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Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y CELLS

Abstract

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.

Abbreviations

ATCC:

American Type Culture Collection

EDTA:

ethylenediaminetetraacetic acid

EGTA:

ethyleneglycoltetraacetic acid

FBS:

fetal bovine serum

GSK3:

glycogen synthase kinase 3

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IP:

immunoprecipitation

MEM:

minimum essential medium

PARP:

poly ADP ribose polymerase

PMSF:

phenylmethylsulfonylfluoride

PUMA:

the p53 upregulated modulator of apoptosis

SDS:

sodium dodecyl sulfate

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Correspondence to Piyajit Watcharasit.

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Ngok-Ngam, P., Watcharasit, P., Thiantanawat, A. et al. Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y CELLS. Cell Mol Biol Lett 18, 58–74 (2013). https://doi.org/10.2478/s11658-012-0039-y

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  • DOI: https://doi.org/10.2478/s11658-012-0039-y

Key words

  • GSK3
  • p53
  • Mitochondrial translocation
  • Apoptosis
  • Bax oligomerization
  • Cytochrome C
  • Phosphorylation
  • Etoposide
  • Doxorubicin
  • Camptothecin