PO-252 Impaired glucose metabolism in human glioma stem cells upon treatment with a cell-penetrating peptide based on CONNEXIN43

Abstract

IntroductionConnexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in glioma stem cells (GSCs). Restoring Cx43 in GSCs reverses their phenotype through the inhibition of c-Src and consequently reduces GSCs tumorigenicity. We have developed a cell-penetrating peptide (TAT-Cx43266–283) containing the region of Cx43 that interacts with c-Src that mimics the effect of Cx43 on GSC phenotype. GSCs reprogram their metabolism to compete for glucose resources through HIF-1-alpha, which can in turn be regulated by c-Src. Therefore, the aim of this work was to study the effect of TAT-Cx43266–283 on GSC metabolism.Material and methodsG166 (human GSCs), Wistar rat organotypic brain slices, neurons and astrocytes from primary culture.2-NBDG uptake: Cells were incubated with 146 µM 2-NBDG for 1 hour, lysed, and supernatant fluorescence intensity was measured by spectrofluorimetry and normalised to mg of protein.GSCs-brain organotypic slice co-cultureFluorescently-dyed human GSCs were injected into rat organotypic brain slices and 2-NBDG uptake was analysed by confocal microscopy.Cell energy analysis were performed on an extracellular flux analyser (Agilent Seahorse XF Technology ) using Mito Stress and Glycolysis Stress kits.Results and discussionsBecause TAT-Cx43266–283 inhibits Src activity in GSCs, we analysed the effect of TAT-Cx43266–283 on the rate of glucose uptake in human GSCs. Our results showed that TAT-Cx43266–283 reduced the uptake of a fluorescent glucose analogue (2-NBDG) into GSCs. Interestingly, TAT-Cx43266–283 did not significantly affect the uptake of glucose in neurons or astrocytes from primary culture, suggesting a specific effect on GSCs. Moreover, experiments using 6-NBDG, a fluorescent glucose analogue that cannot be phosphorylated by HK-2 (whose expression is regulated by HIF-1-a), showed that 6-NBDG uptake does not differ between treated and not treated GSCs. Moreover, we analysed 2-NBDG uptake on a GSCs-brain organotypic slice co-culture. Our results revealed that TAT-Cx43266–283 reduced glucose uptake in tumoral cells when they are within the brain parenchyma. More importantly, data obtained with a cell energy analysis platform (Agilent Seahorse XF Technology) showed impaired metabolism in GSCs treated with TAT-Cx43266–283, but not in neurons or astrocytes.ConclusionIn vitro and ex-vivo experiments revealed that TAT-Cx43266–283 reduces the rate of glucose uptake selectively in human glioma stem cells with the subsequent decrease in metabolic activity and survival.