Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells
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Cell culture and drug treatment procedures
SH-SY5Y (ATCC) and mouse embryonic fibroblast (MEF) Bax−/− and +/+ cells, a gift from Dr M. Serrano (CNIO, Madrid, Spain), were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, 20 units mL−1 penicillin, 5 mg mL−1 streptomycin and 15% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA, USA), as described [25], [39]. Cells were spotted on 35 mm High IbiTreat μ-Dishes (ibidi GmbH, Martinsried, München, Germany) at 2.9 × 105 cells/cm2 and allowed to attach overnight in a
Methadone induces AIF translocation
Translocation of AIF from the mitochondrion to the nucleus has been proposed to be a crucial step in mitochondrial caspases-independent cell death [29]. For this reason, localization of AIF in SH-SY5Y cell cultures treated with 0.5 mM methadone for 24 h was assayed using immunofluorescence and confocal microscopy. Immunofluorescence showed punctuated cytosolic localization of AIF in control cells (Fig. 1A). However, 0.5 mM methadone treatment resulted in a change of AIF localization to nucleus (
Discusion
In this study, we provide supporting evidence that SH-SY5Y cells undergoing methadone-induced death should be classified as type III or necrotic. As previously reported, methadone required high concentrations to activate cell death mechanisms [15], [44], [45], which can be taken in consideration due to methadone probably displays large interindividual variability in its pharmacodynamics. Methadone treatment resulted in AIF translocation from the mitochondria to the nucleus via a process that
Acknowledgments
We are grateful to Noemi Perez Blasco and Jose Ramon Marin Tebar for technical assistance. This work was supported by grant SAF2008-05143-C03-1 from CICYT: Investigación sobre drogodependencias. Ministerio de Sanidad y Consumo (04005-00) and PI2007/55 Consejería de Sanidad from Junta de Comunidades de Castilla-La Mancha (to J.J.), by “CCM Obra Social y Cultural-FISCAM” and “Incorporación de grupos emergentes” FIS CARLOS III (to M.F.G.) and by BFU2008-01328 and TRA2009-0185 from Ministerio de
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