Limits...
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

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526464&req=5

pone.0134896.g006: 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.

Mentions: In NPC cells (CNE2 cells, Fig 6A and 6B), NOX was also activated and p22phox expression was upregulated. These data suggest that NOX activation might also play an important role in malignant transformation and metabolic disorders in NPC cells that lack LMP1 expression. Next, we assessed the effects of DPI on CNE2 cells. Based on MTT assay, DPI treatment (0.1–3 μM) significantly inhibited proliferation in CNE2 cells at 72 h (Fig 6C). Annexin V-PI staining and flow cytometry analysis further confirmed the effect of DPI treatmen t(5 μM) in CNE2 cells, with approximately 90% apoptotic cells at 24 hr (Fig 6D). These findings suggested that LMP1-stimulated NOX activation plays important roles in EBV-related malignant cells. Thus, DPI-mediated inhibition of NOX activity could effectively induce cell death in these cancer 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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526464&req=5

pone.0134896.g006: 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.
Mentions: In NPC cells (CNE2 cells, Fig 6A and 6B), NOX was also activated and p22phox expression was upregulated. These data suggest that NOX activation might also play an important role in malignant transformation and metabolic disorders in NPC cells that lack LMP1 expression. Next, we assessed the effects of DPI on CNE2 cells. Based on MTT assay, DPI treatment (0.1–3 μM) significantly inhibited proliferation in CNE2 cells at 72 h (Fig 6C). Annexin V-PI staining and flow cytometry analysis further confirmed the effect of DPI treatmen t(5 μM) in CNE2 cells, with approximately 90% apoptotic cells at 24 hr (Fig 6D). These findings suggested that LMP1-stimulated NOX activation plays important roles in EBV-related malignant cells. Thus, DPI-mediated inhibition of NOX activity could effectively induce cell death in these cancer 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