Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells

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Abstract

Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5 mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors.

Section snippets

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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