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72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)
Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

06.06. - 09.06.2021

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Tunel labelling assay confirmed induced cell death by methadone in glioblastoma cells and normal astrocytes

Der Tunel Assay bestätigt den durch Methadon induzierten Zelltod in Glioblastomzelllinien und normalen Astrozyten

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  • presenting/speaker Konstantin Brawanski - Medizinische Universität Innsbruck, Universitätsklinik für Neurochirurgie, Innsbruck, Österreich
  • Annette Lohmeier - University Hospital Regensburg, Department of Neurosurgery, Regensburg, Deutschland
  • presenting/speaker Alexander Brawanski - University Hospital Regensburg, Department of Neurosurgery, Regensburg, Deutschland
  • Claudius Thomé - Medizinische Universität Innsbruck, Universitätsklinik für Neurochirurgie, Innsbruck, Österreich
  • Nils Ole Schmidt - University Hospital Regensburg, Department of Neurosurgery, Regensburg, Deutschland
  • Martin Proescholdt - University Hospital Regensburg, Department of Neurosurgery, Regensburg, Deutschland

Deutsche Gesellschaft für Neurochirurgie. 72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie. sine loco [digital], 06.-09.06.2021. Düsseldorf: German Medical Science GMS Publishing House; 2021. DocP127

doi: 10.3205/21dgnc415, urn:nbn:de:0183-21dgnc4158

Published: June 4, 2021

© 2021 Brawanski et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.

Outline

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Objective: Glioblastoma (GBM) is the most frequent primary brain tumor in the adulthood and carries a dismal prognosis. Recently, it has been shown, that D,L-methadone induced apoptosis in GBM cell lines detected by annexin V FACS analysis. To confirm and to assess the executive state of apoptotic cell death with another method, we applied TUEL labelling assay.

Methods: We utilized three different established rat GBM cell lines (9L, F98, S635) and a normal rat astrocytes as a primary culture from Fisher 344 rats. All cell lines were exposed to 5 concentrations of D,L-methadone (0.3, 1.0, 15, 30, 45 µg/ml) alone or in addition to TMZ (100 µM). After 6 days cells were harvested and apoptosis was detected by annexin V FACS analysis and fragmentated DNA was detected by TUNEL labelling assay to confirm apoptosis.

Results: All tested cell lines express of the μ-opioid receptor was similarly; in particular, there was no difference between the GBM cells compared to normal astrocytes. Apoptosis, detected by annexin V FACS, is induced in all cell lines by D,L-methadone alone beginning at 15µg/ml and in addition to TMZ with 0.3µg/ml (9L), respectively with 15µg/ml D,L-methadone (F98, S635, 9L). The tunnel assay showed cell death with defragmented DNA in all cell lines, conforming apoptosis detected by annexin V FACS analysis. The TUNEL labelling experiments confirmed the data of apoptosis. In comparison to GBM cell lines, the astrocytes react refractory against D,L-methadone alone, confirmed in apoptosis and TUNEL labelling assay.

Conclusion: TUNEL labelling experiments confirmed the findings of annexiv V FACS, indicating the induction of apoptotic cell death in higher concentrations of methadone (> 15 mg/ml), with the astrocytes showing the lowest susceptibility.