Skip to main content
Download PDF
  • Research article
  • Published:

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

Cellular & Molecular Biology Letters volume 18, pages 58–74 (2013)Cite this article

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

References

  1. Beurel, E. and Jope, R.S. The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog. Neurobiol. 79 (2006) 173–189.

    Article  PubMed  CAS  Google Scholar 

  2. Kaidanovich-Beilin, O. and Woodgett, J.R. GSK-3: functional insights from cell biology and animal models. Front Mol. Neurosci. 4 (2011) 40.

    Article  PubMed  CAS  Google Scholar 

  3. Gomez-Sintes, R., Hernandez, F., Lucas, J.J. and Avila, J. GSK-3 mouse models to study neuronal apoptosis and neurodegeneration. Front. Mol. Neurosci. 4 (2011) 45.

    Article  PubMed  CAS  Google Scholar 

  4. Watcharasit, P., Bijur, G.N., Zmijewski, J.W., Song, L., Zmijewska, A., Chen, X., Johnson, G.V. and Jope, R.S. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc. Natl. Acad. Sci. USA 99 (2002) 7951–7955.

    Article  PubMed  CAS  Google Scholar 

  5. Qu, L., Huang, S., Baltzis, D., Rivas-Estilla, A.M., Pluquet, O., Hatzoglou, M., Koumenis, C., Taya, Y., Yoshimura, A. and Koromilas, A.E. Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta. Genes Dev. 18 (2004) 261–277.

    Article  PubMed  CAS  Google Scholar 

  6. Song, L., De Sarno, P. and Jope, R.S. Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. J. Biol. Chem. 277 (2002) 44701–44708.

    Article  PubMed  CAS  Google Scholar 

  7. Kim, A.J., Shi, Y., Austin, R.C. and Werstuck, G.H. Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3. J. Cell Sci. 118 (2005) 89–99.

    Article  PubMed  CAS  Google Scholar 

  8. King, T.D., Bijur, G.N. and Jope, R.S. Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res. 919 (2001) 106–114.

    Article  PubMed  CAS  Google Scholar 

  9. King, T.D. and Jope, R.S. Inhibition of glycogen synthase kinase-3 protects cells from intrinsic but not extrinsic oxidative stress. Neuroreport 16 (2005) 597–601.

    Article  PubMed  CAS  Google Scholar 

  10. Pap, M. and Cooper, G.M. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. J. Biol. Chem. 273 (1998) 19929–19932.

    Article  PubMed  CAS  Google Scholar 

  11. Watcharasit, P., Bijur, G.N., Song, L., Zhu, J., Chen, X. and Jope, R.S. Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53. J. Biol. Chem. 278 (2003) 48872–48879.

    Article  PubMed  CAS  Google Scholar 

  12. Turenne, G.A. and Price, B.D. Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53’s transcriptional activity. BMC Cell Biol. 2 (2001) 12.

    Article  PubMed  CAS  Google Scholar 

  13. Greenblatt, M.S., Bennett, W.P., Hollstein, M. and Harris, C.C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54 (1994) 4855–4878.

    PubMed  CAS  Google Scholar 

  14. Harms, K., Nozell, S. and Chen, X. The common and distinct target genes of the p53 family transcription factors. Cell. Mol. Life Sci. 61 (2004) 822–842.

    Article  PubMed  CAS  Google Scholar 

  15. Dumont, P., Leu, J.I., Della Pietra, A.C. III, George, D.L. and Murphy, M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat. Genet. 33 (2003) 357–365.

    Article  PubMed  CAS  Google Scholar 

  16. Marchenko, N., Zaika, A. and Moll, U. Death signal-induced localization of p53 protein to mitochondria A potential role in apoptotic signaling. J. Biol. Chem. 275 (2000) 16202–16212.

    Article  PubMed  CAS  Google Scholar 

  17. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P. and Moll, U.M. p53 has a direct apoptogenic role at the mitochondria. Mol. Cell 11 (2003) 577–590.

    Article  PubMed  CAS  Google Scholar 

  18. Moll, U.M., Wolff, S., Speidel, D. and Deppert, W. Transcription-independent pro-apoptotic functions of p53. Curr. Opin. Cell Biol. 17 (2005) 631–636.

    Article  PubMed  CAS  Google Scholar 

  19. Murphy, M.E., Leu, J.I. and George, D.L. p53 moves to mitochondria: a turn on the path to apoptosis. Cell Cycle 3 (2004) 836–839.

    Article  PubMed  CAS  Google Scholar 

  20. Watcharasit, P., Thiantanawat, A. and Satayavivad, J. GSK3 promotes arsenite-induced apoptosis via facilitation of mitochondria disruption. J. Appl. Toxicol. 28 (2008) 466–474.

    Article  PubMed  CAS  Google Scholar 

  21. Chabner, B.A., Amrein, P.C., Druker, B.J., Michaelson, M.D., Mitsiades, C.S., Goss, P.E., Ryan, D.P., Ramachandra, S., Richardson, P.G., Supko, J.G. and Wilson, W.H. Antineoplastic Agents. in: Goodman and Gilman’s The Pharmacological Basis of Therapeutics (Brunton, L.L., Ed.), 11th Edition, The McGraw-Hill Co. Inc., New York, 2005, 1315–1403.

    Google Scholar 

  22. Chalecka-Franaszek, E. and Chuang, D.M. Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc. Natl. Acad. Sci. USA 96 (1999) 8745–8750.

    Article  PubMed  CAS  Google Scholar 

  23. De Sarno, P., Li, X. and Jope, R.S. Regulation of Akt and glycogen synthase kinase-3 beta phosphorylation by sodium valproate and lithium. Neuropharmacology 43 (2002) 1158–1164.

    Article  PubMed  Google Scholar 

  24. Coghlan, M.P., Culbert, A.A., Cross, D.A., Corcoran, S.L., Yates, J.W., Pearce, N.J., Rausch, O.L., Murphy, G.J., Carter, P.S., Roxbee Cox, L., Mills, D., Brown, M.J., Haigh, D., Ward, R.W., Smith, D.G., Murray, K.J., Reith, A.D. and Holder, J.C. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol. 7 (2000) 793–803.

    Article  PubMed  CAS  Google Scholar 

  25. Erster, S., Mihara, M., Kim, R., Petrenko, O. and Moll, U. In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation. Mol. Cell. Biol. 24 (2004) 6728–6741.

    Article  PubMed  CAS  Google Scholar 

  26. Chen, X., Ko, L.J., Jayaraman, L. and Prives, C. p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev. 10 (1996) 2438–2451.

    Article  PubMed  CAS  Google Scholar 

  27. Haupt, Y., Rowan, S., Shaulian, E., Vousden, K.H. and Oren, M. Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev. 9 (1995) 2170–2183.

    Article  PubMed  CAS  Google Scholar 

  28. Ahn, B.Y., Trinh, D.L., Zajchowski, L.D., Lee, B., Elwi, A.N. and Kim, S.W. Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer. Oncogene 29 (2010) 1155–1166.

    Article  PubMed  CAS  Google Scholar 

  29. O’Connor, P.M., Jackman, J., Bae, I., Myers, T.G., Fan, S., Mutoh, M., Scudiero, D.A., Monks, A., Sausville, E.A., Weinstein, J.N., Friend, S., Fornace, A.J. Jr. and Kohn, K. W. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 57 (1997) 4285–4300.

    PubMed  Google Scholar 

  30. Olsson, A., Manzl, C., Strasser, A. and Villunger, A. How important are post-translational modifications in p53 for selectivity in target-gene transcription and tumour suppression? Cell Death Differ. 14 (2007) 1561–1575.

    Article  PubMed  CAS  Google Scholar 

  31. Vaseva, A.V. and Moll, U.M. The mitochondrial p53 pathway. Biochim. Biophys. Acta 1787 (2009) 414–420.

    Article  PubMed  CAS  Google Scholar 

  32. Chipuk, J., Kuwana, T., Bouchier-Hayes, L., Droin, N., Newmeyer, D., Schuler, M. and Green, D. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303 (2004) 1010–1014.

    Article  PubMed  CAS  Google Scholar 

  33. Marchenko, N.D., Wolff, S., Erster, S., Becker, K. and Moll, U.M. Monoubiquitylation promotes mitochondrial p53 translocation. EMBO J. 26 (2007) 923–934.

    Article  PubMed  CAS  Google Scholar 

  34. David, R. Apoptosis: A lipid trigger of MOMP. Nat. Rev. Mol. Cell Biol. 13 (2012) 208–209.

    Article  PubMed  CAS  Google Scholar 

  35. Moll, U., Marchenko, N. and Zhang, X. p53 and Nur77/TR3-transcription factors that directly target mitochondria for cell death induction. Oncogene 25 (2006) 4725–4743.

    Article  PubMed  CAS  Google Scholar 

  36. Linseman, D.A., Butts, B.D., Precht, T.A., Phelps, R.A., Le, S.S., Laessig, T.A., Bouchard, R.J., Florez-McClure, M.L. and Heidenreich, K.A. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J. Neurosci. 24 (2004) 9993–10002.

    Article  PubMed  CAS  Google Scholar 

  37. Tan, J., Zhuang, L., Leong, H.S., Iyer, N.G., Liu, E.T. and Yu, Q. Pharmacologic modulation of glycogen synthase kinase-3beta promotes p53-dependent apoptosis through a direct Bax-mediated mitochondrial pathway in colorectal cancer cells. Cancer Res. 65 (2005) 9012–9020.

    Article  PubMed  CAS  Google Scholar 

  38. Polakis, P. Wnt signaling and cancer. Genes Dev. 14 (2000) 1837–1851.

    PubMed  CAS  Google Scholar 

  39. Nemajerova, A., Erster, S. and Moll, U.M. The post-translational phosphorylation and acetylation modification profile is not the determining factor in targeting endogenous stress-induced p53 to mitochondria. Cell Death Differ. 12 (2005) 197–200.

    Article  PubMed  CAS  Google Scholar 

  40. Guan, L., Huang, F., Li, Z., Han, B., Jiang, Q., Ren, Y., Yang, Y. and Xu, C. P53 transcription-independent activity mediates selenite-induced acute promyelocytic leukemia NB4 cell apoptosis. BMB Rep. 41 (2008) 745–750.

    Article  PubMed  CAS  Google Scholar 

  41. Park, B.S., Song, Y.S., Yee, S.B., Lee, B.G., Seo, S.Y., Park, Y.C., Kim, J.M., Kim, H. M. and Yoo, Y.H. Phospho-ser 15-p53 translocates into mitochondria and interacts with Bcl-2 and Bcl-xL in eugenol-induced apoptosis. Apoptosis 10 (2005) 193–200.

    Article  PubMed  CAS  Google Scholar 

  42. Bijur, G.N. and Jope, R.S. Glycogen synthase kinase-3 beta is highly activated in nuclei and mitochondria. Neuroreport 14 (2003) 2415–2419.

    Article  PubMed  CAS  Google Scholar 

  43. Talos, F., Petrenko, O., Mena, P. and Moll, U.M. Mitochondrially targeted p53 has tumor suppressor activities in vivo. Cancer Res. 65 (2005) 9971–9981.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Chulabhorn Graduate Institute, 54 Kamphaeng Phet 6 Road, Lak Si, Bangkok, 10210, Thailand

    Patchara Ngok-Ngam, Piyajit Watcharasit, Apinya Thiantanawat & Jutamaad Satayavivad

  2. Laboratory of Pharmacology, Chulabhorn Research Institute, 54 Kamphaeng Phet 6 Road, Lak Si, Bangkok, 10210, Thailand

    Piyajit Watcharasit, Apinya Thiantanawat & Jutamaad Satayavivad

Authors
  1. Patchara Ngok-Ngam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Piyajit Watcharasit
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Apinya Thiantanawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jutamaad Satayavivad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Piyajit Watcharasit.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

Key words

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us