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LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor.

Sun J, Hu C, Zhu Y, Sun R, Fang Y, Fan Y, Xu F - PLoS ONE (2015)

Bottom Line: In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity.Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression.The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, P.R.China.

ABSTRACT
Oxidative stress is thought to contribute to cancer development. Epstein-Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0134896.g002: 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.

Mentions: Next, to avoid the interference from the antioxidant system in NPC cells, we used NP69 and NP69-LMP1 cells to extensively investigate the potential molecular mechanism involved in LMP1-mediated ROS generation. By consuming NAD(P)H and transferring electrons across biological membranes, the NOX family is an important intracellular source of superoxide and downstream ROS [14]. As shown in Fig 2A, compared with parental NP69 cells, LMP1-transformed NP69 cells exhibited a significant increase (nearly 5-fold) in NOX activity, as quantified by lucigenin chemiluminescence in the presence of NAD(P)H using a luminometer. Then, we used a pZIPNeoSV(X)1-LMP1 transient transfection system. As shown in Fig 2A, compared with NP69 cells transfected with empty vector, pZIPNeoSV(X)1-LMP1 transfection also significantly increased NOX activity (approximately 4.6 folds; p < 0.001). To further characterize the effect of NOX on intracellular ROS levels, we treated NP69-LMP1 cells with DPI and N-acetylcysteine (NAC). NAC is an effective antioxidant compound that boosts cellular GSH. Using DCF-DA and flow cytometry, we revealed that LMP1-induced ROS accumulation was reduced by NAC (34% and 83% decrease at 5 and 8 hr, respectively). However, LMP1-induced ROS accumulation was only partly rescued by DPI (22% and 78% decrease at 5 and 8 hr, respectively; Fig 2B). These observations suggest that the observed LMP1-induced ROS accumulation was not exclusively caused by activation of NOX family members. Nevertheless, to further identify which NOX family member plays a vital role in nasopharyngeal epithelial cells, we screened NOX subunit expression profiles in NP69 and NP69-LMP1 cells using RT-PCR. As shown in Fig 2C, among the five catalytic enzymes assessed (NOX1–5), only NOX4 mRNA was expressed at a detectable level in both NP69 and NP69-LMP1 cells. More importantly, mRNA of the regulatory subunit p22phox was significantly upregulated in LMP1-transformed cells compared with parental NP69 cells. Immunoblotting data further confirmed p22phox upregulation in LMP1-overexpressing NP69 cells (Fig 2D). Shiose et al. demonstrated that NOX4 consistently colocalizes with p22phox at the cellular membranes and requires p22phox for its catalytic activity [15]. Together, these data suggest that NOX4 and upregulated p22phox may represent effective targets for the NOX inhibitor DPI in nasopharyngeal epithelial cells.


LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor.

Sun J, Hu C, Zhu Y, Sun R, Fang Y, Fan Y, Xu F - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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pone.0134896.g002: 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.
Mentions: Next, to avoid the interference from the antioxidant system in NPC cells, we used NP69 and NP69-LMP1 cells to extensively investigate the potential molecular mechanism involved in LMP1-mediated ROS generation. By consuming NAD(P)H and transferring electrons across biological membranes, the NOX family is an important intracellular source of superoxide and downstream ROS [14]. As shown in Fig 2A, compared with parental NP69 cells, LMP1-transformed NP69 cells exhibited a significant increase (nearly 5-fold) in NOX activity, as quantified by lucigenin chemiluminescence in the presence of NAD(P)H using a luminometer. Then, we used a pZIPNeoSV(X)1-LMP1 transient transfection system. As shown in Fig 2A, compared with NP69 cells transfected with empty vector, pZIPNeoSV(X)1-LMP1 transfection also significantly increased NOX activity (approximately 4.6 folds; p < 0.001). To further characterize the effect of NOX on intracellular ROS levels, we treated NP69-LMP1 cells with DPI and N-acetylcysteine (NAC). NAC is an effective antioxidant compound that boosts cellular GSH. Using DCF-DA and flow cytometry, we revealed that LMP1-induced ROS accumulation was reduced by NAC (34% and 83% decrease at 5 and 8 hr, respectively). However, LMP1-induced ROS accumulation was only partly rescued by DPI (22% and 78% decrease at 5 and 8 hr, respectively; Fig 2B). These observations suggest that the observed LMP1-induced ROS accumulation was not exclusively caused by activation of NOX family members. Nevertheless, to further identify which NOX family member plays a vital role in nasopharyngeal epithelial cells, we screened NOX subunit expression profiles in NP69 and NP69-LMP1 cells using RT-PCR. As shown in Fig 2C, among the five catalytic enzymes assessed (NOX1–5), only NOX4 mRNA was expressed at a detectable level in both NP69 and NP69-LMP1 cells. More importantly, mRNA of the regulatory subunit p22phox was significantly upregulated in LMP1-transformed cells compared with parental NP69 cells. Immunoblotting data further confirmed p22phox upregulation in LMP1-overexpressing NP69 cells (Fig 2D). Shiose et al. demonstrated that NOX4 consistently colocalizes with p22phox at the cellular membranes and requires p22phox for its catalytic activity [15]. Together, these data suggest that NOX4 and upregulated p22phox may represent effective targets for the NOX inhibitor DPI in nasopharyngeal epithelial cells.

Bottom Line: In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity.Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression.The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, P.R.China.

ABSTRACT
Oxidative stress is thought to contribute to cancer development. Epstein-Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.

No MeSH data available.


Related in: MedlinePlus