2013 Volume 36 Issue 4 Pages 540-547
Ras, a small G-protein, physiologically directs cell proliferation and cell cycle via regulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Dysregulation of Ras/MEK/ERK signaling has been reported to cause tumorigenesis and gliomas. Nobiletin, a citrus flavonoid, has been shown to have anti-tumor cells action. However, it remains elusive whether nobiletin could affect Ras activity. In this study, we provide the first evidence that nobiletin suppresses the proliferation by inhibiting Ras activity in C6 glioma cells, a rat glioma cell line. First, Ras pull-down assay showed that nobiletin inhibits Ras activity in a concentration-dependent manner in C6 cells. Second, farnesyltransferase inhibitor I, a Ras inhibitor, and U0126, a MEK inhibitor, induced an inhibition of the cell proliferation in C6 cells, while the cell proliferation was inhibited by nobiletin as well. Third, western blotting revealed that nobiletin showed inhibitory effects on MEK and ERK phopsphorylation levels in a concentration-dependent manner. Finally, such an inhibitory effect on the level of ERK phosphorylation by nobiletin was appreciably prevented by Gö6976, a selective inhibitor of conventional protein kinase Cs (PKCs) showing Ca2+-sensitivity, while GF109203X, a general inhibitor for PKCs, and BAPTA, a cell-permeable Ca2+ chelator, to a lesser extent, suppressed a reduction of the phosphorylation. These findings suggest that the proliferation of C6 cells is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the cell proliferation by inhibiting Ras activity and MEK/ERK signaling cascade probably via a Ca2+-sensitive PKC-dependent mechanism. Thus, the natural compound has potential to be a therapeutic agent for glioma.
Gliomas is the most common type of primary central nervous system (CNS) tumors and essentially incurable.1) Among gliomas is glioblastoma multiforme (GBM), which is known as a kind of malignant glioma. Integrative genomic studies have revealed that a set of core signaling pathways, i.e. the P53 pathway, the RB pathway, and the receptor tyrosine kinases (RTK) pathway, are commonly activated in GBM due to genetic alteration,2) which helps to fuel the cell proliferation and enhance the cell survival while allowing the tumor cell to escape from cell-cycle checkpoints, senescence, and apoptosis.3–5) In terms of the RTK pathway, it has been reported that in a-frame deletion of epidermal growth factor (EGF) receptor extracellular domain observed in GBM causes ligand-independent transforming activity,6) and that a small G-protein Ras coupled with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), or phosphatidylinositol 3-kinase (PI3 kinase)/Akt signaling cascades show aberrant activation in GBM,7) raising the possibility that both Ras and PI3 kinase signaling cascades might be compelling therapeutic targets for GBM. Moreover, B-Raf mutations causing its constitutive kinase activity have been discovered, although the mutations are present in a very small percentage of gliomas.8) Accordingly, it is suggested that activated Ras/Raf/MEK/ERK signaling may be a critical pathway for the proliferation of glioma.9) But, no drugs that target on these intracellular signaling cascades, thereby preventing glioma proliferation and progression have been so far developed.
It has been reported that nobiletin, a citrus peel polymethoxyflavone, exhibits multiple beneficial pharmacological actions, including anti-inflammatory action in RAW264.7 cells and microglial cells,10,11) neurotrophic, anti-dementia and neuroprotective actions,12–14) anti-tumor effects in human colorectal HT-29 cells, ductal breast carcinoma MDA-MB-435, adenocarcinoma MCF-7 cells and gastric adenocarcioma AGS cells,15–17) and anti-osteoporosis action.18) Furthermore, nobiletin shows an anti-invasive and anti-cell migration effects on human brain tumors19) by inhibition of matrix metalloproteinases 2 and 9 activities. In this study, we have for the first time demonstrated that this natural compound has inhibitory effects on Ras activity and the downstream MEK/ERK signaling to suppress the proliferation of rat glioma C6 cells in culture by using active Ras pull-down assay and Western blot analyses.
Nobiletin was isolated from Citrus depressa as described previously20) (Fig. 1). C6 glioma cells, a rat glioma cell line, was provided from Dr. H. Higashida of Kanazawa University. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissuiseiyaku (Tokyo, Japan). Active Ras Pull-Down and Detection Kit and SuperSignal West Pico Chemiluminescent Substrate were purchased from Thermo Scientific (Waltham, MA, U.S.A.). Anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) (Thr-202/Tyr-204), anti-p44/42 MAPK, anti-phospho-MEK1/2 (Ser217/221), anti-MEK1/2, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473) and anti-Akt antibodies were obtained from Cell Signaling Technology (Beverly, MA, U.S.A.). Protease inhibitor or phosphatase inhibitor cocktails were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Farnesyltransferase (FTase) inhibitor I was obtained from Calbiochem (Darmstadt, Germany). Bovine serum albumin (BSA), BAPTA-AM, GF109203X and Gö6976, and skim milk and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, U.S.A.) and Wako (Tokyo, Japan), respectively. A firefly luciferase reporter plasmid containing cyclic AMP-response element inserted into the upstream of a TATA-like promoter (pTAL) region taken from herpes simplex thymidine kinase promoter (pCRE-Luc), a renilla luciferase vector, phRG-TK, and a dual luciferase assay kit, and LipofectAMINE 2000 were purchased from Clontech, Promega, and Invitrogen, respectively. Other chemicals and drugs were of reagent grade or of the highest quality.
C6 cells culture was carried out as described previously.21) Cells were plated on 100-mm dish and cultured in high-glucose DMEM containing 10% fetal calf serum (FCS) at 37°C in 5% CO2 incubator.
Cell CountingC6 cells were seeded at a density of 5×103 cells/well on 96-well-plate and cultured for 24 h. Cells were then treated with high-glucose DMEM containing 3% FCS and indicated concentrations of nobiletin for 24 or 48 h. Also, cells were treated with 20 µm FTase inhibitor I or 10 µm U0126 for 24 h. Cells were then treated with 0.25% trypsin/5.3 mm ethylenediaminetetraacetic acid (EDTA) for 3 min at 37°C in 5% CO2 incubator to be subjected to cell counting by a hemacytometer. These cell suspensions were poured into the chambers of hemacytometer and cells were counted by microscopy. Cells in the 16 small squares in each of the 4 corners of the hemacytometers were counted.
MTT AssayMTT assay was carried out as described previously,22) except that C6 cells were seeded at a density of 5×103 cells/well on 96-well-plate and cultured for 24 h. Cells were then treated with high-glucose DMEM containing 3% FCS and indicated concentrations of nobiletin for 24 h. MTT of 0.5 mg/mL was added to each well, and cells were incubated at 37°C for 4 h. Medium was replaced with dimethyl sulfoxide. After 15 min, plates were read on the microplate reader (model 680; Bio-Rad Laboratories, Inc., CA, U.S.A.) at a test wavelength of 595 nm.
Pull-Down Assay for Active RasAssay of active Ras was performed using Active Ras Pull-Down and Detection Kit according to the instruction of the manufacturer. C6 cells were seeded at a density of 1×106 cells/100-mm dish and cultured for 2 d to grow to be 80–90% confluent. Cells were then treated with the 3% FCS high-glucose DMEM containing indicated concentrations of nobiletin for 2 min. Cells were gently rinsed with ice-cold Tris-buffered saline (TBS) and scraped with 300 µL of Lysis/Binding/Wash buffer (25 mm Tris–HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 5% glycerol and 1% NP-40) containing protease inhibitor cocktail. After each cell lysate was transferred to a 1.5-mL microcentrifuge tube, the tube was briefly mixed and then incubated on ice for 5 min. The lysate was centrifuged at 16000×g for 15 min at 4°C, and the resulting supernatant was diluted to adjust the concentration to 1000 µg of proteins/500 µL and chilled on ice until pull-down assay. For preparation of positive and negative controls, additional two sister cultures were performed as described above. Cells were collected without treatment to prepare 500 µg of proteins/500 µL from each supernatant. Following each supernatant was added 10 µL of 0.5 m EDTA (pH 8.0), 5 µL of 10 mm guanosine 5′-triphosphate (GTP)γS or 100 mm guanosine 5′-diphosphate (GDP) were added to each sample for preparation of positive and negative controls, respectively. After mixing, the lysates for positive and negative controls were incubated at 30°C for 30 min, to terminate the reactions by cooling on ice and the subsequent addition of 32 µL of 1 m MgCl2. Tested samples, positive and negative controls were incubated with 100 µL of the 50% glutathione resin slurry and 20 µL of GST-Raf1-RBD fusion protein at 4°C for 1 h, with rotation of the reaction mix. After centrifugation for 1 min at 6000×g, at 4°C, glutathione resins were washed three times with Lysis/Binding/Wash buffer for 1 min at 6000×g at 4°C. The glutathione resins were resuspended in 50 µL of 2×sodium dodecyl sulfate (SDS) sample buffer and incubated at room temperature for 2 min. The samples were centrifuged at 6,000×g at room temperature for 1 min to obtain the flow-through fractions and these fractions were heated at 95°C for 5 min. Heated samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western blotting on nitrocellulose membrane and the subsequent detection with anti-Ras antibody.
Western BlottingWestern blotting was carried out as described previously.12,22) For analyses of the levels for phosphorylation of MEK, ERK and Akt, C6 cells were seeded at a density of 5×105 cells/35-mm dish and cultured for 2 d. Cells were treated with the tested concentrations of nobiletin following pretreatment with inhibitors or vehicle for 30 min, and were then lysed with a lysis buffer (10 mm N-(2-hydroxyethyl)piperazine-N′-2-(ethanesulfonic acid)) (HEPES), 1 mm EDTA, 1% SDS including protease and phosphatase inhibitor cocktail. Cell lysates were sonicated and centrifuged at 16000×g for 5 min at 4°C to obtain the supernatant as cell extracts. These extracts were heated at 95°C for 5 min. The extracts were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The blotted membrane was blocked in TBST buffer (150 mm NaCl, 0.05% Tween 20, 10 mm Tris–HCl, pH 7.5) containing 5% skim milk for 1 h at room temperature. The membrane was thereafter incubated with anti-phospho-MEK (Ser217/221) (1 : 2000), anti-phospho-p44/42 MAPK (Thr-202/Tyr-204) (1 : 1000) or anti-phospho-Akt (1 : 1000) antibodies in 5% BSA/TBST buffer overnight at 4°C and horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) for 1 h at room temperature. Blots were then stripped and reprobed with anti-MEK1/2, ERK1/2 or anti-Akt antibodies to verify that equal amount of protein was present in each lane. Immunoreactivities were visualized with SuperSignal West Pico Chemiluminescent Substrate. The band intensities were quantitatively analyzed using SCION image software.
Transient Transfection and Reporter Gene AssayC6 cells were plated on 48-well plates at a density of 1.2×104 cells per well and cultured for 24 h. Cells were then subjected to transfection. Transfection and reporter gene assay were conducted as described previously.23) phRG-TK was co-transfected with pCRE-Luc with LipofectAMINE 2000, and used as an internal control to normalize for variations in transfection efficiency. Following 16-h transfection, cells were treated with the tested concentrations of nobiletin for 5 h. Thereafter, cells were harvested to assay the activities of both firefly and seapansy luciferase by using a dual luciferase assay kit.
Statistical AnalysisStatistical analysis was performed by one-way ANOVA followed by Tukey test. p<0.05 was considered statistically significant.
A small G-protein Ras activates Raf/MEK/ERK signaling cascade, and thereby leads to the cell proliferation in glioma.24) First, we treated C6 rat glioma cells with 10, 30 or 100 µm of nobiletin for 2 min to examine whether the natural compound could affect Ras activity by pull-down assay. Our pull-down assay showed that nobiletin at 30 and 100 µm prevents Ras activity by approximately 50% of that of control C6 cells, with no significantly inhibitory effects on active Ras level at 10 µm (Figs. 2A, B), indicating the concentration-dependency. Ras directs the cell proliferation via Raf/MEK/ERK signaling activation.25) Thus, we tested the effects of FTase inhibitor I, a Ras selective-inhibitor, on the proliferation of C6 cells at a concentration of 20 µm. FTase inhibitor I did inhibit the cell proliferation as treated with this concentration for 24 h (Fig. 3A). Further, it was investigated whether the proliferation of C6 cells is inhibited by treatment with nobiletin which has the activity to inhibit Ras activity in C6 cells as described above. The natural compound actually inhibited the cell proliferation in a concentration-dependent manner when treated with 10, 30 and 100 µm for 24 h; this natural compound prevented the cell proliferation to approximately 50% of that of control cells at 30 µm (Figs. 3A, B), and more potently inhibited it at 100 µm, with no significant effects on the cell proliferation at 10 µm (Fig. 3B). Additionally, 48-h treatment with nobiletin at 10, 30 and 100 µm more effectively inhibited the cell proliferation than that with the natural compound for 24 h (Fig. 3C). The concentration-dependent inhibitory effect of nobiletin on C6 cell proliferation was also observed by using MTT assay (Fig. 3D). Moreover, even when cells were treated with 300 µm nobiletin for 24 h, neither cell shrinkage nor caspase-3 activation (i.e. cleaved caspase-3)26) were detected under the current experimental condition by microscopic observation and western blot analysis (data not shown). Thus, the above observations suggest the concentration- and time-dependent inhibition of C6 cell proliferation by nobiletin, probably without any cell death-inducing effects. Similarly, U0126, a selective inhibitor of MEK which acts downstream of Ras, prevented the cell proliferation at 10 µm (Fig. 3A). In addition, treatment with nobiletin at the concentration of 30 and 100 µm for 24 and 48 h inhibited the proliferation of C6 cells even in culture medium containing 10% FCS as well (data not shown). These results indicate that Ras and the downstream MEK/ERK signaling cascade are required for C6 glioma cell proliferation and that nobiletin reduces Ras activity to inhibit the cell proliferation.
Cells were seeded and cultured as described under Materials and Methods. Cells were then treated with indicated concentrations of nobiletin for 2 min. Pull-down assay with Active Ras pull-down and Detection Kit followed by Western blot analyses with anti-Ras antibody was performed as described under Materials and Methods. GTPγS and GDP were also added to the supernatant of cell lysate from untreated cells to prepare positive and negative controls. (A) Shown were representative results obtained from at least three independent experiments. (B) Data are the mean±S.E.M. (n=3). * p<0.05 versus vehicle control.
Cells were seeded and cultured as described under Materials and Methods. Cells were treated with 30 µm nobiletin, 20 µm FTase inhibitor I or 10 µm U0126 for 24 h (A). Also, cells were treated with nobiletin at indicated concentrations for 24 h (B) or 48 h (C) to be subjected to cell counting. For MTT assay, following seeding and 24-h culture, cells were treated with nobiletin at the indicated concentrations for 24 h. Cell viability is expressed as a percentage against the control viability (100%) in the absence of the natural compound (D). Data are the mean±S.E.M. (n=3). * p<0.05, ** p<0.01 *** p<0.001 versus vehicle control.
MEK/ERK cascade activation is under control of Ras via Raf phosphoryration, and the MEK/ERK signaling and Ras/PI3-kinase/Akt pathway eventually converges upon the cell singling pathway related to the cell proliferation of gliomas, including malignat glioma.5,27) Thus, it was next examined whether nobiletin could affect not only the phosphorylation levels of MEK and ERK, but also those of phosphorylation of both Thr308 and Ser473 of Akt which are required for complete Akt activation through PI3 kinase/PKB-mediated signaling,28) in C6 rat glioma cells by Western blot analyses. Our Western blot analyses revealed that nobiletin diminishes the levels for both phosphorylation sites of Akt (Figs. 4C, D) as well as those of the phosphorylation of MEK and ERK (Figs. 4A, B) in a concentration-dependent manner, as examined in C6 cells treated at the concentrations of 10, 30 and 100 µm for 10 min.
Cells were seeded and cultured as described under Materials and Methods. Cells were then treated with indicated concentrations of nobiletin for 10 min. Western blot analyses with anti-phospho-MEK, anti-phospho-ERK or anti-phospho-Akt (Thr308 or Ser473) antibodies were performed as described under Materials and Methods. Furthermore, the blots for phospho-MEK, phospho-ERK and phospho-Akt were reprobed with anti-MEK1/2, anti-ERK1/2 and anti-Akt antibodies, respectively, to verify that equal amount of protein was present in each lane. Shown were representative results on phospho-MEK (A), phospho-ERK (B), phospho-Akt (Thr308) (C) and phospho-Akt (Ser473) (D) obtained from at least three independent experiments with the data from quantitative analyses. Data are the mean±S.E.M. (n=3). * p<0.05, ** p<0.01, *** p<0.001 versus vehicle control.
PKC acts as one of the upstream regulators of Ras, and is classified into three types: conventional, novel and atypical PKCs.29) It has been reported that nobileltin induces Ca2+-influx in cultured bovine adrenal medullary cells,30) raising the possibility that the inhibitory action of nobiletin might be mediated by Ca2+-influx and Ca2+-sensitive PKCs. To test the possibility, it was examined whether GF109203X, a general inhibitor of PKC isozyms, and Gö6976, a selective inhibitor of conventional PKCs, prevent nobiletin-induced reduction of ERK phosphorylation level in C6 cells. Upon pretreatment with GF109203X and Gö6976 at each concentration of 10 µm for 30 min prior to treatment with nobiletin, Gö6976 appreciably restored nobiletin-reduced phosphorylation of ERK, while GF109203X, to a lesser extent, prevented a decrease in the phosphorylation (Fig. 5A), indicating that conventional PKCs which exhibit Ca2+-sensitivity may at least contribute to such an inhibitory action on ERK phosphorylation in C6 cells. Moreover, we examined the effect of BAPTA-AM, a cell-permeable Ca2+ chelator, on the nobiletin-induced reduction in ERK phosphorylation level in C6 cells. It was shown that BAPTA at a concentration of 20 µm prevented a reduction of ERK phosphorylation induced by nobiletin (Fig. 5B). These findings suggest that nobiletin mechanistically induces activation of Ca2+-sensitive PKC to suppress ERK signaling linked to the proliferation in C6 glioma cells.
Cells were seeded and cultured as described under Materials and Methods. Cells were pretreated with 10 µm GF109203X, 10 µm Gö6976 or 20 µm BAPTA-AM for 30 min, and subsequently treated with 30 µm nobiletin for 10 min. Western blot analysis with the anti-phospho-ERK antibody was performed as described under Materials and Methods. Further, the blots were reprobed with anti-ERK antibody to verify that equal amount of protein was present in each lane. Shown were representative results on ERK phoshorylation obtained from at least three independent experiments. Data are the mean±S.E.M. ** p<0.01 versus vehicle control and # p<0.05, ## p<0.01 versus nobiletin treatment.
It has been shown that an increased intracellular Ca2+ concentration elicits a modest increase in CRE-dependent transcription in C6 cells.31) This natural compound has been reported to induce Ca2+-influx in cultured bovine adrenal medullary cells.30) Accordingly, there is the possibility that nobiletin might induce CRE-dependent transcription in C6 cells. We therefore examined whether CRE-dependent transcription could be upregulated by nobiletin by reporter gene assay. As expected, this natural compound rose CRE-dependent transcription in a concentration-dependent manner in the cell line (Fig. 6).
Cells were seeded and cultured as described under Materials and Methods. Cells were treated with indicated concentrations of nobiletin for 5 h for assay of the activity of CRE-dependent transcription by reporter gene assay. Data are the means±S.E.M. (n=4). * p<0.05, ** p<0.01 versus control.
In the present study, it was evidently shown that nobiletin, a citrus flavonoid, has an inhibitory effect on the level of GTP-Ras or active Ras, which has been documented to give rise to cell proliferation via activation of MEK/ERK signaling and PI3 kinase/Akt pathway in malignant glioma,5,7) in rat glioma C6 cells by pull-down assay. Nobiletin also prevented the proliferation of C6 cells which was sensitive to FTase inhibitor I, a Ras inhibitor, and U0126, a MEK-selective inhibitor. Consistent with the results regarding the cell proliferation inhibition, nobiletin reduced the phoshorylation levels of MEK, ERK and Akt. Moreover, such an inhibitory effect of nobiletin on ERK phosphorylation was prevented by not only GF109203X, a general PKC inhibitor, and BAPTA-AM, a cell-permeable Ca2+ chelator, but also a conventional PKC-selective inhibitor, Gö6976. These findings suggest that C6 cell proliferation is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the glioma cell proliferation at least by an inhibition of Ras activity and Ca2+- and conventional PKC-dependent downregulation of MEK/ERK signaling.
Ras, a small G-protein, transfers multiple extracellular signals from the RTK pathway to the P53 pathway and the RB pathway, which contribute to promotion of cell proliferation in glioma cells.32) The fact leads us to an idea that inhibition of Ras activity might show the inhibitory effect on the proliferation and that Ras could be compelling therapeutic targets for glioma. Indeed, FTase inhibitor I inhibited the cell proliferation in C6 glioma cells. Furthermore, in support of this idea, the current study demonstrates that nobiletin showing the activity to inhibit Ras activity does prevent cell proliferation of C6 glioma cells, although it has been reported that this natural compound has anti-tumor action by inhibition of MMP, induction of apoptosis and arrest of cell cycle.16,33,34)
How does nobiletin inhibit Ras activity and lead to suppression of cell proliferation in C6 cells? Activation of guanine exchange factors (GEFs) promotes the dissociation of GDP from Ras family proteins, which facilitates the exchange of GDP for GTP, and thus stimulates the activity of Ras family proteins. In reverse fashion, activation of GTPase-activating proteins (GAPs) enhances the rate of hydrolysis of Ras family proteins and reduces their activity.35–38) GAPs activity has been shown to be activated by Ca2+ signaling or PKC.39,40) In the present study, it was evidently shown that not only BAPTA but also a conventional PKC-selective inhibitor, Gö6976, do prevent the nobiletin-induced reduction of ERK phosphorylation which occurs downstream of Ras in the glioma cell line, suggesting involvement of a Ca2+-dependent mechanism in this inhibitory action of the natural compound. On the other hand, in the same glioma cell line, the natural compound stimulated CRE-dependent transcription activity which has been reported to be facilitated in response to increased intracellular Ca2+ concentration.31) In addition, it was observed that treatment with nobiletin decreases the levels of MEK and ERK phosphorylation in the glioma cells. As a consequence, the natural compound suppressed the cell proliferation like FTase inhibitor I and U0126. Therefore, it is reasonable to consider that the inhibitory action of nobiletin on Ras activity directing the cell proliferation might require an intracellular Ca2+- and conventional PKC-dependent GAP activation.
In addition to the inhibitory action of nobiletin on Ras/MEK/ERK signaling described above, the natural compound diminished phosphorylation of Thr308 and Ser473 of Akt essential for Akt activation possibly via PI3 kinase-mediated signaling.5,28,41) Such inhibitory effects of nobiletin on MEK activity and Akt phosphorylation are observed in human fibrosarcoma TH-1080 cells42) and human gastric adenocarcinoma AGS cells.17) Thus, it is plausible that the inhibitory actions of nobiletin on the MEK/ERK and Akt signaling pathways appear not to be glioma cell-specific.
In summary, we reported for the first time that nobiletin prevents the activity of Ras and the downstream MEK/ERK signaling to inhibit the proliferation of C6 cells in a Ca2+- and conventional PKC-dependent manner. Our recent study demonstrated that nobiletin can pass through blood brain barrier in animal.43) These findings suggest that nobiletin has a potential therapeutic benefit for glioma. The elucidation of the mechanism underlying the inhibitory action of this natural compound on the proliferation of glioma may give us an insight into therapeutic drug development for the disease.
We thank Dr. H. Higashida (Department of Cellular Neurophysiology, Kanazawa University Graduate School of Medicine) for kindly providing C6 rat glioma cell line.
