ll-O-Tetradecanoylphorbol-lS-acetate-induced Apoptosis in LNCaP Cells Is Mediated through Ceramide Synthase1

Protein kinase C i I'M i activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKCa by 12-0-tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that (/<• novo ceramide generation through the enzyme ceramide synthase is required. TPA induced rapid ceramide generation, which was detecta ble by l h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with (unionism B,, a specific natural inhibitor of ceramide synthase, abrogated both ceramide production and TPA-in duced apoptosis. Ceramide analogues bypassed fumonisin B, inhibition to initiate apoptosis directly. Thus, ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.