Artikel
RNAi of glioma pathogenesis-related protein 1 severely affects survival of glioblastoma cells in vitro and in vivo
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Autoren
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Achim Temme
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Sektion Experimentelle Neurochirurgie und Tumorimmunology, Dresden, Deutschland
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Steffi Ritter
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Sektion Experimentelle Neurochirurgie und Tumorimmunology, Dresden, Deutschland
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Urban Scheuring
- Universitätsklinikum Frankfurt, Klinik für Hämatologie, Onkologie und Infektionskrankheiten, Frankfurt am Main, Deutschland
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Isabell Schau
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Sektion Experimentelle Neurochirurgie und Tumorimmunology, Dresden, Deutschland
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Gabriele Schackert
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Sektion Experimentelle Neurochirurgie und Tumorimmunology, Dresden, Deutschland
| Veröffentlicht: | 18. Juni 2018 |
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Gliederung
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Objective: Glioma pathogenesis-related protein 1 (GliPR1) belongs to the pathogenesis-related protein (PR) superfamily and the cysteine-rich secretory protein (CRISP) family. It is highly expressed in glioblastoma (GBM) and elevated mRNA levels are found in acute myeloid leukemia. It has been proposed that GliPR1 plays a role in the migration and invasion of glioma cells. Furthermore, GliPR1 expression levels are higher in mesenchymal GBM and predict tumor recurrence and poor clinical outcome. We tested the hypotheses whether targeted knockdown of GliPR1 expression using RNAi in vitro and in vivo in a xenograft mouse model may show promising anti-tumor effects in GBM.
Methods: We generated lentiviral vectors encoding shRNA #258 and #301 targeting GliPR1-mRNA. Glioblastoma cell lines U87-MG, A172 and U343-MG were transduced with lentiviral particles. Endogenous GliPR1 protein levels and knockdown efficiencies were analyzed by Western blot analysis. Furthermore, cell morphology, clonal survival and induction of apoptosis were monitored by standard cell culture techniques, Western blot analysis of cleaved caspase 3 and FACS-assisted Annexin V/PI analysis of cells. For survival analysis, U87-MG cells with stable expression of shRNA #258 and transduced with shLuc control, respectively, were stereotactically implanted into the right brain hemisphere of NMRI-Foxn1nu/Foxn1nu mice.
Results: Transduction of encoding shRNA #258 and #301 downregulates steady state levels of GliPR1 protein and significantly decreases clonogenic survival of U87-MG, A172 and U343-MG cells, whereas knockdown of GliPR1 had no effect on cell morphology. Annexin V/PI staining of shRNA #258- and #301-transduced GBM cells revealed a significant increase in the fraction of cells in late apoptosis/necrosis phase when compared to shLuc-transduced controls. However, knockdown of GliPR1 did not result in cleavage of executioner caspase 3. Orthotopic U87-MG xenograft experiments demonstrated a significant longer survival of mice transplanted with glioma cells with stable expression of shRNA #258 targeting GliPR1 when compared to controls.
Conclusion: Our data indicate that knockdown of GliPR1 induces a caspase-3-independent cell death mechanism. Since most GBMs have deregulated apoptosis pathways, RNAi of GliPR1 expression may become a promising alternative approach for glioblastoma treatment.
