Methyl methylthiomethyl sulfoxide (MMTS), neocuproine, sodium ascorbate and GSNO were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SNP was obtained from Beyotime Institute of Biotechnology (Haimen, China). PolyJet™ and Biotin-HPDP were purchased from Thermo fisher Scientific, Inc. (Waltham, MA, USA). Antibodies against Flag (F1084; 1:1,000; Sigma-Aldrich; Merck KGaA), ERK1/2 (ab17942; 1:1,000; Abcam, Cambridge, UK), p-ERK1/2 (sc-81492; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and caspase-3 (GTX110543; 1:1,000; GeneTex, Inc., Irvine, CA, USA) were commercially available.

The U251 glioma cell line was purchased from Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in a cell incubator containing 5% CO₂ under saturated humidity at 37°C. Cells were treated at 37°C with NO donors SNP (0–2 µM) or GSNO (0–500 µM) for 36 h or with SNP (2 µM) or GSNO (500 µM) for different time (0–36 or 48 h) prior to further analysis.

Cell viability was measured using Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). A single cell suspension (5×10³/ml, 100 µl) was seeded into a 96-well plate. Subsequently, 10 µl CCK-8 reagent was added to each well and the plates were incubated for 2 h at 37°C. Finally, the absorbance was measured at 450 nm using a scanning microplate reader. Cell viability at individual time-points was normalized to the untreated group.

Cells were harvested and washed twice with ice-cold PBS, after which they were resuspended in 1X binding buffer [0.01 M HEPES/NaOH (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl₂] at a concentration of 10⁶ cells/ml. Subsequently, 100 µl solution was transferred to a 5 ml cell culture tube and was treated with fluorescein isothiocyanate-conjugated Annexin V apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. The cells were analyzed using flow cytometry (DiVa 8.0.1; BD Biosciences) and a total of 10,000 cells/sample were analyzed to determine the percentage of apoptotic cells.

Total protein was extracted from the cells or tissues, as described previously (13). Protein concentrations were determined by a BCA protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Equal amounts of protein (30 µg) were mixed with SDS sample buffer, separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then incubated with 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in PBS at room temperature for 2 h, and were treated with primary antibodies overnight at 4°C. β-actin (sc-47778; 1:1,000; Santa Cruz Biotechnology, Inc.) was used as a protein-loading control. The next day, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (31460)/mouse (31430) immunoglobulin G (1:4,000; Invitrogen; Thermo fisher Scientific, Inc.) at room temperature for 2 h and were then detected using a standard chemiluminescence detection system (Pierce; Thermo fisher Scientific, Inc.). Band densities were analyzed using ImageJ software (Image J 1.43u; National Institute of Health, Bethesda, MD, USA).

NO levels were determined by Griess assay using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Cell supernatants (100 µl) were thoroughly mixed with reagent I (200 µl). The supernatants were obtained simply by a 200 µl pipette from the cell medium. Subsequently, reagent II (100 µl) was added and mixed for 10 min at room temperature, followed by centrifugation at 1,700 × g for 15 min at room temperature. Finally, 160 µl supernatants were mixed with 80 µl chromogenic reagent for 15 min at room temperature. The optical density of the samples was measured using a spectrophotometer with absorbance set at 550 nm. Sodium nitrite was used as a standard.

Samples were lysed in non-denaturing lysis buffer (25 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, 1% NP-40 and 1X protease inhibitor cocktail, pH 7.4). Protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein lysates (2 mg) were diluted to a final volume of 1.8 ml using HEN buffer (100 mM HEPES, 1 mM EDTA and 0.1 mM neocuproine). Subsequently, 0.2 ml 25% SDS and 20 µl 10% MMTS were added to block free thiols. After removing excess MMTS by acetone precipitation, the S-nitrosothiol (SNO) groups in the samples were reduced to thiols by 30 µl sodium ascorbate (200 mM) and biotinylated by 30 µl Biotin-HPDP (2.5 mg/ml). Finally, the biotinylated proteins were pulled down by streptavidin-agarose beads, and the beads were eluted by SDS loading buffer and subjected to western blot analysis.

Full-length wild-type ERK1 (ERK1^WT) cDNA clone was purchased from Sino Biological, Inc. (Beijing, China) and was subcloned into the 3xFlag vector. The primer sequences used for construction of Flag-ERK1^WT were as follows: Forward, 5′-CCGGAATTCATGGCGGCGGCGGCGGCTCA-3′ and reverse, 5′-CGCGGATCCGGGGGCCTCCAGCACTCCGG-3′. C183A mutant ERK1 (ERK1^C183A) cDNA was obtained by polymerase chain reaction [PCR; Tiangen Biotech (Beijing) Co., Ltd., Beijing, China] with the Flag-ERK1^WT plasmid used as the template, and was then subcloned into the 3xFlag vector. The primer sequences used were as follows: forward, 5′-CCTTAAGATTGCTGATTTCGGCCTGGC-3′ and reverse, 5′-GCCGAAATCAGCAATCTTAAGGTCGCAG-3′. PCR thermocycling was performed as follows: 94°C for 3 min; followed by 35 cycles at 90°C for 30 sec, 60°C for 45 sec and 72°C for 90 sec; 72°C for 10 min; hold at 4°C. The authenticity of the plasmids was confirmed by DNA sequencing. Briefly, when U251 cells reached a confluency of 40–50%, the cell medium was replaced with 2 ml fresh DMEM medium at 30 min before transfection. Plasmid (1 µg) and PolyJet™ reagent (3 µl) were mixed in high glucose DMEM medium at room temperature. The mixture was evenly added into the medium and the cells were incubated at 37°C for 6–8 h before replacing the medium with 2 ml fresh medium. After 48 h, the cells were used for the experiments. Transient transfection was performed using PolyJet™ according to the manufacturer's protocol. Expression of proteins was verified by western blot analysis using ERK1/2 and Flag antibodies.

Human glioma specimens, collected during surgical resection, and noncancerous brain tissues, collected during internal decompression after cerebral trauma, were obtained from the Affiliated Hospital of Xuzhou Medical University (Xuzhou, China). The clinicopathological characteristics of all of the subjects are presented in Table I. Surgically removed tissues were sampled for histological diagnosis, and the remaining tissues were immediately frozen and stored in liquid nitrogen for further analysis. All glioma specimens had a confirmed pathological diagnosis and were classified according to World Health Organization criteria (14). Informed consent was obtained from all subjects, or legal guardians, and the present study was approved by the Medical Ethical Committee of Xuzhou Medical University.

All quantitative data were obtained from at least three independent experiments and are presented as the means ± standard error of the mean [SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA)]. Data between two groups were assessed by Student's t-test, whereas one-way analysis of variance followed by Dunnett post hoc comparison was used to analyze data among three groups or more. P<0.05 was considered to indicate a statistically significant difference.

According to the literature, treatment with the NO donor SNP for 30–40 h significantly inhibits the growth of human glioma cells (15); therefore, the present study conducted a CCK-8 assay to evaluate the concentration-dependent effects of SNP and GSNO on cell survival after 36 h in U251 cells. Cell viability was significantly reduced following treatment with 0.25 and 0.5 mM SNP or 100–200 µM GSNO, and was reduced to a greater extent following treatment with 1 and 2 mM SNP or 250 and 500 µM GSNO (Fig. 1A and B). To determine the appropriate duration of high-dose SNP or GSNO treatment, U251 glioma cells were treated with 2 mM SNP or 500 µM GSNO for 0, 6, 12, 24, 36 and 48 h. Significant inhibition of cell survival was observed when cells were exposed to 2 mM SNP for 36 h or 500 µM GSNO for 36 and 48 h (Fig. 1C and D). These results indicated that high doses of NO donors exert significant inhibitory effects on the growth of glioma cells.

To ascertain whether the NO donors SNP and GSNO could release NO, the Griess method was used to measure NO levels in the supernatant of cultured U251 cells. Significant increases in NO release were detected following treatment with 1 or 2 mM SNP (Fig. 2A) and 250 or 500 µM GSNO (Fig. 2B). These data suggested that SNP and GSNO may breakdown to release NO into the culture supernatant of glioma cells.

The MAPK pathway has been reported to serve a critical role in cell survival (6), and increased phosphorylation of ERK1/2 has been detected in various grades of glioma (7,8). The present study investigated the effects of NO donor treatment on the expression levels of p-ERK1/2 in glioma cells. Initially, a time-dependent assay was performed in U251 glioma cells. The expression levels of p-ERK1/2 were significantly reduced following treatment with 2 mM SNP or 500 µM GSNO treatment for 24 and 36 h (Fig. 3A and B). Subsequently, U251 cells were exposed to various concentrations of SNP or GSNO for 36 h. A concentration-dependent decrease in p-ERK1/2 expression was evident in response to SNP and GSNO treatment. Significant decreases in the expression levels of p-ERK1/2 were observed following treatment with 1 and 2 mM SNP (Fig. 3C). Similarly, p-ERK1/2 expression was significantly reduced following 250 and 500 µM GSNO treatment (Fig. 3D). These data indicated that NO donor treatment, particularly in high concentrations, may attenuate the phosphorylation of ERK1/2 in glioma cells.

To determine whether ERK could be nitrosylated by NO, S-nitrosylation of ERK1/2 (SNO-ERK1/2) was analyzed by a biotin switch assay, followed by western blot analysis. Time- and dose-dependent increases in the levels of SNO-ERK1/2 were detected in response to GSNO treatment of U251 cells. The levels of ERK1/2-SNO were markedly increased at 24 and 36 h, and then returned to control levels at 48 h (Fig. 4A). ERK1/2-SNO was initially detected following treatment with 250 µM GSNO and was amplified with 500 µM GSNO treatment (Fig. 4B). These results suggested that ERK1/2 may be nitrosylated in a time- and dose-dependent manner.

ERK1 has six Cys residues in its FASTA sequence (Fig. 5A). A preliminary computational prediction indicated that Cys¹⁸³ is the most probable site for nitrosylation, according to the previously reported nitrosylation motif (K/R/H/D/E+C+D/E) (16). Therefore, Cys¹⁸³ was replaced with Ala, and the ERK1 mutant plasmid (ERK1^C183A) was constructed. Transfection efficiency of ERK1^WT and ERK1^C183A was verified by western blot analysis using anti-Flag and anti-ERK1/2 antibodies (Fig. 5B). The expression levels of ERK1-SNO were significantly attenuated following transfection of U251 cells with the ERK1^C183A plasmid (Fig. 5B and C). These results suggested that mutation at Cys¹⁸³ may partially prevent S-nitrosylation of ERK1/2 in glioma cells.

To determine the relationship between ERK1/2 nitrosylation and phosphorylation, U251 glioma cells were transfected with either ERK1^WT or ERK1^C183A plasmids, and were then treated with GSNO (500 µM). The results indicated that the expression levels of p-ERK were significantly decreased in the ERK1^WT-transfected cells when GSNO was added (Fig. 6A and B), which was consistent with the previous findings presented in Fig. 3D. However, GSNO failed to reduce p-ERK1/2 levels when U251 cells were transfected with the ERK1^C183A mutant (Fig. 6A and B). These findings indicated that preventing ERK1 S-nitrosylation may increase the phosphorylation of ERK1/2 in glioma cells.

The present study also investigated the effects of ERK1 S-nitrosylation prevention on the survival of glioma cells. In line with the data presented in Fig. 1, cell viability was significantly reduced following treatment with GSNO; however, the reduction in cell viability was reversed by a point mutation at Cys¹⁸³ of ERK1 (Fig. 6C). Furthermore, western blot analysis demonstrated that GSNO treatment induced an increase in cleaved caspase-3 expression; however, this was reversed following transfection with the ERK1^C183A mutant (Fig. 6D and E). In addition, flow cytometric apoptotic assay indicated that the percentage of U251 apoptotic cells transfected with ERK1^C183A mutant was significantly reduced following GSNO treatment compared with in the cells transfected with the ERK1^WT plasmid (Fig. 6F). Taken together, these results suggested that preventing S-nitrosylation of ERK1, via transfection with a ERK1^C183A mutant, partially reversed GSNO-induced decreases in ERK phosphorylation and cell survival in U251 glioma cells.

Western blot analysis was employed to detect p-ERK1/2 and total ERK1/2 levels, and a biotin switch assay followed by western blot analysis was used to measure ERK1/2-SNO levels, in 9 noncancerous brain tissues and 33 glioma specimens (n=11/grade). As presented in Fig. 7, the expression levels of p-ERK1/2 were increased in high-grade glioma tissues, particularly in glioma grade III (Fig. 7A and B), whereas there was a marked reduction in ERK1/2-SNO levels in glioma tissues, which was also evident in glioma grade III (Fig. 7A and C). These data provided in vivo evidence for the possible influence of ERK1/2 S-nitrosylation on ERK1/2 phosphorylation during glioma proliferation.