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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 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).
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pone.0134896.g001: 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).

Mentions: Most cancer cells are under oxidative stress, which is associated with increased metabolic activity and ROS generation. LMP1 is the major transforming protein of EBV and behaves as a classical oncogene in rodent fibroblast transformation assays [12]. In the NP69 immortalized nasopharyngeal epithelial cell line, LMP1 expression induced a morphological change from a typical epithelial cobblestone shape to an elongated and fibroblastoid shape (S1A and S1B Fig). In serum-free keratinocyte medium supplemented with growth factors, NP69-LMP1 cells proliferated rapidly with an average doubling time of 34 h. In contrast, the NP69 cell proliferation rate was considerably reduced with a doubling time of 70 h (i.e., 2-fold longer than NP69-LMP1 cells, S1C Fig). Importantly, LMP1-transformed NP69 cells exhibited significantly increased basal ROS levels (approximately 8- and 10-fold increased) as quantified by flow cytometry using CM-H2DCF-DA and DCF-FM fluorescent probes, which are specific for hydrogen peroxide and nitrogen oxide, respectively (Fig 1A and 1E, fluorescence presented using a logarithmic scale). These data suggested that LMP1 expression causes cellular ROS accumulation 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 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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526464&req=5

pone.0134896.g001: 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).
Mentions: Most cancer cells are under oxidative stress, which is associated with increased metabolic activity and ROS generation. LMP1 is the major transforming protein of EBV and behaves as a classical oncogene in rodent fibroblast transformation assays [12]. In the NP69 immortalized nasopharyngeal epithelial cell line, LMP1 expression induced a morphological change from a typical epithelial cobblestone shape to an elongated and fibroblastoid shape (S1A and S1B Fig). In serum-free keratinocyte medium supplemented with growth factors, NP69-LMP1 cells proliferated rapidly with an average doubling time of 34 h. In contrast, the NP69 cell proliferation rate was considerably reduced with a doubling time of 70 h (i.e., 2-fold longer than NP69-LMP1 cells, S1C Fig). Importantly, LMP1-transformed NP69 cells exhibited significantly increased basal ROS levels (approximately 8- and 10-fold increased) as quantified by flow cytometry using CM-H2DCF-DA and DCF-FM fluorescent probes, which are specific for hydrogen peroxide and nitrogen oxide, respectively (Fig 1A and 1E, fluorescence presented using a logarithmic scale). These data suggested that LMP1 expression causes cellular ROS accumulation 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