Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway

Abstract

GLS-encoded glutaminase promotes tumorigenesis, while GLS2-encoded glutaminase displays tumor-suppressive properties. In glioblastoma (GBM), the most aggressive brain tumor, GLS is highly expressed and in most cases GLS2 is silenced. Previously, it was shown that transfection with a sequence encoding GAB, the main GLS2 isoform, decreased the survival, growth, and ability to migrate of human GBM cells T98G and increased their sensitivity towards an alkylating agent temozolomide (TMZ) and oxidative stress compared to the controls, by a not well-defined mechanism. In this study we report that GAB transfection inhibits growth and increases susceptibility towards TMZ and H2O2-mediated oxidative stress of two other GBM cell lines, U87MG and LN229. We also show that in GAB-transfected cells treated with H2O2, the PI3K/AKT pathway is less induced compared to the pcDNA-transfected counterparts and that pretreatment with PDGF-BB, an activator of AKT, protects GAB-transfected cells from death caused by the H2O2 treatment. In conclusion, our results show that (i) GAB suppresses the malignant phenotype of the GBM cells of different tumorigenic potentials and genetic backgrounds and (ii) the GAB-mediated increase of sensitivity to oxidative stress is causally related to the inhibition of the PI3K/AKT pathway. The upregulation of the GLS2 expression and the inhibition of the PI3K/AKT pathway may become a novel combined therapeutic strategy for anti-glioma preclinical investigations.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation

“…This G2/M arrest is consistent with the observed accumulation of GLS2, p53 and p21 protein levels, in agreement with the functional tumor suppressor role postulated for GLS2 as novel target of the p53 family of tumor suppressors 20,21,43 . Despite these evidences, GLS2 also suppressed the malignant phenotype of human GBM of different tumorigenic potentials and genetic backgrounds, including p53(─) cells 27,40 . Nevertheless, additional data characterizing the nuclear roles of GLS2 are still needed to definitively substantiate a direct link between cell cycle arrest and nuclear translocation of GLS2.…”

“…www.nature.com/scientificreports www.nature.com/scientificreports/ Our current work further suggests a causative link between GLS2 expression and tumor suppression. Transfection of human GBM cells with GLS2 induces a marked change in the cell's transcriptome 27 , leading to a more differentiated and less-malignant phenotype characterized by a strong inhibition of cell proliferation 27,40 (Fig. S4).…”

“…In chemotherapy, efforts to increase G2/M arrest of cancer cells have also been associated with enhanced apoptosis. Interestingly, the generation of reactive oxygen species (ROS) by treatment with oxidizing agents, like arsenic trioxide or hydrogen peroxide, synergizes with GLS2 overexpression to suppress malignant properties of T98G glioma cells 33,40 . Of note, GLS2 evoked in these cells lower c-Myc and Bcl-2 protein levels, as well as a higher expression of the pro-apoptotic Bid protein, which may explain the marked fall in cell number mediated by GLS2 33,40 (Fig.…”

“…Thus, glioma cell lines overexpressing GLS2 undergo significant apoptosis after the G2/M arrest, a response also found in epithelial and hematological malignancies treated with selective chemotherapeutic agents that did not affect normal cell counterparts 51 . In this context, it is noteworthy that stable transfection with GLS2 inhibited cell growth of T98G GBM through downregulation of O6-methylguanine-DNA methyltransferase (MGMT) and, thus, sensitized cells to alkylating agents as temozolomide and carmustine 40,52 . Consequently, GLS2 upregulation may facilitate chemotherapeutic intervention in addition to inhibiting glioma growth.…”

Targeting Glutamine Addiction in Gliomas

“…Human GA is encoded by two genes: GLS encoding the kidney-type KGA and GAC isoforms, and GLS2, encoding the liver-type isoforms, GAB and LGA. The GLS gene expression may be modulated by such oncogenes as MYC, Rho GTPases, and Notch, while the GLS2 gene was reported to be regulated by p53 (references in [23]). Both GLS and GLS2 products are activated by phosphate (therefore the previous name: phosphate-activated glutaminase, PAG); however, GLS2 is activated by lower concentrations of phosphate and to a lesser extent than GLS.…”