Fig 1.
Oncogenic transformation by LMP1 causes increased ROS generation.
A: Basal hydrogen peroxide levels in immortalized nasopharyngeal epithelial cells (NP69 and NP69-LMP1) and NPC cells (C666-1, CNE-2 and SUNE-1) were detected by flow cytometry using DCF-DA. Each histogram is representative of three experiments. Compared to NP69 cells, NP69-LMP1 cells exhibit significantly higher level of hydrogen peroxide (p<0.05). B: Basal protein expression of catalase and superoxide dismutase 2 (SOD2) in immortalized nasopharyngeal epithelial cells (NP69 and NP69-LMP1) and NPC cells (C666-1, CNE-2 and SUNE-1). β-Actin served as a loading control. C: Comparison of total cellular GSH in immortalized nasopharyngeal epithelial cells (NP69 and NP69-LMP1) and NPC cells (C666-1, CNE-2 and SUNE-1) (mean ± SD of three experiments, * p<0.05). D: Basal protein expression of catalase, glutathione peroxidase (GPX) and superoxide dismutase (SOD1 and SOD2) in NP69 and NP69-LMP1 cells. β-Actin served as a loading control. E: Increase in ROS levels in NP69-LMP1 cells detected by flow cytometry using DCF-DA (left panel) and DAF-FM (middle panel). Superoxide was detected using HEt (right panel). Each histogram is representative of three experiments. Compared to NP69 cells, NP69-LMP1 cells exhibit significantly higher level of ROS contents, including hydrogen peroxide and nitrogen oxide (p<0.05).
Fig 2.
Oncogenic transformation by LMP1 causes increased NOX activity.
A: Comparison of NOX activity in NP69 and NP69-LMP1 cells (left panel), NP69-pZIPNeoSV(X)1 and NP69-pZIPNeoSV(X)1-LMP1 transient-transfected cells (right panel) measured by a luminometer using lucigenin in the presence of NADPH (mean ± SD of three experiments; * p < 0.01). B: In NP69-LMP1 cells, ROS level was suppressed by both 0.5 mM NAC (34% at 5 hr and 83% at 8 hr, left panel) and 5 μM DPI (22% at 5 hr and 78% at 8 hr, right panel). ROS level was measured by flow cytometry using DCF-DA. Each histogram is representative of three experiments. C: Expression profile of NOX family subunits by RT-PCR in NP69 and NP69-LMP1 cells. LMP1 induced a significant increase in p22phox expression. β-Actin served as a loading control. Compared to NP69 cells, p22phox expression was significantly higher in NP69-LMP1 cells (*p<0.001). D: Basal protein expression of c-Jun, phosphorylated-c-Jun and p22phox in NP69 and NP69-LMP1 cells. β-Actin served as a loading control.
Fig 3.
Inhibition of the JNK pathway downregulated the expression of p22phox.
A: NP69 cells were transiently transfected with 200 ng/mL empty pZIP-SV(X) plasmid or pZIP-SV(X)-LMP1 plasmid for 48 hr. The increase in ROS in NP69-LMP1 cells was detected by flow cytometry using DCF-DA. Because transient transfection could cause some cells to die and this group of cells could not be stained with DCF-DA, we gated the healthy population to avoid the invalid signal. Each histogram is representative of three experiments. B: NP69 cells were transfected with 200 ng/mL empty pZIP-SV(X) plasmid or 50 to 400 ng/mL pZIP-SV(X)-LMP1 plasmid for 48 hr (upper panel), and NP69 cells were then transfected with 200 ng/mL pZIP-SV(X)-LMP1 plasmid for 24 to 96 hr (middle panel). The expression levels of LMP1 and p22phox in NP69 cells transfected with LMP1 were measured by RT-PCR. NP69 cells were transfected with 200 ng/mL empty pZIP-SV(X) plasmid or 50 to 300 ng/mL pZIP-SV(X)-LMP1 plasmid for 48 hr (lower panel). The phosphorylation of c-Jun and the upregulation of p22phox in LMP1-transfected NP69 cells were detected by an immunoblotting assay. β-Actin served as a loading control. C: Effects of JNK inhibitor SP600125 on the transcription of p22phox and LMP1 in NP69-LMP1 cells (upper panel) and NP69 cells transiently transfected with 200 ng/mL pZIP-SV(X)-LMP1 plasmid for 48 hr (lower panel). β-Actin served as a loading control. D: Effects of SP600125 on the protein expression level of p22phox and phosphorylation of c-Jun in NP69-LMP1 cells. NP69-LMP1 cells were incubated with 10 μM SP600125 for 1 or 2 hr, and cell lysates were analyzed by immunoblotting. β-Actin served as a loading control. E: Effect of SP600125 on the cellular ROS level in NP69-LMP1 cells. Each histogram is representative of three experiments.
Fig 4.
A positive correlation between LMP1 and p22phox expression was detected in nasopharyngeal carcinoma.
A: The distributions of NPC tissue samples and non-NPC tissue samples with LMP1high/ p22phox high, LMP1low/p22phox low, LMP1high/p22phox low and LMP1low/p22phox high were determined with SPSS correlation analysis. B: In a tissue array, the p22phox and LMP1 expression levels were detected in NPC cancer tissues using an IHC assay. P1 and P2 represents two NPC tissues from patients with distinctive p22phox and LMP1 expression pattern. P1 tissue has low p22phox and LMP1 expression level, and P2 has higher LMP1 and p22phox expression level. NPC tissue sample with LMP1 negative and p22phox were served as negative control (magnification × 400).
Table 1.
Analysis of the correlation between LMP1 and p22phox expression NPC tissues and non-cancer tissues.
Fig 5.
LMP1 causes a high glycolytic level and induces vulnerability to DPI.
A: Lactate production in NP69 and NP69-LMP1 cells. Cells were incubated in KSF medium at a density of 5 × 105 cells/mL for 24 hr. The lactate concentration in the medium was measured using an Accutrend Lactate Analyzer as described in Materials and Methods (mean ± SD of three experiments, * p<0.05). B: Comparison of glucose uptake in NP69 and NP69-LMP1 cells for 1 hr. Cells were incubated in glucose-free RPMI 1640 for 2 hr. Glucose uptake was detected by flow cytometry using a fluorescent deoxyglucose analog (2-NBDG). Each histogram is representative of three experiments. C: Oxygen consumption in NP69 and NP69-LMP1 cells (mean ± SD of three experiments, p = 0.5106). D: Comparison of lactate generation (left panel) and glucose consumption (right panel) in NP69-pZIPNeoSV(X)1 and NP69-pZIPNeoSV(X)1-LMP1 transient-transfected cells. Medium lactate and glucose concentration (nmol/L per 105 cells, mean ± SD of three experiments, * p<0.05) were measured as described in Materials and Methods. E: An inhibitor of glucose uptake, 3-bromopyruvate, was used to test the glucose uptake in NP69 and NP69-LMP1 cells. Each histogram is representative of three experiments. F: Effect of combining DPI and 3-bromopyruvate on lactate production in NP69-LMP1 cells. A volume of 30 μM 3-bromopyruvate completely inhibited lactate production in NP69-LMP1 cells. When combined with 3-bromopyruvate, 5 μM DPI treatment could not induce any increase in lactate (mean ± SD of three experiments). * p<0.05, DPI treated group vs untreated control group; ** p<0.05, 3-BrOP treated group alone or combined with DPI vs DPI treated group. G: Treatment with 0.3–30 μM DPI preferentially killed NP69-LMP1 cells compared to NP69 cells (mean ± SD of three experiments). H: Preferential killing of NP69-LMP1 cells by 5 μM DPI was detected by annexin V-PI staining and flow cytometry. The numbers under the plots indicate live cells with both low annexin V staining and low PI staining. Each histogram is representative of three experiments.
Fig 6.
NOX activation makes NPC cells vulnerable to the NOX inhibitor DPI.
A: p22phox mRNA expression level was evaluated in NP69, NP69-LMP1 and CNE2 cells by RT-PCR and real-time quantitative PCR. LMP1 expression was detected in NPC cells and B95.8 B lymphoma cells. β-Actin served as a loading control (mean ± SD of three experiments). Compared to NP69 cells, NP69-LMP1 and CNE2 cells had significantly higher p22phox mRNA expression level (*p<0.01). B: Comparison of NOX activity and the effect of DPI on NOX activity in NP69, NP69-LMP1 and CNE2 cells, measured by a luminometer using lucigenin in the presence of NADPH (mean ± SD of three experiments). Compared to NP69 cells, NP69-LMP1 and CNE2 cells had significantly higher NOX activity (* p < 0.01). DPI treatment could significantly suppress NOX activity in NP69-LMP1 and CNE2 cells (** p<0.01). C: Mortality effect of 0.03–10 μM DPI on CNE2 NPC cells, evaluated using an MTT assay (mean ± SD of three experiments). DPI suppressed CNE2 cells proliferation. D: Mortality effect of 5 μM DPI on CNE2 cells, detected using annexin V-PI staining and flow cytometry. The numbers under the plots indicate the live cells with both low annexin V staining and low PI staining. Each histogram is representative of three experiments.