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

Abstract

In order to explain the contribution of the protein kinase C alpha (PKC alpha) in apoptosis induced by photo-activation of hypericin (Hyp), a small interfering RNA was used for post-transcriptional silencing of pkc alpha gene expression. We have evaluated the influence of Hyp photo-activation on cell death in non-transfected and transfected (PKC alpha(-)) human glioma cells (U-87 MG). No significant differences were detected in cell survival between non-transfected and transfected PKC alpha(-) cells. However, the type of cell death was notably affected by silencing the pkc alpha gene. Photo-activation of Hyp strongly induced apoptosis in non-transfected cells, but the level of necrotic cells in transfected PKC alpha(-) 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 pkc alpha gene and the related decrease of PKC alpha level considerably affects the anti-apoptotic function and the anti-oxidant function of Bcl-2. This implies that PKC alpha, as Bcl-2 kinase, indirectly protects U-87 MG cells against oxidative stress and subsequent cell death.