, Volume 19, Issue 12, pp 1779–1792 | Cite as

Effect of PKCα expression on Bcl-2 phosphorylation and cell death by hypericin

  • Jaroslava Joniova
  • Matus Misuth
  • Franck Sureau
  • Pavol Miskovsky
  • Zuzana NadovaEmail author
Original Paper


In order to explain the contribution of the protein kinase Cα (PKCα) in apoptosis induced by photo-activation of hypericin (Hyp), a small interfering RNA was used for post-transcriptional silencing of pkcα gene expression. We have evaluated the influence of Hyp photo-activation on cell death in non-transfected and transfected (PKCα) human glioma cells (U-87 MG). No significant differences were detected in cell survival between non-transfected and transfected PKCα cells. However, the type of cell death was notably affected by silencing the pkcα gene. Photo-activation of Hyp strongly induced apoptosis in non-transfected cells, but the level of necrotic cells in transfected PKCα cells increased significantly. The differences in cell death after Hyp photo-activation are demonstrated by changes in: (i) reactive oxygen species production, (ii) Bcl-2 phosphorylation on Ser70 (pBcl-2(Ser70)), (iii) cellular distributions of pBcl-2(Ser70) and (iv) cellular distribution of endogenous anti-oxidant glutathione and its co-localization with mitochondria. In summary, we suggest that post-transcriptional silencing of the pkcα gene and the related decrease of PKCα level considerably affects the anti-apoptotic function and the anti-oxidant function of Bcl-2. This implies that PKCα, as Bcl-2 kinase, indirectly protects U-87 MG cells against oxidative stress and subsequent cell death.


Hypericin PKCα Bcl-2 Apoptosis Necrosis Mitochondria ROS GSH 



This work was supported by: the FP7 EU project CELIM 316310, the project of the Slovak Res. and Dev. Agency APVV-0242-11, the project of the Agency of the Ministry of Education of Slovak (Republic for the Structural funds of the European Union: Doktorand (ITMS code: 26110230013) and KVARK (ITMS code: 26110230084) and by the International Program for Scientific Cooperation (PICS N°5398) from the CNRS. This work forms a part of the co-tutoring doctoral study of J. J. (P. J. Safarik University and UPMC).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Mellor H, Parker PJ (1998) The extended protein kinase C superfamily. Biochem J 332:281–292PubMedCentralPubMedGoogle Scholar
  2. 2.
    Nishizuka Y (1995) Protein kinases.5. Protein-kinase-C and lipid signaling for sustained cellular-responses. Faseb Journal 9:484–496PubMedGoogle Scholar
  3. 3.
    Jiffar T, Kurinna S, Suck G et al (2004) PKC alpha mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation. Leukemia 18:505–512PubMedCrossRefGoogle Scholar
  4. 4.
    Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N (2013) Crosstalk between apoptosis, necrosis and autophagy. Biochimica Et Biophysica Acta-Molecular Cell Research 1833:3448–3459CrossRefGoogle Scholar
  5. 5.
    Merino D, Bouillet P (2009) The Bcl-2 family in autoimmune and degenerative disorders. Apoptosis 14:570–583PubMedCrossRefGoogle Scholar
  6. 6.
    Koc M, Nad’ova Z, Truksa J, Ehrlichova M, Kovar J (2005) Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation. Apoptosis 10:381–393PubMedCrossRefGoogle Scholar
  7. 7.
    Ackermann EJ, Taylor JK, Narayana R, Bennett CF (1999) The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem 274:11245–11252PubMedCrossRefGoogle Scholar
  8. 8.
    Ruvolo PP, Deng XM, Carr BH, May WS (1998) A functional role for mitochondrial protein kinase C alpha in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem 273:25436–25442PubMedCrossRefGoogle Scholar
  9. 9.
    Blagosklonny MV, Giannakakou P, el-Deiry WS et al (1997) Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res 57:130–135PubMedGoogle Scholar
  10. 10.
    Voehringer DW (1999) BCL-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity. Free Radic Biol Med 27:945–950PubMedCrossRefGoogle Scholar
  11. 11.
    Franco R, Cidlowski JA (2009) Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ 16:1303–1314PubMedCrossRefGoogle Scholar
  12. 12.
    Circu ML, Aw TY (2008) Glutathione and apoptosis. Free Radical Res 42:689–706CrossRefGoogle Scholar
  13. 13.
    Wilkins HM, Marquardt K, Lash LH, Linseman DA (2012) Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione. Free Radic Biol Med 52:410–419PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Malik F, Kumar A, Bhushan S et al (2007) Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound with a ferin A with concomitant protection by N-acetyl cysteine. Apoptosis 12:2115–2133PubMedCrossRefGoogle Scholar
  15. 15.
    Papa L, Gomes E, Rockwell P (2007) Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death. Apoptosis 12:1389–1405PubMedCrossRefGoogle Scholar
  16. 16.
    Shen S, Zhang Y, Zhang R, Gong X (2013) Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells. Biochem Biophys Res Commun 441:519–524PubMedCrossRefGoogle Scholar
  17. 17.
    Miskovsky P (2002) Hypericin - A new antiviral and antitumor photosensitizer: mechanism of action and interaction with biological macromolecules. Curr Drug Targets 3:55–84PubMedCrossRefGoogle Scholar
  18. 18.
    Kocanova S, Buytaert E, Matroule JY et al (2007) Induction of heme-oxygenase 1 requires the p38(MAPK) and PI3K pathways and suppresses apoptotic cell death following hypericin-mediated photodynamic therapy. Apoptosis 12:731–741PubMedCrossRefGoogle Scholar
  19. 19.
    Huntosova V, Alvarez L, Bryndzova L et al (2010) Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells. Int J Pharm 389:32–40PubMedCrossRefGoogle Scholar
  20. 20.
    Kocanova S, Hornakova T, Hritz J et al (2006) Characterization of the interaction of hypericin with protein kinase c in U-87 MG human glioma cells. Photochem Photobiol 82:720–728PubMedCrossRefGoogle Scholar
  21. 21.
    Dzurova L, Petrovajova D, Nadova Z, Huntosova V, Miskovsky P, Stroffekova K (2014) The role of anti-apoptotic protein kinase Calpha in response to hypericin photodynamic therapy in U-87 MG cells. Photodiagn Photodyn Ther 11:213–226CrossRefGoogle Scholar
  22. 22.
    Petrovajova D, Jancura D, Miskovsky P et al (2013) Monitoring of singlet oxygen luminescence and mitochondrial autofluorescence after illumination of hypericin/mitochondria complex: a time-resolved study. Laser Phys Lett 10:7CrossRefGoogle Scholar
  23. 23.
    Buriankova L, Nadova Z, Jancura D et al (2010) Synchrotron based Fourier-transform infrared microspectroscopy as sensitive technique for the detection of early apoptosis in U-87 MG cells. Laser Phys Lett 7:613–620CrossRefGoogle Scholar
  24. 24.
    Kascakova S, Nadova Z, Mateasik A et al (2008) High level of low-density lipoprotein receptors enhance hypericin uptake by U-87 MG cells in the presence of LDL. Photochem Photobiol 84:120–127PubMedGoogle Scholar
  25. 25.
    Ehrlichova M, Koc M, Truksa J, Naldova Z, Vaclavikova R, Kovarr J (2005) Cell death induced by taxanes in breast cancer cells: cytochrome c is released in resistant but not in sensitive cells. Anticancer Res 25:4215–4224PubMedGoogle Scholar
  26. 26.
    Mandavilli BS, Janes MS. (2010) Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy. Curr Protoc cytom Chapter 9:Unit 9.35–Unit 39.35Google Scholar
  27. 27.
    Huntosova V, Nadova Z, Dzurova L, Jakusova V, Sureau F, Miskovsky P (2012) Cell death response of U87 glioma cells on hypericin photoactivation is mediated by dynamics of hypericin subcellular distribution and its aggregation in cellular organelles. Photochem Photobiol Sci 11:1428–1436PubMedCrossRefGoogle Scholar
  28. 28.
    Adhikary G, Chew YC, Reece EA, Eckert RL (2010) PKC-delta and -eta, MEKK-1, MEK-6, MEK-3, and p38-delta Are Essential Mediators of the Response of Normal Human Epidermal Keratinocytes to Differentiating Agents. J Invest Dermatol 130:2017–2030PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Lahn M, Kohler G, Sundell K et al (2004) Protein kinase C alpha expression in breast and ovarian cancer. Oncology 67:1–10PubMedCrossRefGoogle Scholar
  30. 30.
    Finkel T (2011) Signal transduction by reactive oxygen species. J Cell Biol 194:7–15PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Lindsay J, Esposti MD, Gilmore AP (2011) Bcl-2 proteins and mitochondria-Specificity in membrane targeting for death. Biochim Biophys Acta 1813:532–539PubMedCrossRefGoogle Scholar
  32. 32.
    Ruvolo PP, Deng X, May WS (2001) Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia 15:515–522PubMedCrossRefGoogle Scholar
  33. 33.
    Esteve JM, Mompo J, De La Asuncion JG et al (1999) Oxidative damage to mitochondrial DNA and glutathione oxidation in apoptosis: studies in vivo and in vitro. Faseb J 13:1055–1064PubMedGoogle Scholar
  34. 34.
    Zimmermann AK, Loucks FA, Schroeder EK, Bouchard RJ, Tyler KL, Linseman DA (2007) Glutathione binding to the Bcl-2 homology-3 domain groove - A molecular basis for Bcl-2 antioxidant function at mitochondria. J Biol Chem 282:29296–29304PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Vairetti M, Ferrigno A, Bertone R, Richelmi P, Berte F, Freitas I (2005) Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes. Biochim Biophys Acta 1740:367–374PubMedCrossRefGoogle Scholar
  36. 36.
    Benderdour M, Charron G, Comte B et al (2004) Decreased cardiac mitochondrial NADP(+)-isocitrate dehydrogenase activity and expression: a marker of oxidative stress in hypertrophy development. Am J Physiol Heart Circ Physiol 287:H2122–H2131PubMedCrossRefGoogle Scholar
  37. 37.
    Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251PubMedCrossRefGoogle Scholar
  38. 38.
    Kane DJ, Sarafian TA, Anton R et al (1993) Bcl-2 inhibition of neural death - decreased generation of reactive oxygen species. Science 262:1274–1277PubMedCrossRefGoogle Scholar
  39. 39.
    Voehringer DW, Meyn RE (2000) Redox Aspects of Bcl-2 Function. Antioxid Redox Signal 2:537–550PubMedCrossRefGoogle Scholar
  40. 40.
    Hochman A, Sternin H, Gorodin S et al (1998) Enhanced oxidative stress and altered antioxidants in brains of Bcl-2-deficient mice. J Neurochem 71:741–748PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jaroslava Joniova
    • 1
    • 2
  • Matus Misuth
    • 1
  • Franck Sureau
    • 2
  • Pavol Miskovsky
    • 1
    • 3
  • Zuzana Nadova
    • 1
    • 3
    Email author
  1. 1.Department of Biophysics, Faculty of ScienceUniversity of Pavol Jozef SafarikKosiceSlovak Republic
  2. 2.Laboratoire Jean Perrin LJPCNRS/UPMC Univ Paris 06ParisFrance
  3. 3.Centre for Interdisciplinary BiosciencesUniversity of Pavol Jozef SafarikKosiceSlovak Republic

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