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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

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.
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pone.0134896.g003: 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.

Mentions: LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) and activates a number of signaling pathways, including NF-κB, JNK and STAT. In addition, researchers have recently demonstrated that the JNK pathway is responsible for the specific expression of the NOX regulatory subunit p22phox [16]. Therefore, we hypothesized that LMP1 upregulates the NOX subunit p22phox by activating the JNK pathway. Using TRANSFAC software, we identified a putative AP-1 binding site in the p22phox promoter region. In addition, immunoblotting data indicated that both c-Jun phosphorylation levels and p22phox expression levels were significantly upregulated in NP69-LMP1 cells compared with NP69 cells (Fig 2D). To investigate the molecular mechanism involved in the ellular redox status changes, we used a pZIPNeoSV(X)1-LMP1 transient transfection system. As presented in Fig 3A, compared with NP69 cells transfected with an empty vector, pZIPNeoSV(X)1-LMP1 transfection altered the cellular ROS level, promoting an obvious increase (greater than 2-fold) in cellular hydrogen peroxide concentrations, as quantified by flow cytometry using the DCF-DA fluorescent probe. Furthermore, p22phox mRNA and LMP1 expression were significantly increased (Fig 3B). In addition, along with LMP1 expression, c-Jun phosphorylation and p22phox protein expression were also detected by immunoblotting analysis in NP69 cells transfected with pZIPNeoSV(X)1-LMP1 (Fig 3B). These findings suggested that the JNK pathway was activated by LMP1 and this pathway potentially mediates LMP-induced p22phox upregulation.


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)

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.
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pone.0134896.g003: 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.
Mentions: LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) and activates a number of signaling pathways, including NF-κB, JNK and STAT. In addition, researchers have recently demonstrated that the JNK pathway is responsible for the specific expression of the NOX regulatory subunit p22phox [16]. Therefore, we hypothesized that LMP1 upregulates the NOX subunit p22phox by activating the JNK pathway. Using TRANSFAC software, we identified a putative AP-1 binding site in the p22phox promoter region. In addition, immunoblotting data indicated that both c-Jun phosphorylation levels and p22phox expression levels were significantly upregulated in NP69-LMP1 cells compared with NP69 cells (Fig 2D). To investigate the molecular mechanism involved in the ellular redox status changes, we used a pZIPNeoSV(X)1-LMP1 transient transfection system. As presented in Fig 3A, compared with NP69 cells transfected with an empty vector, pZIPNeoSV(X)1-LMP1 transfection altered the cellular ROS level, promoting an obvious increase (greater than 2-fold) in cellular hydrogen peroxide concentrations, as quantified by flow cytometry using the DCF-DA fluorescent probe. Furthermore, p22phox mRNA and LMP1 expression were significantly increased (Fig 3B). In addition, along with LMP1 expression, c-Jun phosphorylation and p22phox protein expression were also detected by immunoblotting analysis in NP69 cells transfected with pZIPNeoSV(X)1-LMP1 (Fig 3B). These findings suggested that the JNK pathway was activated by LMP1 and this pathway potentially mediates LMP-induced p22phox upregulation.

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