Abstract

In our previous studies, we demonstrated the apoptotic cascades protein kinase C (PKC) δ/c-Jun NH₂-terminal kinase (JNK)/Fas/caspases induced by penta-acetyl geniposide [(Ac)₅GP]. However, the upstream signals mediating PKCδ activation have not yet been clarified. Ceramide, mainly generated from the degradation of sphingomyelin, was hypothesized upstream above PKCδ in (Ac)₅GP-transduced apoptosis. Furthermore, nerve growth factor (NGF)/p75 is supposed to be involved because(Ac)₅GP-induced apoptosis was demonstrated previously in glioma cells. In the present study, (Ac)₅GP was shown to activate neutral sphingomyelinase (N-SMase) immediately, with its maximum at 15 min. The NGF and p75 enhanced by (Ac)₅GP was inhibited when added with GW4869, the N-SMase inhibitor, indicating NGF/p75 as the downstream signals of N-SMase/ceramide. To investigate whether N-SMase is involved in (Ac)₅GP-transduced apoptotic pathway, cells were treated with (Ac)₅GP added with or without GW4869. It showed that N-SMase inhibition blocked FasL expression and caspase 3 activation. Likewise, p75 antagonist peptide attenuated the FasL/caspase 3 expression. The PKCδ translocation induced by (Ac)₅GP was also eliminated by GW4869 and p75 antagonist peptide. To further confirm whether N-SMase activation plays an important role in (Ac)₅GP-induced apoptosis, cells were analyzed the apoptotic rate by 4′, 6-diamidino-2-phenylindole (DAPI) staining. (Ac)₅GP-induced apoptosis was reduced 40 and 80% by 10 and 20 μM GW4869, respectively. It indicated that N-SMase activation is pivotal in (Ac)₅GP-mediated apoptosis. In conclusion, SMase and NGF/p75 are suggested to mediate upstream above PKCδ, thus transducing FasL/caspase cascades in (Ac)₅GP-induced apoptosis.