Artikel
The synthetic ligand Apelin-F13A binds and activates glioblastoma APLNR reducing the pro-invasive side effect of anti-angiogenic therapy
Die Bindung und Aktivierung des APLNR Rezeptors mittels synthetischem Liganden Apelin-F13A führt zur Reduktion des pro-invasiven Nebeneffektes der anti-angiogenen Glioblastom-Therapie
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Veröffentlicht: | 8. Mai 2019 |
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Gliederung
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Objective: Anti-angiogenic therapy of glioblastoma (GBM) blocking vascular endothelial growth factor-A (VEGFA) often resulted in accelerated tumor-cell invasion and upregulation of alternative angiogenic pathways. Here, we studied the impact of different apelin peptides acting on the G-protein coupled receptor APLNR to mediate tumor angiogenesis and glioblastoma cell invasion.
Methods: Towards this aim, we applied the smallest bioactive peptide apelin-13 and its C-terminally mutated alternative apelin-F13A in in vitro migration/invasion assays. Next, we established its mechanistic differences in two different in vitro internalization assays. For functional studies we performed orthotopic implantation of human patient-derived and mouse subtype-specific GBM stem cell cultures and employed pharmacological blockade for apelin / APLNR and VEGFA / VEGFR2-signaling.
Results: Apelin/APLNR are broadly expressed in human GBM and modulating apelin-levels reduced GBM vascularization but also strongly accelerated GBM cell-invasion. Thus we performed two migration assays, a wound healing and a Boyden chamber chemotaxis assay, and confirmed that the depletion of apelin in glioblastoma cells lead to significantly increased invasive behavior of GBM cells. In contrast, application of apelin-13 or apelin-F13A attenuated this increased invasive phenotype. To investigate if both ligands can activate APLNR on GBM cells, we performed two in vitro receptor internalization assays. We found that apelin-13 and apelin-F13A caused the internalization of the APLNR to cytoplasmic and perinuclear regions. Most interestingly, apelin-13 lead to an increase of APLNR localization in the cytoplasm and a massive decrease in the nucleus, apelin-F13A caused exactly the opposite outcome. In a second internalization assay we administered GFP-linked apelin peptides. We found that both, apelin-13 and apelin-F13A were internalized by GBM cells in a sequence specific and dose dependent manner.
Conclusion: Together, these findings imply that the synthetic APLNR ligand apelin-F13A efficiently binds to and activates glioblastoma APLNR to function as a competitive agonist for other APLNR ligands. Our receptor internalization assay revealed that apelin-F13A induces a distinct pattern of intracellular APLNR localization which may explain why only apelin-F13A (but not apelin-13) was able to block the angiogenic effect of intratumoral apelin in addition to its anti-invasive role.