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To study effect of glutamine deprivation on the expression of genes encoding the key proliferation associated factors on a relation to inhibition of inositol requiring enzyme-1 IRE1 in U87 glioma cells was the aim of the research. It was shown that glutamine withdrawal down-regulated the expression of DEK, BRCA1, LIF, and COL6A1 genes in control glioma cells (transfected by empty vector), up-regulated ADGRE5 gene expression, and did not significantly change the expression of TPD52 and GNPDA. Inhibition of ІRE1 signaling enzyme activity modified the effect of glutamine deprivation on the expression of TPD52, BRCA1, LIF, DEK, ADGRE5, and COL6A1 genes: introduces the effect of glutamine deprivation on TPD52 and GNPDA1, reduced – on COL6A1, and enhanced – on ADGRE5, DEK, and BRCA1 in U87 glioma cells. Therefore, glutamine deprivation affect the expression level of most studied genes in U87 glioma cells in relation to the functional activity of IRE1 signaling enzyme, which is responsible for control of cell proliferation and glioma growth.

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.

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 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 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.

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.