PO-438 Internalisation of cell-penetrating peptides based on CONNEXIN43 in glioma stem cells and neural cells

Abstract

Introduction Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in malignant gliomas. These tumours are composed of a heterogeneous population of cells that include many with stem cell-like properties, called glioma stem cells (GSCs). Interestingly, restoring Cx43 in these cells reverses GSC phenotype and reduces their tumorigenicity. This effect is based on the interaction between the carboxy tail of Cx43 and the tyrosine kinase c-Src. A cell-penetrating peptide (CPP) containing residues 266–283 of Cx43 (TAT-Cx43266–283) is able to recruit c-Src inhibitors CSK and PTEN and to inhibit c-Src activity. The aim of this work was to study the internalisation of this CPP into GSCs and other cells of the brain parenchyma. Material and methods Glioma cell lines and primary rat neurons and astrocytes were expanded in adherent culture and incubated with a CPP containing amino acids 266–283 of Cx43 fused to TAT at the N-terminus and to biotin at the C terminus (TAT-Cx43266–283-B) for 5 min at 37°C. The internalisation of the peptide was studied by confocal microscopy. GSCs were microinjected in organotypic brain slice cultures, allowed to engraft into the tissue and incubated with TAT-Cx43266–283-B to study the uptake of the peptide in GSCs exposed to brain tissue interactions. The internalisation of the peptide by the surrounding cells was examined by immunofluorescence. Results and discussions We observed a higher uptake of TAT-Cx43266–283-B in glioma cells and GSCs compared to astrocytes, especially at low concentrations of the peptide (6 µM). We did not find TAT-Cx43266–283-B internalisation by neurons under these experimental conditions. We noted a specific localization of TAT-Cx43266–283-B at the plasma membrane that was not shown by CPPs containing other regions of Cx43. Colocalization studies revealed TAT-Cx43266–283-B colocalization with c-Src, which is myristoilated in its active form, near the plasma membrane. We explored the uptake of TAT-Cx43266–283-B in organotypic slice cultures and found high levels of TAT-Cx43266–283-B internalisation by GSCs engrafted in the tissue. Interestingly, TAT-Cx43266–283-B was also found within the cells of the tumour microenvironment. Conclusion The specific subcellular localization of TAT-Cx43266–283-B at the plasma membrane helps to understand the mechanism by which TAT-Cx43266–283 inhibits c-Src and its consequent antitumorigenic effect. Additionally, the efficient internalisation of the peptide in GSCs increases its therapeutic possibilities.