Potentiation of tumor necrosis factor-α-induced cell death by rottlerin through a cytochrome-c-independent pathway

The protein kinase C (PKC) signal transduction pathway negatively regulates receptor-initiated cell death. In HeLa cells, tumor necrosis factor-α (TNF)-mediated cell death involved mitochondria and was blocked by the overexpression of Bcl-2. The PKC-specific inhibitor bisindolylmaleimide and the PKCδ inhibitor rottlerin enhanced TNF-induced cell death. We have investigated if potentiation of TNF-induced cell death by rottlerin involved amplification of the mitochondrial pathway. TNF induced cleavage of the proapoptotic protein Bid and release of mitochondrial cytochrome c. Rottlerin enhanced activation of caspase-8 and cleavage of Bid. It also enhanced activation of caspase-9 but it did not increase cytochrome c in the cytosol. It, however, increased release of mitochondrial apoptosis-inducing factor (AIF) to the cytosol. Overexpression of Bcl-2 prevented release of both cytochrome c and AIF to the cytosol. Prolonged exposure (≥6 h) of HeLa cells to rottlerin and TNF decreased the level of cytochrome c but not of AIF in the cytosol. These results suggest that rottlerin activates a cytochrome-c-independent cell death pathway to potentiate cell death by TNF.