pubmed.ncbi.nlm.nih.gov

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression - PubMed

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression

Björn Kruspig et al. Cell Mol Life Sci. 2012 Jun.

Abstract

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca(2+) into the cytosol, compromised Ca(2+) buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca(2+) accumulation or chelation of cytosolic Ca(2+) rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1

Effect of MycN downregulation on apoptotic manifestations in Tet21 N cells treated with 10 µg/ml cisplatin for 16 h. a Incubation of Tet21 N cells with 0.1 μg/ml doxycycline blocks expression of MycN oncogene; b switching off MycN suppresses cytochrome c release, caspase-3-like activity (c), number of floating cells (d), processing of caspase-3 and PARP cleavage in response to 10 μg/ml cisplatin (e), black bars: MycN overexpressing cells, white bars: MycN non-expressing Tet21 N cells, *p < 0.05; f analysis of apoptotic morphology in cisplatin-treated MycN(+) and MycN(−) Tet21 N cells; numbers show the percentage of cells with apoptotic nuclei. Number of counted cells: 720 for MycN(+) cells and 450 for MycN(−) cells; g analysis of PS externalization in MycN(+) and MycN(−) Tet21 N cells in response to 10 μg/ml cisplatin; h upper blot: 10 μg/ml cisplatin-stimulated expression of p53 in MycN(+) and MycN(−) cells; middle blot: phosphorylation of p53; lower blot: cisplatin-induced expression of Bak

Fig. 2
Fig. 2

Effect of MycN downregulation on apoptotic manifestations in Tet21 N cells treated with 60 μM α-TOS for 16 h. a α-TOS equally stimulates cell death assessed by the release of cytochrome c (a), caspase-3-like activity (b), the number of floating cells (c), and processing of caspase-3 (d) in MycN(+) (black bars) and MycN(−) (white bars) Tet21 N cells; e α-TOS-induced release of AIF from mitochondria in MycN(+) and MycN(−) Tet21 N cells; f analysis of apoptotic morphology in α-TOS-treated MycN(+) and MycN(−) Tet21 N cells. Numbers show the percentage of cells with apoptotic nuclei. Number of counted cells: 560 for MycN(+) cells and 260 for MycN(−) cells; g upper blot: p53 expression, lower blot: Bak expression in α-TOS treated MycN(+) and MycN(−) Tet21 N cells; h switching off MycN attenuated α-TOS-induced expression of Noxa; i α-TOS-induced cytochrome c release, caspase-3 cleavage, and MycN expression in various NB cells. Lower blot: loading control

Fig. 3
Fig. 3

Alteration of cytosolic and mitochondrial Ca2+ homeostasis in NB cells. a 60 μM α-TOS stimulates Ca2+ transients in MycN(+) and MycN(−) cells. b Chelation of intracellular Ca2+ by BAPTA AM attenuates caspase-3-like activity in Tet21 N cells, *p < 0.05; c ROS accumulation in MycN(+) cells treated with 60 μM α-TOS for 16 h. d Fold increase in ROS content after 16 h incubation of MycN(+) and MycN(−) cells with 60 μM α-TOS, *p < 0.05. The results were calculated as average from at least ten responding cells. e Incubation with 60 μM α-TOS for 16 h decreases the content of SH-groups in NB cells; NAC, 5 mM, *p < 0.05; f NAC and cyclosporin A (5 μM) prevent α-TOS-mediated stimulation of caspase-3-like activity

Fig. 4
Fig. 4

Mitochondrial Ca2+ accumulation in NB cells with different level of MycN expression. a Accumulation of Ca2+ by mitochondria in digitonin-permeabilized MycN(+) cells. Pulses of Ca2+ (20 nmol) were added sequentially until MPT was induced and the accumulated Ca2+ was released; b effect of 60 μM α-TOS and 10 μg/ml cisplatin on mitochondrial Ca2+ capacity (the threshold level of Ca2+ required for MPT induction) in MycN(+) and MycN(−) Tet21 N cells, *p < 0.05

Similar articles

Cited by

References

    1. Cole MD, Henriksson M. 25 years of the c-Myc oncogene. Semin Cancer Biol. 2006;16:241. doi: 10.1016/j.semcancer.2006.08.003. - DOI - PubMed
    1. Fulda S, Lutz W, Schwab M, Debatin KM. MycN sensitizes neuroblastoma cells for drug-induced apoptosis. Oncogene. 1999;18:1479–1486. doi: 10.1038/sj.onc.1202435. - DOI - PubMed
    1. Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta. 2006;1757:639–647. doi: 10.1016/j.bbabio.2006.03.016. - DOI - PubMed
    1. Hagland H, Nikolaisen J, Hodneland LI, Gjertsen BT, Bruserud O, Tronstad KJ. Targeting mitochondria in the treatment of human cancer: a coordinated attack against cancer cell energy metabolism and signalling. Expert Opin Ther Targets. 2007;11:1055–1069. doi: 10.1517/14728222.11.8.1055. - DOI - PubMed
    1. Ralph SJ, Neuzil J. Mitocans, a class of emerging anti-cancer drugs. Mol Nutr Food Res. 2009;53:7–8. doi: 10.1002/mnfr.200890054. - DOI - PubMed

Publication types

MeSH terms

Substances