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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.

The aim of this research was to study the effect of glutamine deprivation on the expression of genes encoding for HTRA1/PRSS11, LONP1/PRSS15, and some cathepsins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1). It was shown that in control glioma cells (transfected by empty vector) glutamine deprivation up-regulated the expression of LONP1, CTSD, CTSF, CTSO, and CTSS genes, down-regulated HTRA1, CTSC, and CTSK gene expressions, and did not significantly change the expression of CTSA, CTSB, and CTSL genes. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified the effect of glutamine deprivation on the expression of HTRA1, LONP1, CTSD, CTSL, CTSO, and CTSS genes: removed the effect of glutamine deprivation on HTRA1 and CTSO genes, introduces on CTSL gene, reduced — on CTSD gene, and enhanced — on LONP1 and CTSS genes. Therefore, glutamine deprivation affect the expression level of most studied genes in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which responsible for control of cell proliferation and tumor growth.

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 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 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.

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.

To study the effect of glucose deprivation 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) was the aim of the research. It was shown that glucose deprivation up-regulated the expression of CTSA, CTSB, CTSD, CTSK, CTSL, CTSO, and LONP1 genes and did not change the expression of CTSC, CTSF, and CTSS genes in control glioma cells (transfected by empty vector). Inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified effect of glucose deprivation on the expression of most studied genes: removed the effect of glucose deprivation on CTSA and CTSO genes, introduces on CTSC and CTSS genes, reduced – on CTSK gene, and enhanced – on CTSL gene. Therefore, glucose deprivation affect 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 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 glucose deprivation on the expression of genes encoding for ubiquitin specific peptidases — USP and autophagy related 7 ATG7 in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1). It was shown that glucose deprivation was downregulated the expression of USP1 and USP10 genes and up-regulated USP4 and USP25 genes in control (transfected by empty vector) glioma cells. At the same time, the expression level of USP14, USP22, and ATG7 genes in these cells did not significantly change upon glucose deprivation condition. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified effect of glucose deprivation on the expression of most studied genes. Therefore, glucose deprivation affected the expression level of most ubiquitin specific peptidases genes in relation to the functional activity of IRE1 enzyme, which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress.

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 EIF2AK1 (eukaryotic translation initiation factor 2-alpha kinase 1) and AIFM1 (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 EIF2AK1 and AIFM1 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, EIF2AK1, and AIFM1 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.

We have studied the expression of genes encoding proliferation related factors and enzymes such as IL13RA2, KRT18, CD24, ING1, ING2, MYL9, BET1, TRAPPC3, ENDOG, POLG, TSFM, and MTIF2 in U87 glioma cells upon glucose and glutamine deprivation in relation to inhibition of inositol requiring enzyme 1, a central mediator of endoplasmic reticulum stress. It was shown that glutamine deprivation leads to up-regulation of the expression of BET1, MYL9, and MTIF2 genes and down-regulation of CD24, ING2, ENDOG, POLG, and TSFM genes in control (with native IRE1) glioma cells. At the same time, glucose deprivation enhances the expression of MYL9 gene only and decreases – ING1, ING2, and MTIF2 genes in control glioma cells. Thus, effect of glucose and glutamine deprivation on gene expressions in glioma cells is gene-specific. Inhibition of inositol requiring enzyme 1 by dnIRE1 significantly modifies the effect of both glutamine and glucose deprivation on the expression of most studied genes with different direction and magnitude, especially for ING2, CD24, and MTIF2 genes. Present study demonstrates that IRE1 knockdown modifies glucose and glutamine deprivation effects on the expression of proliferation related genes and possibly contributes to slower tumor growth of these glioma cells after inhibition of IRE1 signaling enzyme.

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.

Inhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulum stress, significantly decreases glioma cell proliferation and tumor growth. We have studied the expression of TNFα-related genes and effect of glucose deprivation on these gene expressions in U87 glioma cells overexpressing dominant-negative IRE1 defective in both kinase and endonuclease (dn-IRE1) activity of IRE1 with hopes of elucidating its contribution to IRE1 mediated glioma growth. We have demonstrated that glucose deprivation condition leads to down-regulation of the expression of TNFRSF11B, TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes and up-regulation of TNFRSF10B/TRAILR2/DR5 gene at the mRNA level in control glioma cells. At the same time, the expression of TNFRSF21/DR6, TNFAIP1, TNFAIP3, TRADD, and CD70/TNFSF7 genes in control glioma cells is resistant to glucose deprivation condition. The inhibition of IRE1 modifies the effect of glucose deprivation on LITAF, TNFRSF21, TNFRSF11B, and TRADD gene expressions and induces sensitivity to glucose deprivation condition the expression of TNFRSF10B, TNFRSF1A, and CD70 genes. We have also demonstrated that the expression of all studied genes is affected in glioma cells by inhibition of IRE1, except TNFRSF1A gene, as compared to control glioma cells. Moreover, the changes in the expression of TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes induced by glucose deprivation condition have opposite orientation to that induced by inhibition of IRE1. The present study demonstrates that fine-tuning of the expression of TNFα-induced proteins and TNF receptor superfamily genes, which related to cell death and proliferation, is regulated by IRE1, an effector of endoplasmic reticulum stress, as well as depends on glucose deprivation in gene specific manner. Thus, the inhibition of kinase and endoribonuclease activity of IRE1 correlates with deregulation of TNFα-induced protein genes and TNF receptor superfamily genes in gene specific manner and thus slower the 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 F1), 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 ERN1 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.

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.

Hypoxia is one of powerful inducers of expression of a large group of genes, including genes which control glycolysis, angiogenesis and proliferation supportingcell surviving at low oxygen condition. However, in tumor cells hypoxia was observed at normal oxygen tension condition as a result of its decreased utilization. Moreover, hypoxia is an obligate component of malignant tumor growth and substantially controls glycolysis, angiogenesis and proliferation processes. Data concerning molecular mechanisms of activation of hypoxia inducible transcription factor HIF in cells at hypoxia and in malignant tumors, as well as its role of in the regulation of gene expressions have been analyzed. The mechanisms of interaction of the transcription factor HIF with specific hypoxic regulatory elements in the promoter region of genes activated by hypoxia were examined.

The angiogenesis is an important component of tumor growth and tightly associated with hypoxia. The expression level of genes related to regulation of angiogenesis (BAI2, SPARC, TIMP1, TIMP2, TIMP3, TIMP4, THBS1, THBS2, ADAMTS5 and FGF2) in glioma U87cells and cells with suppressed function of signaling enzyme ERN1, a major mediator of the endoplasmic reticulum stress by qPCR, was studied. We have shown that the expression of genes encoding BAI2, SPARC, TIMP2, TIMP3, THBS1 and THBS2 is strongly increased in glioma cells with ERN1 signaling enzyme loss of function, being more intense for TIMP2, TIMP3 and THBS1 genes. At the same time, the expression of genes encoding TIMP1, TIMP4, ADAMTS5 and FGF2 is significantly decreased with more strong effect for ADAMTS5 and TIMP4 genes. At hypoxia, the expression of most of studied genes in both glioma cell types is affected. Hypoxia induced the expression of TIMP1, TIMP3 and ADAMTS5 genes both in control glioma cells and cells with ERN1 enzyme loss of function. However, the effect of hypoxia towards TIMP2 gene expression was observed only in control glioma cells. At the same time, the expression of genes encoding BAI2, SPARC, THBS1, THBS2, ADAMTS5 and FGF2 is decreased under hypoxia action, but its expression mostly depended on ERN1 signaling enzyme function. The results of this study provide strong evidence that suppression of ERN1 signaling enzyme function, as well as hypoxia, changes the expression of genes related to regulation of angiogenesis in glioma cells. It is possible that changes in the expression of these genes contribute to the suppression of glioma cells’ proliferation by blockade of ERN1 signaling enzyme functioning.

Accumulating evidence raises the hypothesis that dysregulation of different intrinsic mechanisms which control most metabolic processes are involved in the development of obesity, metabolic syndrome and type 2 diabetes mellitus, the most profound public health problems. Angiogenesis is an important component of different proliferative processes, in particular, fat tissue growth. Moreover, dysregulation of molecular components of the angiogenesis system can contributes to the development of diabetic complications. We have studied the expression levels of genes related to regulation of angiogenesis (TIMP1, TIMP2, TIMP3, TIMP4, THBS1, THBS2, THBS3, ADAMTS5, LUM, DCN, IL6 and ZEB1) in subcutaneous adipose tissue of obese individuals as well as obese patients with impaired glucose tolerance (prediabetic) and type 2 diabetes using real-time quantitative PCR. We have shown that the expression level of most of these genes with anti-angiogenic properties significantly increases in subcutaneous adipose tissue of obese individuals versus lean patients, being more intense for TIMP1, TIMP2, THBS2 and LUM genes. Increased expression level of TIMP1, TIMP2, TIMP3, ADAMTS5 as well as THBS1 and THBS2 in adipose tissue was found in obese patients with impaired glucose tolerance. At the same time, expression of genes which encode for TIMP1 and TIMP2 strongly decreased in adipose tissue of obese individuals with type 2 diabetes versus subjects with glucose intolerance. Results of this study provide strong evidence that expression of genes mostly related to suppression of angiogenesis is dysregulated in adipose tissue of obese individuals as well as in obese patients with glucose intolerance and type 2 diabetes. It is possible that these changes in the expression of TIMP and THBS genes in adipose tissue in obesity as well as in obese individuals with impaired glucose tolerance and type 2 diabetes can contribute to fat tissue storage, insulin resistance and the development of diabetic complications.

Endoplasmic reticulum stress, as well as hypoxia and ischemia, are important factors for tumor neovascularization and growth. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. High glucose metabolism of cancer cells is caused by a combination of hypoxia-responsive transcription factors, activation of oncogenic proteins and the loss of tumor suppressor function and is realized in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) and hexokinase 2. We have studied the effect of hypoxia and ischemia on the expression of PFKFB and hexokinase genes in glioma cell line U87 under knockdown of endoplasmic reticulum–nuclei-1 (ERN1) sensing and signaling enzyme. It was shown that loss of the signaling enzyme ERN1 function leads to an increase in the expression levels of HK1, HK2, PFKFB3 and PFKFB4 mRNA. Moreover, the expression levels of all studied genes increase under hypoxia in control and ERN1-deficient glioma cells; however knockdown of ERN1 suppresses the effect of hypoxia. Besides, HK2 and PFKFB4 are more sensitive to hypoxia than HK1 and PFKFB3. Glucose or glutamine deprivation conditions have different effects on the expression levels of these genes and its effect depends mainly on ERN1 function. Expression levels of alternative splice variants of PFKFB3 and PFKFB4 mRNA change at used experimental conditions in a fashion similar to the basic PFKFB variants. Thus, the expression of hexokinase and PFKFB genes is mainly dependent on ERN1 signaling enzyme function in normal, hypoxic and ischemic conditions.

The main molecular components of circadian clock system are proteins which play a significant role in the control of both metabolism and malignant tumor growth. Endoplasmic reticulum stress as well as hypoxia and ischemia are important factors for tumor neovascularization and growth. We have studied the expression of circadian genes in the glioma cell line U87 under knockdown of endoplasmic reticulum–nuclei-1 (ERN1) sensing and signaling enzyme. It was shown that blockade of ERN1 leads to increase in the expression levels of PER1, PER3 and CLOCK mRNA; but the CRY1, PER2, BMAL1, BMAL2 and DEC2 mRNA levels are decreased. Moreover, the expression levels of most of the studied genes are increased under glucose or glutamine deprivation conditions both in control and ERN1-deficient glioma cells; however knockdown of ERN1 modifies the effect of these ischemic conditions. Hypoxia has different effects on the expression levels of circadian genes and these effects are dependent on ERN1 function. Hypoxia induces the expression of PER1, BMAL1 and DEC2 and decreased – PER3, CLOCK, CRY1 and BMAL2 mRNA in control glioma cells, while in genetically modified cells it induces the expression of BMAL1 mRNA only. Thus, the expression of circadian genes is dependent on ERN1 signaling enzyme function in normal, hypoxic and ischaemic conditions.

Hypoxia and ischemia, as well as endoplasmic reticulum stress, are principal factors which are necessary for tumor neovascularization and growth. We have studied the effect of blocking the endoplasmic reticulum – nuclei-1, the main signaling enzyme of stressing of the endoplasmic reticulum, on the expression of several retinoblastoma-related genes which play a significant role in the control of cell cycling, proliferation and apoptosis in glioma cell line U87. We have also studied the effect of hypoxia and ischemic conditions (glucose or glutamine deprivation) on the expression of these genes in control glioma cells and in subline of these cells with suppressed function of endoplasmic reticulum – nuclei-1 sensing and signaling enzyme. It was shown that blocking of endoplasmic reticulum – nuclei-1 signaling enzyme leads to increase in the expression levels of several retinoblastoma binding protein (RBBP) genes: RBBP2H1 (RDM5B), RBBP4 and RBBP8. Moreover, the expression levels of most of studied genes are significantly decreased under glucose or glutamine deprivation conditions both in control and endoplasmic reticulum – nuclei-1-deficient glioma cells. However, the expression levels of RBBP2H1 and RBBP2 (RDM5A) mRNA are increased in glutamine deprivation conditions in both tested cell types. The expression levels of RBBP4, RBBP7 and RBBP8 are decreased but such levels of RBBP2 and RBBP2H1 are increased in both tested cell types under the hypoxic conditions. Thus, the expression of most retinoblastoma-related genes is dependent on the endoplasmic reticulum – nuclei-1 signaling enzyme function in normal, hypoxic and ischaemic conditions.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is a key regulatory enzyme of glucose metabolism. This bifunctional enzyme controls glycolysis and gluconeogenesis as well as glucose phosphorylation and metabolism. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) plays significant role in the glycolysis regulation in the heart as well as in some other organs (kidney, brain and lung). Results of this investigation clearly demonstrated that in the glioma cells as well as in some mouse organs are presented several new unique alternative splice variants of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2. These PFKFB-2 splice variants are a result of deletions or/and inserts in catalytic regions of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase as well as in С-terminal regulatory region. Alternative splice variants of PFKFB-2 from glioma and mouse cells contain different inserts after 2nd exon but mouse splice variant has also deletion in 6-phosphofructo-2-kinase region and insert in C-terminus. Another splice variants of mouse PFKFB-2 have deletions in 6-phosphofructo-2-kinase or fructose-2,6-bisphosphatase regions, since one splice variant has deletion of all catalytic domains. In conclusion, this study provides evidences that alternative splicing of PFKFB-2 mRNA possibly has a significant role in glycolysis regulation via creation of monofunctional isoforms of this enzyme.

Most of physiological and biochemical processes in living organisms have circa­dian character. Disturbance of the regulation of circadian processes leads to developing different pathological processes and cancer, in particular. In this work, we have studied the effect of silver nanoparticles on the expression of very important regulatory genes which control the circadian processes: Per1, Clock and BMal1. We have shown that the level of Per1 expression significantly increased in the liver and lung but decreased in the testes. The level of Clock mRNA expression also increased in liver and lung but decreased in kidney. The Clock mRNA expression in testis and heart was observed at late period of silver nanoparticles action. Thus, the expression of Per1, Clock and BMal1 mRNA significantly changed in tissue specific manner in rats treated with silver nanoparticles. Disturbance of these genes expression can destroy cellular signal pathways and leads to developing pathological processes. The results of our investigation clearly demonstrated that silver nanoparticles have significant effect on the regulatory mechanisms which control cell metabolism via circadian gene expression. Expression of circadian genes can be a sensitive test for the detection of silver nanoparticles action on the organisms.

A bifunctional PFKFB enzyme is a key regulator of glycolysis. Four different genes encode the synthesis of multiple PFKFB isoforms. We analyzed data concerning structural organization of PFKFB genes and alternative splice variants of PFKFB mRNA, as well as expression and molecular mechanisms of regulation of different PFKFB isoforms in malignant tumors and at hypoxia, and mechanisms of hormonal regulation. Hypoxia inducible factor (HIF)-dependent mechanisms of regulation of the expression of different PFKFB variants are presented and discussed.

Methyl tertial butyl ether, an oxygenated compound of gasoline, is a toxic and ecolo­gically dangerous chemical compound with an ability to introduce a variety of neurotoxic, allergic and respiratory diseases, as well as cancer and leukemia. We used RT/PCR or quan­titative PCR to study the effect of methyl tertial butyl ether (everyday injection for one month) on the expression of mRNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF), which are important for tumor growth and metastasis in the lung and liver. The level of PFKFB-3 and VEGF mRNA expression was increased in the liver in methyl tertial butyl ether treated rats with respect to the control animals. However, methyl tertial butyl ether had opposite ef­fect on the expression of PFKFB-3 and VEGF mRNA in the lung: with strong induction of PFKFB-3 and suppression of VEGF mRNA expression. Moreover, alternative splicing of PFKFB-3 mRNA was changed in methyl tertial butyl ether treated rats in organ specific manner. Thus, methyl tertial butyl ether induces alternative splicing of PFKFB-3 mRNA accompanied by appearing of variants with longer C-terminus in the liver, and with shorter C-terminus in the lung. The results of this investigation clearly demonstrate that methyl tertial butyl ether affects in organ-specific manner the expression and alternative spli­cing of mRNA coding for PFKFB-3 and VEGF, key regulatory factors of glycolysis, and angiogenesis.

Expression of vascular endothelial growth factor (VEGF), glucose transporter Glut1 and two members of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family (PFKFB-3 and PFKFB-4) in lung, colon and gastric cancers was studied. We have shown that expression of VEGF and glucose transporter Glut1 mRNA significantly increased in lung and colon cancers and much less – in gastric cancers comparing with corresponding normal tissues counterparts. However, expression of mRNA of glucose transporter Glut1 was increased in all tested cancers more distinctly as compared with mRNA for VEGF. Moreover, we have shown that PFKFB-3 and -4 mRNA were overexpressed in different cancers, but much more marked changes were observed in the lung and colon. PFKFB-4 protein level was also increased in different cancers being more clearly induced in the lung cancer. However, normal tissue counterparts from colon and stomach have much higher levels of PFKFB-4 protein, as compared to the lung. Thus, our data demonstrated that VEGF, glucose transporter Glut1, PFKFB-3 and PFKFB-4, known HIF-dependent genes, are overexpressed in malignant tumors from lung, colon and stomach and thus can participate in regulation of tumor growth.

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.