iridoids and Glioma

Oleuropein, the main phenolic compound of secoiridoids, has been proven to have inhibitory effects on various types of cancers. However, the antitumor effects and related mechanisms in glioma remain unclear. In the present study, U251 and A172 cells were used to assess the effects of oleuropein. Using cell viability assay, we found that oleuropein greatly inhibited the viability of the U251 and A172 cells. Additionally, flow cytometric apoptosis assay indicated that oleuropein induced the apoptosis of the two cell lines. Consistently, the inhibitory effects of oleuropein on migration and invasion were also observed in vitro. In regards to the mechanism, we found that oleuropein significantly decreased phosphorylation of AKT (p-AKT), accompanied by upregulation of Bax and downregulation of Bcl-2. We also found that there was a decrease in the expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 after treatment with oleuropein. Furthermore, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, LY294002, enhanced the pro-apoptotic and anti-invasive effects induced by oleuropein, which suggested that oleuropein suppressed the growth and invasion of glioma cells via inhibition of AKT activity. Taken together, our results indicated that treatment with oleuropein may be an effective therapy for malignant glioma through suppression of tumor proliferation and invasion by inhibition of the AKT signaling pathway.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Flow Cytometry; Glioma; Humans; Iridoid Glucosides; Iridoids; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Signal Transduction

Penta-acetyl geniposide [(Ac)(5)GP], an acetylated geniposide product from Gardenia fructus, has been known to have hepatoprotective properties and recent studies have revealed its anti-proliferative and apoptotic effect on C6 glioma cells. In this study, we first report the anti-metastastic effect of (Ac)(5)GP in the rat neuroblastoma line: C6 glioma cells. First (Ac)(5)GP exhibited an inhibitory effect on abilities of adhesion and motility by cell-matrix adhesion assay, wound healing assay and Boyden chamber assay. Second, the decreasing activity of matrix metalloproteinase-2 (MMP-2) was noted by gelatin zymography assay. Further analysis with semi-quantitative RT-PCR showed the mRNA levels of MMP-2 and membrane type I matrix metalloproteinase (MT1-MMP) were significantly reduced, while the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) was elevated by (Ac)(5)GP treatment. Further (Ac)(5)GP also exerted an inhibitory effect on phosphoinositide 3-kinase (PI3K) protein expression, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and inhibition of activation of transcription factor nuclear factor kappa B (NF-kappaB), c-Fos, c-Jun. These findings proved (Ac)(5)GP is highly likely to be a inhibiting cancer migration agent to be further developed in the future.

Topics: Animals; Base Sequence; Cell Line, Tumor; DNA Primers; Electrophoresis, Polyacrylamide Gel; Extracellular Signal-Regulated MAP Kinases; Glioma; Glucosides; Iridoid Glucosides; Iridoids; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Protease Inhibitors; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac)(5)GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac)(5)GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac)(5)GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac)(5)GP maximally increases AP-1 and NF-kappaB DNA binding at 6h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-kappaB, suggesting these MAPKs are involved in (Ac)(5)GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac)(5)GP, with or without AP-1 or NF-kappaB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac)(5)GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-kappaB inhibitor PDTC; NF-kappaB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac)(5)GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac)(5)GP in chemoprevention or as an anti-tumor agent.

Topics: Animals; Anthracenes; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Apoptosis; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Flavonoids; Gardenia; Glioma; Glucosides; Imidazoles; Immunoblotting; Iridoid Glucosides; Iridoids; Keratolytic Agents; Masoprocol; Mitogen-Activated Protein Kinases; Molecular Structure; Nerve Growth Factor; NF-kappa B; Peptide Fragments; Pyridines; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factor AP-1; Transcriptional Activation

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.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Enzyme Activation; Fas Ligand Protein; Glioma; Iridoids; Membrane Glycoproteins; Models, Biological; Nerve Growth Factor; Protein Kinase C-delta; Protein Transport; Pyrans; Rats; Receptor, Nerve Growth Factor; Signal Transduction; Sphingomyelin Phosphodiesterase; Tumor Cells, Cultured; Tumor Necrosis Factors

In our previous study, penta-acetyl geniposide ((AC)(5)GP) is suggested to induce tumor cell apoptosis through the specific activation of PKCdelta. However, the downstream signal pathway of PKCdelta has not yet been investigated. It was shown that JNK may play an important role in the regulation of apoptosis and could be a possible downstream signal of PKCdelta isoforms. In the present study, we investigate whether JNK is involved in (AC)(5)GP induced apoptosis. The result reveals that (AC)(5)GP induces JNK activation and c-Jun phosphorylation thus stimulating the expression of Fas-L and Fas. Using SP600125 to block JNK activation shows that (AC)(5)GP-mediated apoptosis and related proteins expression are attenuated. Furthermore, we find that the (AC)(5)GP induces apoptosis through the activation of JNK/Jun/Fas L/Fas/caspase 8/caspase 3, a mitochondria-independent pathway. The JNK pathway is suggested to be the downstream signal of PKCdelta, since rottlerin impedes (AC)(5)GP-induced JNK activation. Therefore, (AC)(5)GP mediates cell death via activation of PKCdelta/JNK/FasL cascade signaling.

Topics: Animals; Apoptosis; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Fas Ligand Protein; Gardenia; Glioma; Glucosides; Iridoid Glucosides; Iridoids; JNK Mitogen-Activated Protein Kinases; Membrane Glycoproteins; Mitochondria; Phosphorylation; Protein Kinase C; Pyrans; Rats; Signal Transduction; Taiwan; Up-Regulation

Penta-acetyl geniposide, (Ac)5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G0/gap phase 1 (G1) by (Ac)5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac)5-GP in C6 glioma cells. The inhibitory effect of (Ac)5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G0/G1 phase was associated with a marked decrease in the protein expression of cyclin D1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac)5-GP, arresting cells at the G0/G1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D1/cdk 4 and the phosphorylation of Rb.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Drugs, Chinese Herbal; Glioma; Glucosides; Iridoid Glucosides; Iridoids; Phosphorylation; Plant Extracts; Pyrans; Rats; Retinoblastoma Protein; Tumor Suppressor Protein p53

Experimental data are provided which demonstrate the inhibitory effects of penta-acetyl geniposide, (Ac)5-GP, on the growth and development of C-6 glioma cells inoculated into rats. In the pretreatment experiments, (Ac)5-GP prolonged the latency period of T50 (time for 50% tumour incidence). At week 7, the growth inhibition was 41% with 5 mg/kg and 75% with 10 mg/kg of (Ac)5-GP. In the post-treatment experiments, growth inhibition was less. No significant hepatotoxic effects were observed in the treated group, indicated by the constant levels of serum enzymes (e.g. asparate aminotransferase). We suggest that (Ac)5-GP is a potent chemopreventive agent on glioma cells.

Topics: Animals; Cell Division; Cells, Cultured; Drugs, Chinese Herbal; Glioma; Glucosides; Iridoid Glucosides; Iridoids; Liver; Male; Pyrans; Rats; Rats, Wistar