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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 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.