR61/IGFBP10 gene expression at the mRNA level. At the same time, the expression of IGFBP6 and IGFBP7 genes in control glioma cells was resistant to glutamine deprivation. It was also shown that the inhibition of IRE1 modifies the effect of glutamine deprivation on the expression of all studied genes. Thus, the inhibition of IRE1 signaling enzyme enhances the effect of glutamine deprivation on the expression of CYR61 and WISP1 genes and suppresses effect of the deprivation on WISP2 gene expression in glioma cells. Moreover, the inhibition of IRE1 introduces sensitivity of the expression of IGFBP6 and IGFBP7 genes to glutamine deprivation and removes this sensitivity to NOV gene. We have also demonstrated that the expression of all studied genes in glioma cells growing with glutamine is regulated by IRE1 signaling enzyme, because the inhibition of IRE1 significantly down-regulates IGFBP6 and NOV genes and up-regulates IGFBP7, CYR61, WISP1, and WISP2 genes as compared to control glioma cells. The present study demonstrates that glutamine deprivation condition affects most studied IGFBP and WISP gene expressions in relation to IRE1 signaling enzyme function and possibly contributes to slower glioma cell proliferation upon inhibition of IRE1.

We have studied hypoxic regulation of the expression of genes encoded GADD (growth arrest and DNA damage) family proteins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. We have shown that hypoxia up-regulates the expression of GADD34, GADD45A, GADD45B, and GADD153 genes, which are related to cell proliferation and apoptosis, in control (transfected by empty vector) glioma cells in gene specific manner. At the same time, the expression level of EIF2AK 1 (eukaryotic translation initiation factor 2-alpha kinase 1) and AI FM1 (apoptosis inducing factor, mitochondria associated 1) genes in these cells is down-regulated upon hypoxic condition. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells enhances the effect of hypoxia on these genes expression, except EIF2AK 1 and AI FM1 genes. Furthermore, the expression of all studied genes in ІRE1 knockdown cells is significantly decreased upon normoxic condition, except GADD45B gene, which expression level is strongly up-regulated. Therefore, the expression level of genes encoding GADD34, GADD45A, GADD45B, GADD153, EIF2AK 1, and AI FM1 is affected by hypoxia and by inhibition of IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner and correlates with suppression of glioma cell proliferation upon inhibition of the IRE1 enzyme function.

Objective. The aim of the present study was to investigate the effect of hypoxia on the expression of genes encoding insulin-like growth factors (IGF1 and IGF2), their receptor (IGF1R), binding protein-4 (IGFBP4), and stanniocalcin 2 (STC2) in U87 glioma cells in relation to inhibition of endoplasmic reticulum stress signaling mediated by IRE1 (inositol requiring enzyme 1) for evaluation of their possible significance in the control of tumor growth. Methods. The expression of IGF1, IGF2, IGF1R, IGFBP4, and STC2 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia was studied by qPCR. Results. The expression of IGF1 and IGF2 genes is down-regulated in glioma cells without IRE1 signaling enzyme function in comparison with the control cells. At the same time, the expression of IGF1R, IGFBP4, and STC2 genes was up-regulated in glioma cells upon inhibition of IRE1, with more significant changes for IGFBP4 and STC2 genes. We also showed that hypoxia does not change significantly the expression of IGF1, IGF2, and IGF1R genes but up-regulated IGFBP4 and STC2 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells does not change significantly the effect of hypoxia on the expression of IGF1, IGF1R, and IGFBP4 genes but introduces sensitivity of IGF2 gene to hypoxic condition. Thus, the expression of IGF2 gene is resistant to hypoxia only in control glioma cells and significantly down-regulated in cells without functional activity of IRE1 signaling enzyme, which is central mediator of the unfolded protein response and an important component of the tumor growth as well as metabolic diseases. Conclusions. Results of this study demonstrate that the expression of IGF1 and IGF1R genes is resistant to hypoxic condition both in control U87 glioma cells and cells without IRE1 signaling enzyme function. However, hypoxia significantly up-regulates the expression of IGFBP4 gene independently on the inhibition of IRE1 enzyme. These data show that proteins encoded by these genes are resistant to hypoxia except IGFBP4 and participate in the regulation of metabolic and proliferative processes through IRE1 signaling.

The effect of single-walled carbon nanotubes (SWCNTs) on the expression level of several genes, related to cell cycle control, cell proliferation and migration in human glioma cell line U87 was investigated. The exposure of human U87 glioma cells to single-walled carbon nanotubes in different concentrations during 24 of 48 h affects the expression of CCND2 (Cyclin D2), DTNA (Dystrobrevin alpha), PARVB (parvin beta), DUSP1 (dual specificity phosphatase 1), PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3) and PFKFB4 genes at mRNA level, as well as the expression of alternative splice variants of DTNA, PFKFB3 and PFKFB4 transcripts. In case of CCND2 gene a strong, time and dose-dependent suppressive effect was observed. The effect of single-walled carbon nanotubes on the expression level of PFKFB3, PFKFB4, and PARVB was also suppressive but less prominent. At the same time, the expression of DUSP1 and DTNA genes is up-regulated in cells treated by single-walled carbon nanotubes. Thus, single-walled carbon nanotubes exert anti-proliferative properties in U87 glioma cells, as they down-regulate the expression of proliferation-related genes (CCND2, PARVB, PFKFB3, and PFKFB4) and up-regulate DUSP1 and DTNA genes, which control apoptosis.

Obesity and its metabolic complications are one of the most profound public health problems and result from interactions between genes and environmental. The development of obesity is tightly connected with dysregulation of intrinsic gene expression mechanisms controlling majority of metabolic processes, which are essential for regulation many physiological functions, including insulin sensitivity, cellular proliferation and angiogenesis. Our objective was to evaluate if expression of angiogenesis related genes VEGF-A, CYR61, PDGFC, FGF1, FGF2, FGFR2, FGFRL1, E2F8, BAI2, HIF1A, and EPAS1 at mRNA level in adipose tissue could participate in the development of obesity and metabolic complications. We have shown that expression level of VEGF-A, PDGFC, FGF2, and FGFRL1 genes is decreased in adipose tissue of obese men with normal glucose tolerance (NGT) versus a group of control subjects. At the same time, in this group of obese individuals a significant up-regulation of CYR61, FGF1, FGFR2, E2F8, BAI2, and HIF1A gene expressions was observed. Impaired glucose tolerance (IGT) in obese patients associates with down-regulation of CYR61 and FGFR2 mRNA and up-regulations of E2F8, FGF1, FGF2, VEGF-A and its splice variant 189 mRNA expressions in adipose tissue versus obese (NGT) individuals. Thus, our data demonstrate that the expression of almost all studied genes is affected in subcutaneous adipose tissue of obese individuals with NGT and that glucose intolerance is associated with gene-specific changes in the expression of E2F8, FGF1, FGF2, VEGF-A, CYR61 and FGFR2 mRNAs. The data presented here provides evidence that VEGF-A, CYR61, PDGFC, FGF1, FGF2, FGFR2, FGFRL1, E2F8, BAI2, and HIF1A genes are possibly involved in the development of obesity and its complications.

The effect of single-walled carbon nanotubes (SWCNTs) on the expression of a subset of immune response, apoptosis and cell proliferation –associated genes was studied in normal human astrocytes (line NHA/TS). In the cells treated with SWCNTs (2, 10 and 50 ng/ml of medium for 24 h) we observed a strong dose-dependent down-regulation of the expression of a cell surface glycoproteins HLA-DRA (major histocompatibility complex, class II, DR alpha) and HLA-DRB1. At the same time, the expression of HLA-F (major histocompatibility complex, class I, F), LMNB1 (lamin B1), and HTRA1 (high temperature requirement A1) genes as well as the level of miR-190b and miR-7 was up-regulated in NHA/TS subjected to different concentrations of SWCNTs. After 24 h of treatment with SWCNTs we detected a dose-dependent suppression of PHLDA2 (pleckstrin homology-like domain, family A, member 2) gene expression in these cells. Obtained data show that SWCNTs may affect an immune response, in particular through suppression of HLA-DRA and HLA-DRB1 gene expressions and that miR-190b and miR-7 possibly participated in this suppression. Deregulation of lamin B1 expression indicates the possibility of alterations in genome stability following treatment of astrocytes with SWCNTs. Thus, more caution is needed in biomedical application of SWCNTs.

We have studied hypoxic regulation of the expression of different insulin-like growth factor binding­ protein genes in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth. We have de­monstrated that hypoxia leads to up-regulation of the expression of IGFBP6, IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulation – of IGFBP9/NOV gene at the mRNA level in control glioma cells, being more significant changes for IGFBP10/CYR61 and WISP2 genes. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes: eliminates sensitivity to hypoxia the expression of IGFBP7 and IGFBP9/NOV genes, suppresses effect of hypoxia on IGFBP6, IGFBP10/CYR61, and WISP2 genes, and slightly enhances hypoxic regulation of WISP1 gene expression in glioma cells. We have also demonstrated that the expression of all studied genes in glioma cells is regulated by IRE1 signaling enzyme upon normoxic condition, because inhibition of IRE1 significantly up-regulates IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulates IGFBP6 and IGFBP9/NOV genes as compared to control glioma cells. The present study demonstrates that hypoxia, which contributes to tumor growth, affects all studied IGFBP and WISP gene expressions and that inhibition of IRE1 preferentially abolishes or suppresses the hypoxic regulation of these gene expressions and thus possibly contributes to slower glioma growth. Moreover, inhibition of IRE1, which correlates with suppression of cell proliferation and glioma growth, is down-regulated expression of pro-proliferative IGFBP genes, attesting to the fact that endoplasmic reticulum stress is a necessary component of malignant tumor growth.

Hypoxia as well as the endoplasmic reticulum stress are important factors of malignant tumor growth and control of the expression of genes, which regulate numerous metabolic processes and cell proliferation. Furthermore, blockade of ERN1 (endoplasmic reticulum to nucleus 1) suppresses cell proliferation and tumor growth. We studied the effect of hypoxia on the expression of genes encoding the transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F), and HOXC6 (homeobox C6) in U87 glioma cells with and without ERN1 signaling enzyme function. We have established that hypoxia enhances the expression of HOXC6, E2F8, ATF3, and EPAS1 genes but does not change TBX3 and FOXF1 gene expression in glioma cells with ERNI function. At the same time, the expression level of all studied genes is strongly decreased, except for TBX3 gene, in glioma cells without ERN1 function. Moreover, the inhibition of ERN1 signaling enzyme function significantly modifies the effect of hypoxia on the expression of these transcription factor genes. removes or introduces this regulation as well as changes a direction or magnitude of hypoxic regulation. Present study demonstrates that fine-tuning of the expression of proliferation related genes depends upon hypoxia and ERN1-mediated endoplasmic reticulum stress signaling and correlates with slower proliferation rate of glioma cells without ERN1 function.

Objective The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells. Methods The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation. Results The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes. Conclusions The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that inhibition of IRE1 signaling enzyme function may significantly affect the expression of all studied genes in the presence of glucose as well as modify the effect of glucose deprivation on the expression of most studied genes. These data also show that proteins encoded by these genes may participate in the regulation of metabolic and proliferative processes via IGF/INS receptors and possibly other signaling pathways as well, via IRE1 signaling, which is a central mediator of the unfolded protein response and an important component of the tumor growth and metabolic diseases.

We have studied the effect of hypoxia on the expression level of mRNA of the basic enzymes of pentose-phosphate cycle (G6PD, TKT, TALDO1, PGLS and RPIA) and glucose-6-phosphate isomerase (GPI) in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme 1). It was shown that hypoxia leads to up-regulation of the expression of GPI and PGLS genes and to down-regulation of TALDO1 and RPIA genes in control glioma cells. Changes for GPI gene were more significant than for other genes. At the same time, inhibition of IRE1 modified the effect of hypoxia on the expression of all studied genes. In particular, it increased sensitivity to hypoxia of G6PD and TKT genes expression and suppressed the effect of hypoxia on the expression of GPI and RPIA genes. Additionally, inhibition of IRE1 eliminated hypoxic regulation of PGLS gene and did not change significantly effect of hypoxia on the expression of TALDO1 gene in glioma cells. Present study demonstrated that hypoxia, which often contributes to tumor growth, affects the expression of most studied genes and inhibition of IRE1 modified the hypoxic regulation of pentose-phosphate cycle gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation warrant further investigation.

Tumor necrosis factor (TNF) superfamily receptors and TNF apoptosis inducing ligands play an important role in the realization of TNF function and control tumor growth. The TNF-related pathways are controlled by endoplasmic reticulum stress signaling, which has a crucial role in the control of cell proliferation and tumor growth. Furthermore, the inhibition of IRE1 (inositol requiring enzyme-1), which is a central mediator of endoplasmic reticulum stress sand mainly responsible for cell proliferation and apoptosis, leads to suppression of tumor growth through specific changes in the expression of genes encoding transcription factors, tumor suppressors, angiogenesis and apoptosis related proteins, including TNF superfamily receptors and TNF apoptosis inducing ligands. Therefore, changes in the expression level of TNF-related genes encoding TNF superfamily receptors and apoptosis inducing ligands possibly reflect metabolic reprogramming of cancer cells upon inhibition of IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation.

Activation of pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growth. Phosphoribosyl pyrophosphate synthetase (PRPS) is a key enzyme of this pathway and plays a central role in the synthesis of purines and pyrimidines. Hypoxia as well as ERN1 (from endoplasmic reticulum to nuclei-1) mediated endoplasmic reticulum stress response-signalling pathway is linked to the proliferation because the blockade of ERN1 suppresses tumor growth, including glioma. We studied the expression of different PRPS genes in glioma cells with ERN1 knockdown under hypoxic condition. It was shown that hypoxia decreases the expression of PRPS1 and PRPS2 genes in both types of glioma cells, being more pronounced in cells without ERN1 function, but PRPSAP1 and PRPSAP2 gene expressions are suppressed by hypoxia only in glioma cells with blockade of ERN1. Moreover, the blockade of endoribonuclease activity of ERN1 does not affect the expression of PRPS1 and PRPS2 as well as PPRS-associated protein genes in U87 glioma cells. At the same time, the induction of endoplasmic reticulum stress by tunicamycin in glioma cells with suppressed activity of ERN1 endoribonuclease decreases the expression level of PRPS1 and PRPS2 genes only. Results of this investigation clearly demonstrated that the expression of different genes encoding subunits of PRPS enzyme is affected by hypoxia in U87 glioma cells, but the effect of hypoxia is modified by suppression of endoplasmic reticulum stress signaling enzyme ERN1.

The endoplasmic reticulum stress is an important factor of tumor growth and is induced in cancer cells. We have studied the effect of ERN1 knockdown as well as hypoxia on the expression of genes encoding­ factors, which control cell proliferation, in U87 glioma cells. It was shown that the complete blockade of ERN1 enzyme function leads to an increase of the PLAT (tissue plasminogen activator), CCN2 (CCN family member 2), and ITGB1 (integrin β-1) as well as to a decrease of PLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), and SLURP1 (secreted LY6/PLAUR domain containing 1) mRNA expressions. Moreover, we have shown that hypoxia does not affect the expression level of ITGB1 mRNA, but increases that of CCN2, PLAUR, SLURP1, and PLAT mRNA and decreases the expression level of only PLAU mRNA in control glioma cells. At the same time, in ERN1 knockdown glioma cells the expression level of PLAU, PLAUR, and SLURP1 mRNA is decreased under hypoxia, but PLAT and ITGB1 mRNA expression levels are increased under these experimental conditions. Thus, results of this study have shown that the expression level of all studied genes is affected by ERN1 knockdown as well as by hypoxia and that the effect of hypoxia mostly depends on ERN1 signaling enzyme function.

Endoplasmic reticulum stress and hypoxia are necessary components of malignant tumors growth and suppression of ERN1 (from endoplasmic reticulum to nuclei-1) signalling pathway, which is linked to the apoptosis and cell death processes, significantly decreases proliferative processes. Glioma cells with ERN1 knockdown were used in order to investigate the effect of ERN1 blockade on the expression of TP53, MDM2, PERP, and USP7 genes and its hypoxic regulation. We have studied the expression of TP53 (tumor protein 53), MDM2 (TP53 E3 ubiquitin protein ligase homolog), PERP (TP53 apoptosis effector), and USP7 (ubiquitin specific peptidase 7) genes, which are related to cell proliferation and apoptosis, in glioma cells with ERN1 knockdown under hypoxic condition. It was shown that blockade of ERN1 gene function in U87 glioma cells intensified the expression of TP53 and USP7 genes, but decreased the expression of MDM2 and PERP genes. Thus, an enhanced expression of TP53 gene in ERN1 knockdown glioma cells correlates with the decreased level of ubiquitin ligase MDM2 and increased expression level of USP7 which deubiquitinates TP53 and MDM2 and induces TP53-dependent cell growth repression and apoptosis. At the same time, the expression levels of TP53, MDM2, and USP7 genes do not change significantly in glioma cells with suppression of endoribonuclease activity only, but PERP gene expression is strongly increased. Moreover, the expression of TP53 and UPS7 genes is decreased in hypoxic conditions in control glioma cells only; however, MDM2 and PERP gene expressions are increased in both cell types, being more significant in ERN1 knockdown cells. Thus, the expression of genes encoding TP53 and related to TP53 factors depends upon the endoplasmic reticulum stress signaling as well as on hypoxia, and correlates with suppression of glioma growth under ERN1 knockdown.

We have studied the effect of acute L-glutamine deprivation on the expression oftumor protein 53 (TP53)-related genes such as TOPORS (topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein ligase), TP53BPI (TP53 bindingprotein 1), TP53TG1 (TP53 inducible gene 1), SESN1 (p53 regulatedPA26 nuclear protein), NME6 (NME/NM23 nucleoside diphosphate kinase 6), and ZMAT3 (zinc finger Matrin-type 3) in glioma cells with ERN1 knockdown. It was shown that blockade of ERN1 finction in U87 glioma cells is induced the expression of RYBP and SESN1 genes, but decreased the expression of TP53BP1, TP53TG1, TOPORS, NME6, genes. Moreover, the expression levels ofRYBPI SESN1, TP53BP1, and ZMAT3 genes is increased in control glioma cells by L-glutamine deprivation condition but blockade of ERN1 signaling enzyme function significantly enhances this effect on the expression all of these genes. At the same time, the ERN1 knockdown eliminates the response TP53TG1 and TOPORS genes to L-glutamine deprivation condition. Results of this investigation clearly demonstrate that the expression most of genes encoding TP53-related factors depends upon acute L-glutamine deprivation condition as well as upon ERN1, the major signaling system of the endoplasmic reticulum stress.

Insulin-like growth factor binding proteins play an important role in the regulation of cell proliferation and malignant tumor growth. It was shown that blockade of both enzymatic functions of sensor and signaling enzyme ERN1 (from endoplasmic reticulum to nuclei-1), the major component of endoplasmic reticulum stress signaling, decreases the expression level of IGFBP1, IGFBP2 and IGF2BP3 genes in U87 glioma cell. The decreased level of these gene expressions in glioma cells with ERN1 signaling enzyme loss of function correlates with suppression of cell proliferation. It was shown that glutamine deprivation condition leads to enhance the expression of IGFBP1 gene, but did not change significantly the expression of IGFBP2 and IGF2BP3 genes in both types of glioma cells. Moreover, this effect of glutamine deprivation did not depend from suppression of ERN1 enzyme function. At the same time, the expression of IGFBP2 and IGF2BP3 genes is decreased in glucose deprivation condition in both types of glioma cells and blockade of ERN1 signaling enzyme enhanced this effect. Thus, results of this investigation demonstrated that the expression of IGFBP1, IGFBP2 and IGF2BP3 genes in U87 glioma cells is dependent from signaling enzyme ERN1 and is changed in glutamine and glucose deprivation conditions, but only effect of glucose deprivation was depended of ERN1 signaling enzyme function. Moreover, the decreasing of IGFBP1, IGFBP2 and IGF2BP3 gene expressions in glioma cells with blockade of both enzymatic activities of ERN1 is possibly related to suppression of these cells proliferation.

Hypoxia is one of the inductors of the expression of a large group of genes, which control glycolysis and proliferation processes in low oxygen conditions or as a result of low oxygen consumption. Moreover, hypoxia is one of the factors which induce the endoplasmic reticulum stress which, like hypoxia, is an obligatory component of malignant tumor growth and is connected with cytoplasm and nuclei through three sensor and signalling systems: PERK, ATF6 (Ta) ERN1. The suppression of ERNI, the main sensing and signalling enzyme of endoplasmic reticulum stress, leads to a decrease of tumor growth and changes the character of hypoxic regulation of many genes responsible for the control of proliferation and glycolysis. ERN1 sensing and signalling system controls the expression of a large set of genes, which are dependent on endoplasmic reticulum stress as well as hypoxia. Moreover, this signalling pathway is an important factor of malignant tumor growth.

The endoplasmic reticulum is a dynamic intracellular organelle with exquisite sensitivity to alterations in homeostasis, and provides stringent quality control systems to ensure that the only correctly folded proteins transit to the Golgi and unfolded or misfolded proteins are retained and ultimately degraded. The endoplasmic reticulum stress represents the unfolded protein response to cope with the accumulation of unfolded or misfolded proteins and is required to maintain the functional integrity of the endoplasmic reticulum. The endoplasmic reticulum stress is a fundamental phenomenon which provides a secure protection of the cells from different factors. This stress provides a wide spectrum of physiological roles in diverse developmental and metabolic processes, especially for professional secretory cells with high-level secretory protein synthesis, such as pancreatic beta cells, hepatocytes and osteoblasts and is required throughout the entire life. The endoplasmic reticulum stress and hypoxia are the obligate components of malignant tumor growth, are interconnected and activate angiogenesis via growth and metabolism control. The endoplasmic reticulum stress is mediated by three by three sensor and signaling pathways (PERK, ATF6 and ERN1), besides that blockade one (ERN1) leads to a decrease of tumor growth through suppression of angiogenesis and proliferation. The data concerning the interaction of signaling enzyme ERN1 and pro- and anti-angiogenic gene expressions is analyzed.

This study was undertaken to examine the effect of cerium dioxide nanoparticles on the expression levels of genes, encoded transcription and growth factors, which are important for control of cell proliferation and survival in human astrocytes. Exposure of astrocytes with cerium dioxide nanoparticles (0.17 and 0.34 mg/ml of medium) during 20 h significantly decreases the expression level of transcription factors TBX3, E2F8 and FOXF1 mRNAs, being stronger for higher doses of nanoparticles. Moreover, the expression levels of IGFBP1 and IGFBP2 as well as LIF mRNA also significantly decreased in astrocytes after treatment with cerium dioxide nanoparticles. It is possible that the observed changes in the expression profile of the studied genes in astrocytes treated with cerium dioxide nanoparticles are associated with its toxic effect mediated by endoplasmic reticulum stress sensor-signaling systems in part through the bifunctional membrane enzyme ERN1. Thus, cerium dioxide nanoparticles have a significant effect on important regulatory mechanisms, which control cell proliferation and survival in human astrocytes. Die vorliegende Studie untersucht den Einfluss von Cerdioxid-Nanopartikeln auf die Expression von Transkriptions- und Wachstumsfaktoren humaner Astrozyten, die Proliferation und Uberleben regulieren. Eine Exposition humaner Astrozyten mit Cerdioxid-Nanopartikeln (0,17 und 0,34 mg/ml) fur 20 Stunden in vitro fuhrte zu einer signifikanten Herabregulierung der Transkriptionsfaktoren TBX3, E2F8 und FOXF1. Dieser Effekt nahm mit zunehmender Cerdioxid-Konzentration zu. Die Expression von IGFBP1, IGFBP2 und LIF mRNA nahm hingegen signifikant ab. Die toxische Wirkung von Cerdioxid wird offenbar durch Stress-Sensor-Systeme im endoplasmatischen Retikulum unter Beteiligung des bifunktionalen Transmembranproteins ERN-1 vermittelt. Es konnte gezeigt werden, dass Cerdioxid-Nanopartikel eine entscheidende Rolle in Regulationsprozessen spielen, welche Wachstum und Uberleben humaner Astrozyten kontrollieren.

The effect of C60 fullerene on the expression levels of genes, related to endoplasmic reticulum stress and cell proliferation, in human astrocytes was investigated. The exposure of human astrocytes to C60 fullerene during 24 or 72 h increases the expression levels of transcription factor E2F8, insulin-like growth factor binding protein-2, and tumor suppressor SMAD mRNAs in human astrocytes. At the same time, the expression levels of HSD17B2, PBEF1, HSPB8 mRNA decrease in cells treated with C60 fullerene, being more intense for HSD17B2. Moreover, C60 fullerene increases the proliferation rate of astrocytes. Thus, C60 fullerene affects the expression of genes which control proliferation. Der Einfluss von Fulleren C60 auf die Genexpression in Bezug auf den Stress des endoplasmatischen Retikulums und die Zellproliferation in humanen Astrozyten wurde untersucht. Unter Einwirkung von Fulleren C60 fur 24 und 72 h stieg die Expression des Transkriptionsfaktors E2F8, des Insulin-ahnlichen Wachstumsfaktors von Protein-2, und des Tumorsuppressors SMAD mRNA in humanen Astrozyten. Im Gegensatz dazu sank das Expressionsniveau der HSD17B2-, PBEF1-und HSPB8 mRNA in Zellen, die mit Fulleren C60 behandelt wurden, am starksten bei HSD17B2. Daruber hinaus erhohte Fulleren C60 die Proliferationsgeschwindigkeit der Astrozyten und beeinflusst daher die Expression der proliferationssteuernden Gene.

We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.

Objective. The aim of the present investigation was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoding glucocorticoid receptor (NR3C1) and some related proteins (SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, NNT) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of the glioma growth. Methods. The expression of NR3C1,SGK1,SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by quantitative polymerase chain reaction. Results. Inhibition of IRE1 signaling enzyme function up-regulates the expression of NR3C1, SGK1, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in comparison with the control glioma cells, with more significant changes for NR3C1, SGK1, and NNT genes. At the same time, the expression of SGK3 gene is strongly down-regulated in glioma cells upon inhibition of IRE1. We have also shown that hypoxia increases the expression of NR3C1, SGK1, NCOA2, ARHGAP35, and NNT genes but decreases SGK3 and NCOA1 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in U87 glioma cells enhances the eff ect of hypoxia on the expression of SGK1, SGK3, and NNT genes, but decreases the sensitivity of NR3C1 gene to hypoxic condition. Furthermore, the expression of NCOA1 gene is resistant to hypoxia in control glioma cells, but NCOA2 and ARHGAP35 genes are resistant to this condition in glioma cells without functional activity of IRE1 signaling enzyme. Conclusions. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NR3C1, SGK1, SGK3, NCOA1, NCOA2, ARHGAP35, and NNT genes in U87 glioma cells in gene specific manner and that all these genes are regulated by hypoxia preferentially through IRE1 signaling pathway of the endoplasmic reticulum stress.

The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or glucose deprivation conditions as model of ischemia. The blockade of function of the ERN1 enzyme, which is a major sensor of endoplasmic reticulum stress, leads to a decrease of the VEGFA, VEGFB and VEGFC mRNA expression level. The level of VEGFA proteins also decreases at this experimental condition in the cytosolic fraction, but increases in the nuclear fraction. Hypoxia does not affect VEGFC and increases the expression level of VEGFA and VEGFB mRNA in both used cell types, however, the change was much less profound in cells with suppressed function of ERN1. The expression level of VEGFC mRNA decreases in both used cell types in glutamine deprivation condition, however, the change was more profound in control glioma cells. At the same time, the expression level of VEGFA mRNA increases and VEGFB—decreases in gluta-mine deprivation condition in control glioma cells only. Exposure of glioma cells to glucose deprivation condition increases VEGFB mRNA expression level in both used cell types; however, VEGFA—in control glioma cells only and VEGFC—in cells with ERN1 signaling enzyme loss of function only. Thus, the results of this study clearly demonstrated the down-regulation of the expression of all three VEGF genes in glioma cells with ERN1 loss of function which correlates to the suppressed angiogenesis and proliferation rate of these cells. Moreover, the effect of hy-poxia and glutamine or glucose deprivation condition on the expression level of all VEGF genes is different and mainly depends on ERN1 signaling enzyme function.