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It was shown previously that inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) pathway, a central mediator of the unfolded protein response, leads to suppression of tumor growth through down-regulation of key pro-proliferative and up-regulation of tumor suppressor factors and modifies the sensitivity of these genes to glucose and glutamine deprivation. However, the executive mechanisms of ERN1 mediated control of glioma cell proliferation are not yet known. The goal of this study was to estimate the effect of glucose and glutamine deprivations on expression of cancer related genes in glioma U87 cells at ERN1 signaling inhibition for evaluation of their possible significance in ERN1 mediated control of glioma cell proliferation. We have studied the effect of glucose and glutamine deprivations on the expression level of cancer related genes encoding TMED10 (transmembrane p24 trafficking protein 10), MYL9 (myosin, light chain 9, regulatory), SPOCK1 (sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1), CUL4A (cullin 4A), and CUL4B in U87 glioma control cells and cells with ERN1 knockdown. It was shown that at glucose deprivation the expression level of MYL9, SPOCK1 and CUL4B genes was significantly up-regulated in control glioma cells. ERN1 knockdown modified the sensitivity to glucose deprivation of all studied genes except TMED10 gene. At glutamine deprivation the expression of MYL9, CUL4A and CUL4B genes was shown to be up-regulated in control glioma cells. The sensitivity of MYL9, TMED10 and CUL4B gene expression to glutamine deprivation in glioma cells with ERN1 knockdown was significantly modified, while CUL4A and SPOCK1 gene expression did not respond to ERN1 inhibition. The present study demonstrates that glucose and glutamine deprivation affected the expression of the most studied genes in a specific manner and that inhibition of ERN1 signaling preferentially modified their expression at glucose and glutamine deprivation.

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.

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.