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【关键词】 penta-acetyl
In our previous studies, we demonstrated the apoptotic cascades protein kinase C (PKC) /c-Jun NH2-terminal kinase (JNK)/Fas/caspases induced by penta-acetyl geniposide [(Ac)5GP]. However, the upstream signals mediating PKC activation have not yet been clarified. Ceramide, mainly generated from the degradation of sphingomyelin, was hypothesized upstream above PKC 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 PKC 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 PKC, thus transducing FasL/caspase cascades in (Ac)5GP-induced apoptosis.
Penta-acetyl geniposide [1-(-D-2',3',4',6'-tetraacetyl-glucopyranosyloxy)-1,4a,5,7a-tetrahydro-7-(acetomethyl)-cyclopentapyran-4-carboxylic acid methyl ester; (Ac)5GP, Fig. 1] is an herbal derivative prepared from Gardenia geniposide. It has been suggested that (Ac)5GP plays more potent roles than geniposide in chemoprevention (Peng et al., 2005a). In addition, (Ac)5GP possesses the antitumor effect on apoptosis and growth arrest (Wang et al., 1993; Chang et al., 2002, 2004).
Fig. 1. Structure of (Ac)5GP.
In our previous studies, we demonstrated that (Ac)5GP may transduce the apoptotic signals through PKC activation and the downstream cascades of JNK/Jun phosphorylation, FasL/Fas elevation, and the subsequent activation of caspase 8 and caspase 3 (Peng et al., 2004, 2005b). PKC seems to play the pivotal role in (Ac)5GP-mediated apoptosis of tumor cells. However, the upstream signals above PKC have not yet been clarified.
There exist different kinds of lipids in cell membrane, which could be released and transduce the secondary signals while cells were being stimulated. Among all of them, one derivative of sphingolipid, ceramide, showed to be highly involved in apoptotic pathways (Obeid et al., 1993). The generation of ceramide mainly depends on the degradation of sphingomyelin, which is catalyzed by sphingomyelinase (SMase) (Jaffrezou et al., 1996). Ceramide regulates a variety of cell functions, including growth arrest, differentiation, aging, apoptosis, and immune response (Hannun, 1996). Obeid et al. (1993) showed that ceramide analog, such as TNF-, induced DNA fragmentation in leukemia cells (Obeid et al., 1993). Defective sphingomyelin/ceramide signaling showed resistance to radiation therapy in Burkitt's lymphoma, breast carcinoma, and myeloid leukemia cells (Cai et al., 1997; Michael et al., 1997; Bruno et al., 1998).
It was suggested that ceramide could mediate apoptotic signals via PKC isoforms. Ceramide translocated PKC and - (from membrane) to the cytosol, thus induced the apoptosis of leukemia cells (Sawai et al., 1997). In prostate cancer cell lines, ceramide generation was found only in PKC (+) cells. Ceramide translocated PKC to mitochondria, inducing the subsequent release of cytochrome c and activation of caspase 9, thus further amplifying the ceramide-mediated mitochondrial amplification loop (Sumitomo et al., 2002). Ceramide has been shown to translocate PKC to Golgi apparatus, and then transduce the tyrosine phosphorylation, activation, and apoptotic signals (Kajimoto et al., 2004). Therefore, ceramide might be hypothesized as the possible upstream signal of PKC in (Ac)5GP-induced apoptosis.
In the previous studies, apoptotic signals induced by (Ac)5GP were demonstrated in C6 glioma cells, which is derived from the nervous system (Benda et al., 1968). It has been shown that nerve growth factor (NGF) regulates the growth, differentiation, and apoptosis of glioma cells (Rabin et al., 1998). The biological functions of NGF are mediated by its high-affinity receptor, trkA, and the low-affinity receptor p75NTR (Chao and Hempstead, 1995). trkA contains a cytosol tyrosine kinase domain, which is not found in the wide-expressed p75 receptor. The collaboration of trkA and p75 potentiate functional responses to neurotrophins (Hantzopoulos et al., 1994). It was also reported that p75 plays an important role in apoptotic pathway (Rabizadeh et al., 1993). The antibody to the extracellular domain of p75 NGF receptor (NGFR) mediated apoptosis in sympathetic neurons (Freidin, 2001), whereas antagonist to p75 neurotrophin receptor retarded apoptosis-driven hair follicle involution (Botchkarev et al., 2003). It was suggested that p75 NGFR has some functional similarities to the superfamily of TNF- receptors, Fas and CD40 (Smith et al., 1994).
Ceramide and SMase have been reported as capable of inducing NGF synthesis in primary astrocyte cultures, implying that there may exist the cross-talks between ceramide and NGFR signals in nervous system (Galve-Roperh et al., 1997). Thus, in the present study, we investigated whether ceramide/SMase and NGF/NGFR are involved in (Ac)5GP-mediated programmed death in glioma cells and clarified the upstream transduction pathway above PKC.
Chemicals. Minimal essential medium, fetal calf serum, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Amplex Red Sphingomyelinase kit is a product of Molecular Probes (Carlsbad, CA). SDS, bis-acrylamide, ammonium persulfate, TEMED, and nitrocellulose paper were from Bio-Rad (Hercules, CA). PKC antibody was purchased from BD Transduction Laboratories (Lexington, KY). Triton X-100, Tris base, -actin antibody, nonhydroxy fatty acid ceramide, and DAPI were from Sigma (St. Louis, MO). GW4869, a reported specific inhibitor for N-SMase, was also purchased from Sigma (Luberto et al., 2002). Antibodies of NGF, NGFR p75, Fas-L, and caspase 3 were from Santa Cruz Biotechnology (Santa Cruz, CA). High-performance thin-layer chromatography (HPTLC) silica gel 60 plate was from Merck (Whitehouse Station, NJ). Lipofectamine was from Invitrogen (Carlsbad, CA). p75 antagonist peptide YCDIKGKECY (cysteine-cysteine bond resulting in cyclization is indicated by the underline), which has been reported as specific to NGF p75 receptor, was synthesized by Protech Technology (Reno, NV) with a minimum 90% purity (Li et al., 2005).
(Ac)5GP Preparation. (Ac)5GP was isolated and acetylated from Gardenia fructus with the same procedure used in our previous study (Wang et al., 1992). The stock solution of (Ac)5GP was prepared in dimethyl sulfoxide, protected from light, and stored at -20°C. Before use, the (Ac)5GP solution was freshly prepared in the medium at the desired concentration.
Cell Culture and Treatment. The rat C6 glioma cell line was originally derived from an N-nitrosomethylurea-induced rat brain tumor (Benda et al., 1968). C6 cells were maintained in minimal essential medium supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), at 37°C in a humidified atmosphere with 5% CO2. Cells were seeded at a density of 5 x 105 onto each 10-mm Petri dish 24 h before drug treatment.
DAPI Staining. We used DAPI staining to assess morphological changes in the chromatin structure of C6 glioma cells undergoing apoptosis. Cells were trypsinized, mounted on glass slides, and fixed in 4% paraformaldehyde for 30 min followed by staining with 1 µg/ml DAPI for 30 min. Apoptosis was characterized by chromatin condensation and fragmentation when examined by fluorescence microscopy. The incidence of apoptosis in each preparation was analyzed by counting 300 cells and determining the percentage of apoptotic cells.
Cell Fraction and PKC Translocation. Subcellular fractionation was performed as follows. Cells were washed with phosphate-buffered saline and added with cold buffer consisting of 20 mM Tris, 0.03 mM Na3VO4, 2 mM MgCl2 ·6H2O, 2 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 250 mM sucrose, and 10 µg/ml leupeptin, and then they were scraped into suspension and lysed by a homogenizer (Eyela Nazelax; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) on ice. The lysate was centrifuged at 50,000 rpm at 4°C for 1 h. The supernatant was collected and used as the cytosol fraction. The pellet was solubilized with 20 mM Tris, 0.03 mM Na3VO4, 5 mM MgCl2 ·6H2O, 2 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 5 mM NaF, 10 µg/ml leupeptin, and 0.1% Triton X-100 at 4°C and mixed by vortex every 10 min to extract soluble membrane proteins. One hour later, samples were recentrifuged at 50,000 rpm at 4°C for 1 h, and the supernatant was used as a membrane fraction. PKC expressed in the two fractions were then analyzed with immunoblot and densitometer.
Electrophoresis and Immunoblotting. Cells were harvested into lysis buffer containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% -mercaptoethanol and then lysed by sonication. Equal amounts of protein (10 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis and transfected onto nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin with 0.05% Tween 20 in phosphate-buffered saline and then incubated with the first antibody for 1 h. Membranes were then washed three times with 0.05% Tween 20 in phosphate-buffered saline and incubated with the secondary antibody conjugated to anti-mouse horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Bands were visualized by using the enhanced chemiluminescence Western blotting detection system. Protein quantitation was determined by densitometry using AlphaImager Series 2200 software (Alpha Innotech, San Leandro, CA).
Antisense Knockout Assay. Sense (5'-CCTCTTACCTCAGTTACAATTTATA-3') and antisense (5'-GCCGCAGAGAAAAGTTGTGCTTCAT-3') oligonucleotides for N-SMase were synthesized by Mission Biotech (Taipei, Taiwan) (GenBank accession number BC091139). Cells were plated at 70% density 24 h before antisense oligonucleotide treatment. The cells were washed and then incubated with antisense oligonucleotide (5 µM) in serum-free minimal essential medium containing 10 µg/ml Lipofectamine at 37°C. The medium was changed to 5% fetal bovine serum minimal essential medium 6 h later before culturing at 37°C for 24 h.
SMase Activity Assay. SMase activity was analyzed with Amplex Red Sphingomyelinase Assay Kit. Cells were washed with phosphate-buffered saline and pelleted by centrifugation at 1500g for 10 min at 4°C. The cell pellet was resuspended and lysed in buffer containing 1% Triton X-100, 1 µg/ml aprotinin, 1 mM EDTA, and 100 µg/ml phenylmethylsulfonyl fluoride for 60 min on ice and then centrifuged at 17,000g for 10 min at 4°C to remove the nuclei. The protein concentration in the supernatant was measured with BioRad Protein Assay. For analyzing the activity of N-SMase, the supernatant was diluted with 1x reaction buffer containing 0.1 M Tris-HCl and 10 mM MgCl2, pH 7.4, and then inoculated into the 96-well plate as 100 µl/well. The total protein amount of each well was approximately 25 µg. The solution was added with 100 µl of working solution (containing 8 U/ml alkaline phosphatase, 0.2 U/ml choline oxidase, 2 U/ml horseradish peroxidase, and 0.5 mM sphingomyelin) and incubated for 45 min at 37°C. The H2O2 generated thus reacted with Amplex Red to elicit the fluorescent resorufin. The fluorescent intensity was measured with Fluorescence Multi-Well Reader (HTS 7000) immediately at excitation/emission levels of 571/585 nm. On the other hand, acid sphingomyelinase (A-SMase) activity was analyzed with two-step assay. First, we used 50 mM sodium acetate (not 1x reaction buffer) to dilute the supernatant and inoculate 100 µl to each well of the 96-well plate. To generate phosphorylcholine and ceramide, 10 µl of 5 mM sphingomyelin solution was added to each well and then incubated for 1 h at 37°C. Working solution (100 µl) containing 8 U/ml alkaline phosphatase, 0.2 U/ml choline oxidase, and 2 U/ml horseradish peroxidase was then added to each well to react with phosphorylcholine for 45 min at 37°C. H2O2 generated from the two-step procedure then reacted with Amplex Red to elicit resorufin, which was measured with the Fluorescence Multi-Well Reader as mentioned above.
Quantitative Measurement of Ceramide Content. Cells were harvested into lysis buffer containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% -mercaptoethanol and then lysed by sonication. Lipids were extracted as the following procedures. In brief, 1 ml of cell lysate was suspended with 1.25 ml of chloroform and 2.5 ml of methanol, and then vortexed and incubated overnight at 4°C. After centrifugation for 5 min at 1700g, 1.25 ml of chloroform and 1.25 ml of 0.88% KCl were added to the supernatant. The tubes were vortexed and centrifuged for 5 min at 1700g. The organic lower phase was transferred into a new tube and evaporated under nitrogen. The lipids were dissolved into chloroform-methanol [2:1 (v/v)] and analyzed by HPTLC on an HPTLC silica gel 60 plate using dichloromethane-methanol-glacial acetic acid [100:2:5 (v/v)] for ceramides. Individual lipid classes were visualized by dipping each plate into CuSO4 (3%)/H3PO4 (8%) and then charring the plate on a heat block at 180°C for 10 min. Densitometric scanning of the bands and evaluation of the data were done with LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). In addition to three replicates of samples, nonhydroxy fatty acid ceramide was added to each plate as the lipid standard.
Fig. 2. SMase activated by (Ac)5GP. A, cells were treated with 0.6 mM (Ac)5GP for the indicated time. SMase activity was analyzed and was directly proportional to the fluorescence emitted. Data were presented as mean ± S.D., n = 3; *, P < 0.05; **, P < 0.005; ***, P < 0.001 compared with the control. B, cells were treated with 0.6 mM (Ac)5GP with or without 20 µM GW4869 for the indicated time. Ceramide levels are shown as percentages of the control group at 0 h. Std., standard nonhydroxy fatty acid ceramide. Data are presented as mean ± S.D., n = 3; *, P < 0.05; **, P < 0.005; ***, P < 0.001 compared with the control.
Statistical Analysis. Statistical differences were evaluated using the unpaired t test and were considered significant at the P < 0.05 level.
(Ac)5GP Induces N-SMase Activation. C6 glioma cells were treated with 0.6 mM (Ac)5GP at different time points until 3 h. SMase activity was presented as the percentage of fluorescence. Figure 2A showed that N-SMase reached the maximal activity at 15 min and then declined within 2 h, whereas A-SMase was not activated by (Ac)5GP. The N-SMase activity showed to be up-regulated approximately 70% by (Ac)5GP. Figure 2B showed the change of ceramide level. (Ac)5GP increased approximately 4-fold of ceramide within 2 h. Addition of GW4869 blocked the increase of ceramide induced by (Ac)5GP.
NGF/p75 Signals Downstream of N-SMase. C6 cells were treated with 0.6 mM (Ac)5GP at different time points. The protein level of NGF increased 1.5-fold at 6 h and more than 2-fold at 24 h (Fig. 3A). p75 also showed to be elevated by (Ac)5GP. At 9 h, p75 increased 2-fold compared with the control. Similar results were observed in ceramide-treated cells, implying that NGF and p75 could be downstream of ceramide generation (Fig. 3B). Figure 3C showed that the elevation of NGF and p75 by (Ac)5GP was reduced by GW4869, the N-SMase inhibitor. These data indicate NGF/p75 as the downstream signals of (Ac)5GP-induced N-SMase activation.
Fig. 3. NGF and p75 levels elevated by (Ac)5GP and downstream of the SMase signal in C6 cells. A, cells were treated with 0.6 mM (Ac)5GP at the indicated time. B, cells were treated with 30 µM ceramide at the indicated time. C, cells were added with 20 µM GW4869 for 1 h and then treated with 0.6 mM (Ac)5GP at the indicated time. The expression of NGF and p75 was analyzed by Western blot and densitometer. Three independent experiments were conducted, which showed similar pattern of changes.
N-SMase Activation and NGF/p75 Interaction is Involved in (Ac)5GP-Transduced FasL/Caspase 3 Cascades. To investigate whether N-SMase activation is involved in (Ac)5GP-transduced FasL/caspase 3 pathway, cells were treated with 0.6 mM (Ac)5GP added with or without GW4869 for 24 h. Figure 4A shows that (Ac)5GP enhanced the amount of FasL. It also increased the protein level and activation of caspase 3. N-SMase inhibition dose-dependently blocked FasL expression and caspase 3 activation induced by (Ac)5GP. In Fig. 4B, p75 antagonist peptide also is shown to reduce (Ac)5GP-mediated FasL/caspase cascades. Thus (Ac)5GP seems to transduce the death signals via N-SMase and NGF/p75.
Fig. 4. SMase and p75 mediating FasL expression and caspase 3 activation induced by (Ac)5GP in C6 cells. A, cells were treated with 0.6 mM (Ac)5GP with or without pretreatment of 10 or 20 µM GW4869. B, cells were added with or without 50 or 100 µM p75 antagonist peptide for 1 h and then treated with 0.6 mM (Ac)5GP for 24 h. Three independent experiments were conducted with the similar change.
N-SMase Activation and NGF/p75 Interaction Mediate PKC Translocation Induced by (Ac)5GP. Cells were treated with 0.6 mM (Ac)5GP with or without GW4869 or p75 antagonist for 9 h. Figure 5A showed that N-SMase inhibition reduced the translocation of PKC. Likewise, blocking the NGF/p75 interaction by peptide antagonist inhibited PKC translocation dose-dependently (Fig. 5B). Figures 4 and 5 demonstrate that N-SMase and NGF/p75 are involved in (Ac)5GP-transduced PKC activation and the downstream FasL/caspase cascades, which were reported in the previous study.
Fig. 5. SMase and p75 mediating the PKC translocation induced by (Ac)5GP in C6 cells. A, cells were treated with 0.6 mM (Ac)5GP with or without pretreatment of 10 or 20 µM GW4869. B, cells were treated with 0.6 mM (Ac)5GP with or without pretreatment of 50 or 100 µM p75 antagonist peptide. Translocation of PKC was analyzed as the protein level exist in different cell fractions. As shown in the bar graph, data are presented as mean ± S.D., n = 3; *, P < 0.005; **, P < 0.001 compared with the control. #, P < 0.05; ##, P < 0.005 compared with the cells treated with (Ac)5GP only.
(Ac)5GP Induces Apoptosis through N-SMase Activation. To further confirm that N-SMase activation plays an important role in (Ac)5GP-induced apoptosis, cells were treated with 0.6 mM (Ac)5GP with or without GW4869 for 24 h. The apoptotic rate was then analyzed by DAPI staining. Figure 6A, c and d, shows that (Ac)5GP induced apoptosis as ceramide-treated, whereas N-SMase inhibition dose-dependently eliminated the apoptosis induced by (Ac)5GP (Fig. 6A, e and f). As shown in the bar graph, (Ac)5GP-induced apoptosis was reduced 40 and 80% by 10 and 20 µM GW4869, respectively, indicating that N-SMase activation is critical in (Ac)5GP-mediated apoptosis. The pivotal role of N-SMase was further confirmed by antisense knockout assay (Fig. 6B). The antisense oligonucleotide of N-SMase eliminated more than 80% of apoptosis induced by (Ac)5GP (Fig. 6B, d and f).
Fig. 6. SMase mediating (Ac)5GP-induced apoptosis in C6 cells. Apoptosis rate was analyzed by DAPI staining. A, cells treated with vehicle only (a), 20 µM GW4869 (b), 30 µM ceramide (c), 0.6 mM (Ac)5GP (d), 0.6 mM (Ac)5GP added with 10 µM GW4869 (e), and 0.6 mM (Ac)5GP added with 20 µM GW4869 (f). As shown in the bar graph, data were presented as mean ± S.D., n = 3; *, P < 0.001 compared with the control. #, P < 0.05; ##, P < 0.005 compared with the cells treated with (Ac)5GP only. B, cells treated with Lipofectamine as the control group (a), 5 µM sense oligonucleotide (b), 5 µM antisense oligonucleotide (c), 0.6 mM (Ac)5GP (d), 0.6 mM (Ac)5GP added with 5 µM sense oligonucleotide (e), and 0.6 mM (Ac)5GP added with 5 µM antisense oligonucleotide (f). As shown in the bar graph, data are presented as mean ± S.D., n = 3; *, P < 0.001 compared with the control. #, P < 0.05; ##, P < 0.005 compared with the cells treated with (Ac)5GP only.
In the present study, we demonstrated that N-SMase plays a critical role as the upstream signal above PKC in (Ac)5GP-induced apoptotic pathway. SMases are divided into the classes of 1) A-SMase, which is popular in lysosome; 2) zincdependent acid sphingomyelinase, which exists in the serum; 3) N-SMase, which was found in nervous system and mediates the TNF- signaling; 4) Mg2+-independent neutral sphingomyelinase, which was found in myelin sheath; and 5) alkaline sphingomyelinase, which exits in biliary and gastrointestinal tracts (Levade and Jaffrezou, 1999). Because C6 glioma cell is the cell line tested in the experiment, the SMase possibly involved could be N-SMase and/or A-SMase. Our data suggest that N-SMase, but not A-SMase, is activated and mediates the death signals in (Ac)5GP-induced apoptosis.
Generation of oxidative stress has been reported as a critical event for death-inducing agent, including TNF-, Fas, viral infections, and chemotherapeutic drugs. It has been found that depletion of GSH precedes the onset of apoptotic signals induced by various agents (Buttke and Sandstrom, 1994; Macho et al., 1997). Liu and Hannun observed that N-SMase from human leukemic cells was inhibited by GSH, suggesting that N-SMase may be a direct target transmitting the effect of GSH depletion (Liu et al., 1998). In the present study, we also found that GSH depletion is involved in (Ac)5GP-induced N-SMase activation (see Supplementary Data S1). GSH depleted rapidly within 15 to 30 min, accompanying with the SMase activation shown in Fig. 2. The (Ac)5GP-induced depletion of GSH was not altered by GW4869, the noncompetitive inhibitor of N-SMase, implicating that GSH was upstream of N-SMase. The addition of GSH reduced the downstream apoptotic signals (PKC/Fas L/caspases) induced by (Ac)5GP, just like the phenomenon observed in cells treated with GW4869. However, using thiobarbituric acidreactive substances analysis, no oxidative stress was found after (Ac)5GP treatment (see Supplementary Data S1). It might be supposed that in C6 glioma cells, (Ac)5GP transduced N-SMase activation and the downstream apoptotic signals not via the oxidative stress. The impeding effect of GSH may be caused by the structural specificity or other indirect mechanism (Liu and Hannun, 1997).
(Ac)5GP increased the expression of p75 within 9 h, whereas the protein amount was not altered after 12 h. The rapid elevation of p75 might be regulated in translational or post-translational level. However, it may also result from the attenuation of p75 degradation, which is supposed to occur in physiological condition. On the other hand, NGF level increased gradually even after 24 h of (Ac)5GP treatment. It describes that NGF exerts different actions via binding with its receptors trkA or p75. trkA transduces NGF signals for survival and differentiation, whereas p75 induces the cell death (Rabizadeh et al., 1993). The consistent elevation of NGF shown in the present study could be supposed as amplification of the apoptotic signals, whereas the rebound effect for cell survival might be included. Thus, the stable expression of p75 should be viewed as the key point in NGF-mediated apoptosis induced by (Ac)5GP.
The cyclic peptide YCDIKGKECY has been shown to be a specific antagonist to p75 receptor (Longo et al., 1997; Li et al., 2005). Early crystallographic studies revealed that three hydrophilic -loops were contained in NGF surface, which constituted the likely domain for receptor binding. One of the hairpin loops was identified to be formed by NGF 29 to 35 region (McDonald et al., 1991). Ibanetz et al. (1992) found that NGF mutants modified in the 29 to 36 loop region inhibited the binding of p75. Subsequent study suggested that the cyclized peptide dimer exerted the survival-promoting and NGF-inhibitory activity, whereas the linear peptide was inactive (Longo et al., 1997). The short peptide interferes with the action of NGF because it lacks three-dimensional conformation, which may be necessary to activate NGF receptor.
Many previous studies reported that NGF regulated various cell responses via ceramide generation or SMase activation. NGF transduced the cell death of hippocampal neurons through the activation of N-SMase (Brann et al., 2002). NGF/p75 induced differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves (Hirata et al., 2001). It also indicated that SMase is involved in NGF-mediated outgrowth of nerve cells (Brann et al., 1999). In the present study, we demonstrated NGF signaling downstream of SMase. Similar results were also reported by Golve-Roperch et al. (1997), who suggested that SMase/ceramide induces NGF synthesis via activation of MAP kinase. Taken together, some complicated cross-talks are predicted to exist between SMase and NGF, which may coordinate to promote the death signals.
The apoptotic pathway induced by (Ac)5GP is shown in Fig. 7. (Ac)5GP transduced SMase activation and NGF/p75 expression, thus mediating the PKC/JNK/Fas/caspase death signals. In fact, SMase/ceramide may modulate a variety of downstream targets, including kinase, phosphatase, and transcription factors. SMase and ceramide were reported to activate MAP kinase in HL-60 cells (Raines et al., 1993). It has been described that Raf phosphorylation is involved in SMase/ceramide-stimulated MAP kinase activation (Huwiler et al., 1996). SMase pathway has been suggested to control NGF synthesis via MAP kinase activation in astrocyte cultures (Galve-Roperh et al., 1997). In our undergoing investigation, we also found that MAP kinase is involved in NGF/p75 expression (data not shown). Whether MAP kinase is involved downstream of (Ac)5GP-activated SMase, hence mediating the transcription of NGF/p75, will be further investigated in our future study.
Fig. 7. The apoptotic pathway induced by (Ac)5GP in C6 glioma cells. Signals downstream PKC were reported in our previous studies (Peng et al., 2004).
In C6 glioma cells, (Ac)5GP transduces apoptotic signals through the activation of SMase and downstream expression of NGF and p75, thus mediating PKC translocation and the following JNK/Fas/caspase cascades. These results indicate that (Ac)5GP could potentially be developed as an antitumor drug.
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; PKC, protein kinase C ; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; TNF-, tumor necrosis factor-; HPTLC, high-performance thin-layer chromatography; TEMED, N,N,N',N'-tetramethylethylenediamine; DAPI, 4',6-diamidino-2-phenylindole; GSH, glutathione; GW4869, C30H28N6O6 ·2HCl.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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作者单位：Department of Nursing, Hungkuang University, Sha Lu, Taichung, Taiwan (C.-H.P.); Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan (C.-N.H.); and Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (S.-P.H., C.-J.W.)