PO-129 Transfection with liver-type glutaminase (GAB) sensitiseshuman glioblastoma cell lines to hydrogen peroxide by downregulation of the PI3K/AKT pathway

Abstract

IntroductionGlutamine (Gln) plays a crucial role in the metabolism of neoplastic cells and its elevated catabolism is observed in tumours of different origin. Glutaminase (GA, EC 3.5.1.2) is an enzyme metabolising Gln to glutamate (Glu) and ammonia. The following GA isoforms are known at present: i) kidney-type isoforms, KGA and GAC, encoded by GLS and ii) liver-type isoforms, GAB and LGA, encoded by GLS2. GA isoforms play opposite roles in tumorigenesis. In glioblastoma (GBM), an incurable brain tumour, GLS is highly expressed, while GLS2 is hardly detectable. Previous studies showed that transfection of human GBM cells (T98G, U87MG and LN229) with a GAB sequence decreased their survival, proliferation index and sensitised them to hydrogen peroxide (H2O2). H2O2 treatment has been shown to activate the PI3K/AKT signalling pathway which is upregulated in GBM. Here we tested the hypothesis that GAB transfection sensitises GBM cells to H2O2 treatment via downregulation of the PI3K/AKT cascade.Material and methodsHuman GBM cell lines T98G, U87MG and LN229 transfected with GAB sequence (herein named -GAB) or an empty vector (herein named -pcDNA) were used in this study. The protein levels were measured by Western blot. Mitochondrial activity was assessed by MTT test. Apoptosis was determined using the Caspase-Glo 3/7 Assay Systems (Promega).Results and discussionsUpon H2O2 treatment, TGAB, UGAB and LNGAB cells presented diminished phosphorylation level of proteins belonging to the PI3K/AKT cascade: PI3K, PDK1, AKT, as compared to their –pcDNA counterparts. TGAB and UGAB cells showed also lowered phosphorylation level of NFκβ and higher apoptotic rates compared to pcDNA cells. Pretreatment with PDGF, an activator of AKT phosphorylation, reduced H2O2-evoked GAB cell death compared to the vehicle-treated counterparts.ConclusionIn conclusion, downregulation of the PI3K/AKT signalling pathway by GAB transfection, an effect likewise observed in T98G, U87MG and LN229 cells, contributes to increased cell sensitivity to H2O2 treatment. Combination of GAB upregulation and treatment with oxidising agents is a potential therapeutic strategy against GBM.Supported by the National Science Centre grant No 2016/23/N/NZ5/01428.