Penta-acetyl geniposide induce apoptosis in C6 glioma cells by modulating the activation of neutral sphingomyelinase-induced p75 nerve growth factor receptor and protein kinase Cdelta pathway.
In our previous studies, we demonstrated the apoptotic cascades protein kinase C (PKC) delta/c-Jun NH2-terminal kinase (JNK)/Fas/caspases induced by penta-acetyl geniposide [(Ac)5GP]. However, the upstream signals mediating PKCdelta activation have not yet been clarified. Ceramide, mainly generated from the degradation of sphingomyelin, was hypothesized upstream above PKCdelta 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 PKCdelta 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 microM 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 PKCdelta, thus transducing FasL/caspase cascades in (Ac)5GP-induced apoptosis.
Peng
Chiung-Huei
CH
Department of Nursing, Hungkuang University, Sha Lu, Taichung, Taiwan.
Huang
Chien-Ning
CN
Hsu
Shu-Ping
SP
Wang
Chau-Jong
CJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein; D005910:Glioma / Q000473:pathology*; D039823:Iridoids / Q000737:chemistry / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D020932:Nerve Growth Factor / Q000494:pharmacology; D051745:Protein Kinase C-delta / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011714:Pyrans / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D048069:Tumor Necrosis Factors / Q000378:metabolism
D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D039823:Iridoids; D008562:Membrane Glycoproteins; D011714:Pyrans; D020800:Receptor, Nerve Growth Factor; D048069:Tumor Necrosis Factors; C007835:geniposide; D020932:Nerve Growth Factor; D051745:Protein Kinase C-delta; D013108:Sphingomyelin Phosphodiesterase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases