Rat glioma C6 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin; Gibco-BRL). The cell lines were maintained at 37°C in a humidified air atmosphere with 5% CO₂.

To detect cell survival subsequent to treatment, a standard cell proliferation assay was performed (MTT reduction assay) as in a previous study (18). Cells were seeded at a density of 1×10⁴ cells/well in 96-well plates and incubated with rat recombinant TNF-α (specific activity 2×10⁷ U/mg) for 24 h (TNF-α concentrations: 0, 1×10⁴, 1×10⁵, 2×10⁵ and 5×10⁵ U/L) (Sigma-Aldrich). MTT (20 μl; Amresco, Solon, OH, USA) was added to each well and the cells were incubated at 37°C for 4 h. Subsequently, 150 μl dimethyl sulfoxide (Sigma-Aldrich) was added. The optical density (OD) was then measured at a wavelength of 570 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Vantaa, Finland). The inhibitory rate of tumor cell proliferation (%) was measured with the following formula: (OD value of the control group − OD value of the test group) × 100/OD value of the control group. Based on the IC₅₀ of TNF-α in C6 cells, one concentration of TNF-α was selected for the experiments that followed.

The morphology of apoptotic cells was measured using an H-800 transmission electron microscope (Hitachi, Ltd., Tokyo, Japan). Subsequent to treatment, the C6 cells were fixed with glutaraldehyde (Sigma-Aldrich) for 1 h, washed twice with phosphate-buffered saline (PBS; Gibco-BRL) and then suspended with osmic acid (Sigma-Aldrich). Following a 1-h resting period, the cells were gradually dehydrated with acetone (Sigma-Aldrich) and embedded in resin (PRIMASET PT-30; Lonza Japan Ltd, Chiba, Japan). The cells were then placed onto slides and observed using TEM (JEM-2000EX; JEOL, Ltd, Tokyo, Japan).

Apoptosis was analyzed by flow cytometry. Subsequent to treatment, 1×10⁶ glioma C6 cells were prepared into a cell suspension by trypsinization (Sigma-Aldrich), and washed twice with ice-cold PBS. Cells were fixed with 70% ethanol (Sigma-Aldrich) at 4°C overnight, then were treated with propidium iodide (Sigma-Aldrich) and RNase A (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 40 min. Subsequently, samples were analyzed with a FACSCalibur flow cytometry system (BD Biosciences, San Jose, CA, USA). The sub-G₁ population, representing apoptotic cells, was scored using the hypodiploid DNA content, which was quantified using ModFit LT software, version 3.2 (Verity Software House, Inc., Topsham, ME, USA); the percentages of DNA fragmentation reflecting apoptotic cells were determined by measuring the fraction of nuclei containing a hypodiploid DNA (sub-G1 peak), which based on >5,000 cells analyzed by flow cytometry.

For the detection of the p38MAPK (1:500; sc-166357; mouse anti-rat monoclonal antibodies), phosphorylated-p38MAPK (1:500; sc-166182; mouse anti-rat polyclonal antibodies), JNK (1:500; sc-7345; mouse anti-rat monoclonal antibodies) and phosphorylated-JNK (1:500; sc-6254; mouse anti-rat monoclonal antibodies) proteins (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), the whole-cell extracts were lysed in lysis buffer (Sigma-Aldrich) and then clarified by centrifugation at 12,000 × g for 10 min at 4°C. The volume of protein was normalized by the Bradford method, as previously described (19). Equal amounts of total protein were loaded onto 10–15% SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for electrophoresis (Bio-Rad Laboratories, Inc.) and the separated proteins were subsequently electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% nonfat milk in PBS with Tween-20 (Sigma-Aldrich), and probed with various primary antibodies (as described above) overnight at 4°C, then secondary antibodies (goat anti-mouse Immunoglobulin G-horseradish peroxidase; 1:2,000; sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h. The membranes were subsequently washed and the bands were examined using a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.) with the enhanced chemiluminescence (ECL) western blot detection system (Amersham Life Science, Arlington Heights, IL, USA). The densities of the bands were determined by densitometry using Quantity One 4.5 software (Bio-Rad Laboratories, Inc.).

To investigate whether MAPK influences TNF-α-induced apoptosis, C6 cells were also treated with TNF-α in the presence of a specific inhibitor of p38MAPK (SB202190) or JNK inhibitor (SP600125) (10 μmol/L; Calbiochem, San Diego, CA, USA) for 2 h. TNF-α was then added for the indicated experimental groups.

Transient transfection was carried out using Lipofectin (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. During the logarithmic growth phase, C6 cells were transferred into a 6-well plate at a density of 2×10⁸cells/well and were incubated for 24 h. C6 cells were washed with Serum-free medium (0.8 ml; Gibco-BRL) prior to the addition of 2 μg pCMV5-p38MAPK (Promega Corp.) and 10 μl Lipofectin, which were dissolved in 100 μl serum-free medium, and then rested for 30 min at room temperature. Following 12-h incubation, 1 ml medium with 15% serum was added and then incubated for 24 h. The control was transfected with pCMV5 plasmid (Addgene, Inc., Cambridge, MA, USA) and non-transfected C6 cells. Subsequent to transfection, certain cells were fixed for TEM observation. The cell cycle distribution was analyzed by flow cytometry, and the expression of p38MAPK was detected by western blot analysis as described in the above methods.

The rat soluble TNF-α (sTNF-α) ELISA kit (Invitrogen Life Technologies) was used to perform the ELISA in accordance with the manufacturer’s instructions. Following p38MAPK transfection, the culture supernatants were collected. The coating TNF-α goat anti-rat polyclonal antibody (1:100; sc-1349; Santa Cruz Biotechnology, Inc.) was added into an ELISA plate (Invitrogen Life Technologies) at 100 μl/well for 48 h at 4°C. Subsequently, the plate was washed with PBS containing 0.05% Tween-20 three times. A total of 100 μl/well of the detected sample, negative control and standard sample were added at proportional dilutions with PBS (1:1), seperately. Following incubation at 37°C for 1 h, the plate was washed three times. A total of 100 μl/well of the horseradish peroxidase-labeled donkey anti-goat monoclonal antibody (1:100; sc-2020; Santa Cruz Biotechnology, Inc.) was added, then following incubation at 37°C for 1 h, the plate was washed three times with PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich). A total of 100 μl/well 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS; Sigma-Aldrich) was added and 15 min later, the OD was detected by the ELISA reader (MR 5000; Dynatech Laboratories, Chantilly, VA, USA) at a wavelength of 410 nm. The standard curve was drawn and the concentrations of sTNF-α in the sample were calculated.

Cells in each group were prepared into a cell suspension using trypsin and washed with PBS containing 1% BSA; the final concentration was 1×10⁶/ml. A total of 2 μl rabbit-anti-rat membrane TNF-α polyclonal antibody (1:1,000; 3707S; Cell Signaling Technology, Inc., Beverly, MA, USA) or TNFRI polyclonal antibody (1:1,000; 13377S; Cell Signaling Technology, Inc.) was added into 0.5 ml cell suspension. The cell suspension was shaken every 5 min for 30 min, at 37°C. Subsequent to washing with PBS containing 1% BSA, goat-anti-rabbit monoclonal immunoglobulin G (1:2,000; 7074S; Cell Signaling Technology) labeled by fluorescein isothiocyanate was added and washed with PBS containing 1% BSA three times. Subsequent to the addition of 1 ml 4% paraformaldehyde (Sigma-Aldrich), the cells were observed by fluorescence microscopy (DP90; Olympus, Tokyo, Japan) and detected by flow cytometry using a FACS LSR II system (Becton Dickinson, San Jose, CA, USA).

All data are presented as the mean ± standard deviation of at least three independent experiments. Statistical differences between the means were analyzed by the independent-samples t-test. Rates were compared using the χ² test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA).

The MTT assay was used to detect cell viability of glioma C6 cells. Subsequent to treatment with TNF-α, the proliferation of C6 cells was inhibited in a dose-dependent manner. The inhibitory rates of different concentrations of TNF-α on C6 cells are described in Table I. The IC₅₀ of TNF-α in C6 cells was 2.53×10⁵ U/L, and this concentration was used for the subsequent experiments. Following treatment of glioma cells with SB202190 and TNF-α simultaneously, the inhibitory rate reduced to 9.849±0.675% (Fig. 1). However, following simultaneous treatment with SP600125 and TNF-α, the inhibitory rate was similar to that of TNF-α treatment alone (41.615±1.236% vs. 44.518±1.305%). These results suggest that TNF-α is antiproliferative in a dose-dependent manner in glioma cells, and that this effect of TNF-α can be partly blocked by the inhibition of p38MAPK with SB202190, but not by inhibition of JNK with SP600125.

To investigate the mechanisms associated with a TNF-α-induced reduction in cell proliferation, the effects of TNF-α treatment on cell cycle arrest and apoptosis were examined. Apoptosis was observed by TEM and quantified by flow cytometry. In the TNF-α and SP600125-treated groups, a number of the cells exhibited clear apoptotic characteristics, including pyknosis, chromatin condensation and formation of apoptotic bodies, when observed by TEM (Fig. 2A). Few apoptotic cells were observed in the control and SB202190-treated groups, whereas in the TNF-α- and SP600125-treated groups, the proportion of cells in the G₁ phase was increased and the proportions in the S and G₂ phases were reduced, and clear apoptotic peaks emerged (Fig 2B–E). The apoptotic rates in the TNF-α- and SP600125-treated groups were 37.5 and 34.1%, respectively (Fig. 2B and C). There was a smaller apoptotic peak in the control and SB202190-treated groups, in which the apoptotic rates were 7 and 16.9%, respectively (Fig. 2D and E). The apoptotic rates of the TNF-α- and SP600125-treated groups were significantly different from that of the control or SB202190-treated group (P<0.01; data not shown). The results of the current study demonstrate that TNF-α-induced apoptosis may be partly blocked by the inhibition of p38MAPK, but not by inhibition of JNK.

To investigate the potential involvement of MAPK in TNF-α-induced apoptosis, the activation states of p38MAPK and JNK were investigated using western blot analysis with antibodies specific to the phosphorylated forms of these kinases. Subsequent to 2×10⁵ U/L TNF-α treatment, clear activation of phosphorylated-p38MAPK was observed (Fig. 3). In contrast, induction of JNK phosphorylation was not observed in any group. In addition, no phosphorylated-p38MAPK band was detected in the SB202190-treated or control group (Fig. 3). These data suggest that p38MAPK phosphorylation, but not JNK phosphorylation, may be involved in the mediation of TNF-α-induced apoptosis.

To investigate the role of p38MAPK in TNF-α-induced apoptosis, transient transfection was carried out using Lipofectin. During the 24-h pCMV5-p38MAPK transfection, the asteroid-shaped cells became bipolar or round in shape, with reduced sizes and increased intracellular bubbles. After 24 h, a number of the dead cells floated in the medium. There were no distinct alterations in the non-transfected and pCMV5 groups. In addition, a number of cells demonstrated clear apoptotic alterations, including pyknosis, chromatin condensation and formation of apoptotic bodies (Fig. 4A). Cell cycle analysis identified a clear apoptotic peak in the pCMV5-p38MAPK group and the apoptotic index was 31.2% (Fig. 4B). Western blot analysis demonstrated that a distinct immunoreactive band at 38 kDa was present in the pCMV5-p38MAPK group. No band was present in the non-transfected or pCMV5 groups (Fig. 4C). Additionally, the sTNF-α concentration was analyzed subsequent to gene transfection. The results demonstrated that the concentration of sTNF-α in the culture supernatant of the pCMV5-p38MAPK group increased and reached 43.4 pg/ml following treatment for 24 h, but there was no clear alteration in the pCMV5 and non-transfected groups (data not shown).

The expression rates of membrane TNF-α in the pCMV5, pCMV5-p38MAPK and control (non-transfected) groups were 4.079±0.532, 6.218±0.601 and 3.845±0.482%, respectively, and were not significantly different. The expression rates of membrane TNFRI in the pCMV5, pCMV5-p38MAPK and control groups were 5.422±0.551, 14.906±0.627 and 3.633±0.404%, respectively. Out of these, the level in the pCMV5-p38MAPK group was significantly higher than that of the other two groups (P<0.01; Fig. 5).