Artikel
The Disintegrin-Metalloproteinase ADAM8 as a therapeutic target in highly invasive gliomas
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Veröffentlicht: | 4. Juni 2012 |
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Gliederung
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Objective: ADAMs are zinc-dependent metalloproteases with important roles in tumor cell growth, cell migration and tumor angiogenesis. We explored the functional role of a member of this family, ADAM8, in gliomas by generating U87 glioma cell lines with a knockdown of ADAM8 and pharmacological inhibition of ADAM8 using a novel and specific ADAM8 inhibitory peptide to assess in vivo drugability of ADAM8.
Methods: A stable knockdown of ADAM8 in U87 was generated by transfection of an shRNA construct and cell clones were selected. ADAM8 knockdown cells were compared to wild type U87 cells by scratch and invasion assays, fluorescence protease activity assays, proteomics and intracellular signalling assays. Cells were injected stereotactically and the effect of an inhibitory ADAM8 peptide was assessed in cell culture and in vivo.
Results: Knockdown of ADAM8 caused a significant reduction in migration and invasion behaviour of U87 cells. As a result of ADAM8 knockdown, proteolytic release of a number of membrane proteins involved in cell migration was markedly reduced, including CD44, JAM-B and osteopontin, molecules with essential functions for glioma cell migration. In addition, ADAM8 mediated intracellular signalling was decreased in ADAM8 knockdown cells, i.e. ERK1/2 phophorylation and integrin β1 signalling. In vivo, reduced migration was seen as decreased number of brain-infiltrating cells. In cell culture, application of an ADAM8 inhibitory peptide in submicromolar concentrations caused a significant reduction of glioma invasiveness, to a similar extent as the ADAM8 knockdown.
Conclusions: ADAM8 is a drug target in highly invasive glioblastoma cells, since knockdown or inhibition causes a significant reduction of cellular migration and invasiveness, hallmarks of high grade gliomas. The pathophysiological effect of ADAM8 is given by proteolysis of important mediators of cell migration and by induction of integrin/ERK signalling. Our results provide a good basis for further studies to treat glioma by application of ADAM8 inhibitory drugs.