Oxamate sodium was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). CNE-1 and CNE-2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cell types were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco), supplemented with 10% fetal calf serum (FCS), 5 mmol/l L-glutamine, 5 mmol/l non-essential amino acids, 100 U/ml penicillin and 100 U/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37ºC under 5% CO₂.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed in 96-well plates to test cell viability. Cells were seeded at 10⁴/well and treated with different concentrations of oxamate sodium (0–100 mmol/l) for 24, 48 and 72 h, respectively. Then 20 μl of MTT solution (5 mg/l) was added to each well, and the plates were incubated at 37ºC for another 4 h. The supernatant was discarded, and 150 μl dimethyl sulfoxide was added for 10 min. The absorbance was measured using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 570 nm. The cell viability is expressed as the percentage of untreated control cells.

Cells pretreated with 20 mmol/l oxamate sodium for 24 h were irradiated at 0–9 Gy with 6 MV X-rays from a linear accelerator (Siemens, Munich, Germany), with a dose rate of 200 cGy/min. The cells were then seeded at different densities according to the IR dose. After 14 days of incubation at 37ºC, the cells were fixed, stained with Giemsa and counted. Colonies were scored only when containing ≥50 cells. Experiments were conducted in triplicate. Survival fractions were fitted into a linear quadratic model using GraphPad Prism software (version 5.0).

Intracellular LDH activity was determined using an LDH activity assay kit (BioVision, Tucson, AZ, USA). The assay is based on an enzymatic coupling reaction in which LDH reduces NAD to NADH, which then reacts with a specific probe to generate a color (λ_max = 450 nm). Cells treated with different concentrations of oxamate were harvested and lysed for protein to measure the LDH activity. Results were normalized to the protein concentrations of the cell lysate, and the protein concentrations were determined using a BCA protein assay kit (Beyotime, Haimen, China).

Intracellular ATP was measured using a firefly luciferase-based ATP assay kit (Beyotime) following the manufacturer’s instructions. Briefly, cells were cultured in DMEM with different concentrations of oxamate sodium (0, 20, 50 and 100 mmol/l). After 24 h, cells were lysed and centrifuged at 12,000 × g for 5 min. The supernatant (100 μl) was transferred to a 24-well plate mixed with 100 μl ATP detection working dilution. Luminescence was measured by a microplate reader (Bio-Tek Instruments, Inc.). The protein concentration of each group was also determined using a BCA protein assay kit. The relative ATP level is expressed as ATP value/protein value.

Cells were collected at 24 h after treatment with different concentrations of oxamate sodium, then fixed with 70% ethanol and stored overnight at 4ºC. After centrifugation and washing with 1X PBS twice, the fixed cells were stained with 500 μl of propidium iodine (PI) (10 μg/ml; Sigma-Aldrich) for 30 min in the dark. Cell cycle distribution was analyzed using 10,000 cells by flow cytometry with the FACSCalibur system (Becton-Dickinson, San Jose, CA, USA).

Apoptosis was tested using the Annexin V-FITC apoptosis kit (BD Biosciences, San Jose, CA, USA). Cells treated with different concentrations of oxamate sodium for 48 h were harvested and stained with Annexin V/PI for 30 min. The apoptotic fraction was detected by a flow cytometer with the FACSCalibur system.

ROS content was tested using an ROS detection kit (Beyotime), based on the intracellular peroxide-dependent oxidation of DCFH-DA to form the fluorescent compound 2′,7′-dichlorofluorescein (DCF). Cells treated with 0, 20, 50 and 100 mmol/l oxamate sodium were collected and incubated with 1 ml DCFH-DA (20 μM) for 30 min. Fluorescence intensity was detected by flow cytometry.

Cells were harvested and suspended in IP lysing buffer (Beyotime) containing protease inhibitor (25 mg/ml; Roche, Mannheim, Germany). The protein concentration was measured using the BCA protein assay kit. Then 50 μg of total protein was separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in PBS with 5% non-fat dry milk for 45 min, the membranes were incubated with relevant antibodies for 1 h. Signals were detected using an ECL western blotting kit (Beyotime). The following antibodies were utilized: anti-cyclin B1 mouse antibody (1:500; Santa Cruz Biotechnology), anti-CDK1 mouse antibody (1:500; Santa Cruz Biotechnology), anti-Bcl-2 mouse antibody (1:500; Santa Cruz Biotechnology, anti-Bax mouse antibody (1:500; Santa Cruz Biotechnology), anti-caspase-3 rabbit antibody (1:1,000; Cell Signaling Technology), anti-cleaved-caspase-3 rabbit antibody (1:1,000; Cell Signaling Technology), anti-β-actin antibody (1:2,000; Santa Cruz Biotechnology), anti-mouse antibody (1:1,000; Santa Cruz Biotechnology) and anti-rabbit antibody (1:1,000; Santa Cruz Biotechnology).

Female Balb/c nude mice weighing 18–22 g at 4–6 weeks of age were purchased from the Institute of Laboratory Animal Sciences (Shanghai, China) and were raised under specific pathogen-free environments. CNE-1 cells (1×10⁶) suspended in 100 μl of 1:1 DMEM culture and Matrigel (BD Biosciences) were injected subcutaneously into the flanks of the nude mice. When tumor sizes reached ~100 mm³, 24 mice were randomly divided into 4 groups (n=6 mice/group) and treated with PBS, oxamate, irradiation, or oxamate combined with irradiation, respectively. Oxamate was intraperitoneally injected at 750 mg/kg daily for 3 weeks, and mice were irradiated with 3.3 Gy X-rays for a consecutive 3 days (total dose of 9.9 Gy) 2 h after injection of oxamate from the second day of drug administration using a 6-MV linear accelerator (Siemens). Tumor sizes and mouse body weights were measured every 3 days for 5 weeks. Tumor volumes were calculated using the formula: Volume = length × width²/2.

Each experiment was repeated three times and performed in triplicate. Data are expressed as the means ± standard deviation (SD). Differences between two groups were assessed by the two-tailed Student’s t-test. All of the statistical analyses were performed using SPSS version 17.0. A P-value <0.05 was considered to indicate a statistically significant result.

First, to explore the effect of oxamate on cell proliferation in NPC cells, MTT assays were conducted. CNE-1 and CNE-2 cancer cells were treated with different doses of oxamate for 24–72 h. As shown in Fig. 1A, treatment with increasing concentrations of oxamate inhibited cell proliferation in a dose- and time-dependent manner in both NPC cancer cell lines. The IC₅₀ values were 74.6, 32.4 and 17.8 mmol/l and 62.3, 44.5, 31.6 mmol/l at 24, 48 and 72 h in the CNE-1 and CNE-2 cancer cells, respectively. Then, to confirm the LDH inhibition effect of oxamate as previously reported (18,19), a commercially available kit was used to determine the intracellular LDH enzyme activity after treatment with different doses of oxamate. The results showed that oxamate significantly decreased LDH activity (Fig. 1B), which indicated that oxamate was an inhibitor of human LDH enzyme and provided evidence of the LDH inhibitory effect by oxamate for the subsequent experiments. Fig. 1C shows that oxamate markedly reduced the intracellular ATP levels. Relative ATP levels in the 20, 50 and 100 mmol/l oxamate-treated groups were 87.3±5.2, 51.3±8.5 and 32.7±4.1%, respectively, when compared to the untreated control group (100±7.0%) (P=0.21, 0.025 and 0.007) in the CNE-1 cell line, and 84.3±5.0, 47.6±8.3 and 27±5.3%, when compared to the untreated control group (100±6.2%) (P=0.002, 0.001 and <0.001) in the CNE-2 cell line. The results demonstrated that LDH inhibition by oxamate disturbed energy metabolism and decreased ATP production in the NPC cells.

Since a great majority of anticancer agents act on the cell cycle and its checkpoint (23), alterations in cell cycle distribution after oxamate treatment were investigated in NPC cancer cells. Cells were exposed to 0, 20, 50 and 100 mmol/l oxamate for 24 h, and flow cytometry was used to analyze the cell cycle changes after PI staining. As shown in Fig. 2A, there was a dose-dependent increase in the numbers of CNE-1 and CNE-2 cells in the G2/M phase after a 24-h treatment with oxamate at concentrations of 0, 20, 50 and 100 mmol/l. The percentages of cells in the G2/M phase were 18.8±2.1, 26.9±1.6, 35.4±2.3, 43.6±3.6, respectively, in the CNE-1 cells and 13.0±3.2, 19.7±2.7, 21.5±2.3 and 26.1±1.1%, in the CNE-2 cells, respectively. Meanwhile, the proportions of cells in the G₀/G₁ phase were decreased. However, the S phase fractions did not change significantly in both cell lines. In addition, it was noteworthy that there was a significant increase in the percentage of cells in the sub-G₁ phase after treatment with oxamate.

To further certify the G₂/M arrest induced by oxamate and to understand the mechanisms, western blot analysis was used to examine the changes in expression of proteins related to G₂/M transition. As shown in Fig. 2B, the expression levels of cyclin B1 and CDK1 significantly decreased in both the CNE-1 and CNE-2 cells after treatment with oxamate, which suggests that oxamate induces G₂/M arrest by modulating the expression of cyclin B1 and CDK1.

We demonstrated that LDH inhibition by oxamate impaired cell growth and increased the sub-G₁ fraction in both cell lines. To confirm that oxmate treatment induces apoptosis, cells were exposed to different concentrations of oxamate for the prolonged time of 48 h, and then Annexin V/PI staining and flow cytometric analysis were performed. After the 48-h treatment with oxamate, the percentages of early and late apoptotic cells were increased in a dose-dependent manner (Fig. 3A). Next, western blot analysis was utilized to determine the changes in expression of apoptosis-related proteins. As shown in Fig. 3B, the expression of pro-apoptotic Bax and cleaved-caspase-3 was significantly enhanced, while the anti-apoptotic signals of Bcl-2 and pro-caspase-3 were reduced notably after treatment with different concentrations of oxamate for 48 h. The results indicate that oxamate induces apoptosis via caspase-3 activation and the mitochondrial pathway in NPC cells.

To further explore the mechanisms involving the inhibitory effect induced by oxamate in NPC cells, we determined the changes in ROS levels after oxamate treatment, which play an important role in the mitochondrial apoptotic pathway. As shown in Fig. 4A, oxamate dose-dependently enhanced ROS levels in both NPC cell lines. ROS levels were increased to 1.3-, 2.4- and 3.1-fold (P<0.01) after treatment with 20, 50 and 100 mmol/l oxamate for 24 h, when compared to the untreated control group. Similarly, there was an 1.5- to 3.3-fold increase (P<0.01) in the CNE-2 cells. To examine the role of ROS generation in the oxamate-induced growth inhibitory effect, oxamate-treated cells were incubated simultaneously with 10 mM N-acetylcysteine, a specific scavenger of ROS. As shown in Fig. 4B, pre-treatment with NAC significantly blocked the growth inhibitory effect in both CNE-1 and CNE-2 cell lines, indicating that ROS generation contributes partially to the anti-proliferative effect induced by oxamate.

Radiotherapy is the main treatment for NPC patients at present. Thus, we examined whether LDH inhibition by oxamate influences the sensitivity of NPC cells to ionizing radiation. Firstly, Annexin-V/PI staining was conducted to observe the effect of oxamate on the apoptosis induced by irradiation within a short period of time, and then colony formation assays were performed to evaluate the long-term effects of the combined treatment. As shown in Fig. 5A the combination of irradiation and oxamate synergistically enhanced the apoptosis rates in both NPC cancer cells, when compared to either treatment alone. Furthermore, oxamate enhanced the IR-induced inhibition of clonogenic survival in NPC cells (Fig. 5B). The sensitivity enhancement ratios (SERs) were 1.26 and 1.35 in CNE-1 and CNE-2 cells, respectively, as determined by analyzing the linear quadratic models. These results revealed that oxamate increased the radiosensitivity in NPC cells in vitro, and the effects were similar in both CNE-1 and CNE-2 cells.

Finally, we examined whether oxamate inhibits tumor growth in vivo and its effects when combined with irradiation treatment. As shown in Fig. 6A, oxamate effectively delayed tumor growth in vivo. Moreover, combined treatment with oxamate and irradiation significantly improved the growth inhibitory effect when compared to either oxamate alone or irradiation alone. As shown in Fig. 6B that inhibition by oxamate had little influence on the body weights of the mice. A small decrease in body weights of the mice was noted following treatment with oxamate and irradiation. However, the decrease had no statistical significance when compared to the mice treated with oxamate alone.