Expression of hexokinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase genes in ERN1 knockdown glioma U87 cells: effect of hypoxia and glutamine or glucose deprivation

D O Minchenko,O H Minchenko,E V Khomenko,T V Bakalets,R Y Marunych

doi:10.30970/sbi.0503.172

Abstract

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. Keywords: mRNA expression, HK1, HK2, PFKFB3, PFKFB4, glioma cells, ERN1 knockdown, hypoxia, glucose and glutamine deprivation.

Highlights

High rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors
The level of suppression of the enzymatic activity of ERN1 was estimated by analysis of the expression of XBP1 alternative splice variant (XBP1s) and its splice variant, shorter isoform (XBP1s), a key transcription factor in ERN1 signaling, in U87 glioma cells that overexpress a dominant-negative construct of endoplasmic reticulum–nuclei-1 compared with the control glioma cells transfected with a vector
We have found that HK1, hexokinase 2 (HK2), PFKFB3 and PFKFB4 genes are expressed in the human glioma cell line U87 and the levels of their expression mainly depend upon the

Summary

Introduction

High rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors. H. Minchenko proteins and the loss of tumor suppressor function. Over-expression of HIF-1α or HIF2α and MYC, activation of RAS and loss of TP53 and/or other tumor suppressor functions each have been found to stimulate glycolysis in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) and hexokinases (HK) [1, 2]. PFKFB3 and PFKFB4 are hypoxia responsive enzymes and overexpressed in different cancer tissues [6,7,8,9]. Overexpression of these enzymes is an obligatory factor of tumor cell glycolysis [10, 11]. Several alternative splice variants for PFKFB3 and PFKFB4 were identified in tumor cells which possibly have significance in cancer growth [13,14,15]

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