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.
Footnotes
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This work was supported by the National Science Council, Taiwan under NSC 94-2320-B-241-007 and the Chung Shan Medical University, Taiwan under CSMC 92-OM-B-024.
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ABBREVIATIONS: (Ac)5GP, penta-acetyl geniposide; SMase, sphingomyelinase; N-SMase, neutral sphingomyelinase; A-SMase, acid sphingo-myelinase; NGF, nerve growth factor; NGFR, nerve growth factor receptor; PKCδ, protein kinase C δ; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; TNF-α, tumor necrosis factor-α; HPTLC, high-performance thin-layer chromatography; TEMED, N,N,N′,N′-tetramethylethylenediamine; DAPI, 4′,6-diamidino-2-phenylindole; GSH, glutathione; GW4869, C30H28N6O6 ·2HCl.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received January 5, 2006.
- Accepted June 7, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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