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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.

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.

Вивчено вплив дефіциту глюкози на експресію генів, які кодують специфічні до убіквітину пептидази (USP) та протеїн 7, що стосується автофагії (ATG7), у клітинах гліоми лінії U87 за умов пригнічення inositol requiring enzyme-1 (IRE1). Показано, що дефіцит глюкози знижує експресію генів USP1 та USP10 і підвищує USP4 та USP25 у контрольних (трансфікованих пустим вектором) клітинах гліоми. Водночас рівень експресії генів USP14, USP22 та ATG7 у цих клітинах істотно не змінюється. Пригнічення функції сигнального ензиму IRE1 у клітинах гліоми лінії U87 модифікує ефект дефіциту глюкози на експресію більшості досліджених генів. Таким чином, дефіцит глюкози змінює рівень експресії більшості генів специфічних до убіквітину пептидаз залежно від функціональної активності ензиму IRE1, що контролює процеси проліферації та росту пухлин як центральний медіатор стресу ендоплазматичного ретикулума.Изучено влияние дефицита глюкозы на экспрессию генов, кодирующих специфические к убиквитину пептидазы (USP) и протеин 7, что имеет отношение к аутофагии (ATG7), в клетках глиомы линии U87 при угнетении inositol requiring enzyme-1 (IRE1). Показано, что дефицит глюкозы снижает экспрессию генов USP1 и USP10 и повышает USP4 и USP25 в контрольных (трансфецированных пустым вектором) клетках глиомы. В то же время уровень экспрессии генов USP14, USP22 и ATG7 в этих клетках существенно не изменяется. Угнетение функции сигнального энзима IRE1 в клетках глиомы линии U87 модифицирует эффект дефицита глюкозы на экспрессию большинства изученных генов. Таким образом, дефицит глюкозы изменяет уровень экспрессии большинства генов специфических к убиквитину пептидаз в зависимости от функциональной активности энзима IRE1, который контролирует процессы пролиферации и роста опухолей в качестве центрального медиатора стресса эндоплазматического ретикулума.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 down-regulated 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.